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A 10-year surveillance of antimicrobial susceptibility patterns in Shigella sonnei isolates circulating in Jiangsu Province, China

Highlights • Long-term multicentre surveillance of antimicrobial susceptibility patterns in Shigella sonnei.• Epidemic clones and integron types and resistance gene cassettes were characterised.• PFGE indicated large-scale clonal transmission among different cities occurred several times during 10 years.• Class 1, 2 and atypical class 1 integrons were detected in S. sonnei.• High prevalence of integrons and gene cassettes was related to the increasing antimicrobial resistance.

Introduction
Shigella is a major gastrointestinal pathogen accounting for 5-10% of diarrhoeal diseases or bacillary dysentery throughout the world [1]. In China, up to 1.7 million episodes of shigellosis occurred each year, with up to 0.20 million patients hospitalised [2]. Among the four species of Shigella (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), S. sonnei has been the predominant species in developed countries and is often the second most prevalent species in developing countries for many years [3][4][5]. Recently, S. sonnei has become increasingly important due to the growing rate of this species in many developing countries [6,7].
Antimicrobial treatment is usually recommended for shigellosis as it can reduce the duration of symptoms and the severity of illness by shortening the period of pathogen excretion [8]. However, owing to long-term overuse of antimicrobials, drugresistant and multidrug-resistant (MDR) Shigella isolates prevail worldwide [9,10]. Evidence from China [11], Bangladesh [5], Belgium [12], Gabon [13] and the USA [14] indicated that China has a much higher prevalence of resistance to most commonly used antimicrobials.
The close correlation between antimicrobial resistance and the presence of integrons containing gene cassettes has been frequently reported in studies from various countries and regions [10,15,16]. However, limited information is available on the following three aspects: the regional level of the antimicrobial resistance profile; the genotype level of the dominating endemic strains; and long-term surveillance and comparison of integrons and gene cassettes.

Sample collection
A total of 340 S. sonnei were collected between 2002 and 2011 from outpatients and inpatients in hospitals throughout Jiangsu Province. Jiangsu Province, a populous and prosperous province in China, was divided into three regions comprised of 13 provincial cities, the South (Nanjing, Suzhou, Wuxi, Changzhou and Zhenjiang), the Centre (Yangzhou, Taizhou and Nantong) and the North (Xuzhou, Suqian, Huai'an, Yancheng and Lianyungang). As shown in Table 1, South Jiangsu exhibited the highest isolation rate of S. sonnei (186/340; 54.7%), followed by the Centre (84/340; 24.7%) and the North (70/340; 20.6%).
All strains were identified by API system (bioMérieux, Marcyl'Étoile, France) and were confirmed as S. sonnei by slide agglutination with Shigella antisera (Lanzhou Institute of Biological Products, Lanzhou, China).
In view of the small sample size (n = 21 strains) and different antimicrobials used in antimicrobial susceptibility testing in 2002-2005, this study only compared the resistance rates of S. sonnei collected in 2006-2011.

Pulsed-field gel electrophoresis (PFGE)
To determine DNA fingerprinting profiles, the 340 S. sonnei strains were analysed by PFGE according to the US Centers for Disease Control and Prevention (CDC) PulseNet protocol [18] and were digested with the restriction enzyme Xbal (Takara, Dalian, China). Electrophoresis was performed with a CHEF-DR II system (Bio-Rad, Hercules, CA) under the following conditions: switching time from 2.2 s to 54.2 s at 6 V for 17 h with an angle of 120 . Salmonella enterica serovar Braenderup H9812 was used as a molecular weight standard. PFGE banding patterns were compared using BioNumerics software v.4.0 (Applied Maths, Sint-Martens-Latem, Belgium) based on the Dice coefficient. Dendrograms were constructed by the unweighted-pair group method with arithmetic mean (UPGMA), with a position tolerance of 1% and optimisation of 0.5%. Strains sharing 90% similarity in DNA fingerprinting profiles were defined as a cluster [19].

Identification of integrons and gene cassettes
Class 1, 2 and 3 integrons were detected by PCR using degenerate primers hep35 and hep36 targeted to conserved regions of integrase genes intI1, intI2 and intI3 [20]. PCR products were analysed by restriction fragment length polymorphism (RFLP) to classify integrons [21]. Atypical class 1 integrons were also screened in S. sonnei using primers int1f and is16 [20].
The variable region of class 1 and 2 integrons was amplified using the primers 5 0 CS1/3 0 CS1 and 5 0 CS2/3 0 CS2, respectively, as described previously [22]. PCR products of variable regions were digested by the restriction enzyme HinfI (Takara). Same-sized amplicons with the same RFLP pattern were considered as one type. At least one representative of each type of amplicon was sequenced to identify the gene cassettes. Sequence analysis and alignment was performed using BLAST programs available at the National Center for Biotechnology Information (NCBI) website.

Statistical analysis
Statistical analysis was performed with Stata v.7.0 software (StataCorp LP, College Station, TX). Pearson's x 2 test was used to analyse the antimicrobial resistance of isolates from different regions. Fisher's exact test was applied to compare antimicrobial resistance between integron-positive and integron-negative groups. A P-value of <0.05 was considered statistically significant.

Resistance trends from 2006 to 2011
A wide of spectrum of antimicrobial resistance was observed in 319 S. sonnei strains isolated from 2006 to 2011 ( Table 2). The resistance rates to ampicillin (67.7%), nalidixic acid (75.2%), tetracycline (73.7%) and SXT (68.7%) remained at a high level during 6 years, whereas resistance to cefotaxime and norfloxacin was relatively lower (17.9% and 21.0%, respectively). Of the nine antimicrobials tested, a sharp resistance change was observed for gentamicin, with a three-fold increase from 26.3% in 2006 to 78.1% Table 1 Source of Shigella sonnei strains (n = 340), by year and by city, 2002-2011.

Characteristic
No. of strains % in 2011. A decrease in resistance to AMC (from 52.6% to 18.8%) was observed.

Regional distribution of resistance rates
Of the 319 S. sonnei strains isolated from 2006 to 2011, 176 were collected from South Jiangsu, 78 from the Centre and 65 from the North ( Table 3). The proportions of resistant strains from these three regions showed a significant difference for eight of the nine antimicrobials tested, the only exception being tetracycline. Resistance to ampicillin, cefalotin, cefotaxime and norfloxacin was higher in Centre Jiangsu, whilst resistance to gentamicin, nalidixic acid and SXT was higher in the South. The North isolates showed low rates of antimicrobial resistance.
During 10 years, similar clonal transmission among different cities was revealed several times in Jiangsu Province. The dendrograms of PFGE patterns of all strains are displayed in Supplementary material.

Integrons detected in Shigella sonnei
The detection rate of integrons in S. sonnei was generally high ( To determine the role of class 1 integron in drug resistance, strains were divided into two groups (class 1 integron-positive and class 1 integron-negative). Resistance rates to the nine antimicrobials in the class 1 integron-positive group were significantly higher than the class 1 integron-negative group (P < 0.05) ( Table 5).
Only one amplicon of 2.0 kb in size was identified in class 2 integrons, which carried dfrA1-sat1-aadA1. The gene cassette array bla OXA -30 -aadA1 was detected in a 2.4-kb amplicon of an atypical class 1 integron.

Discussion
In this study, only 40 (11.8%) of 340 S. sonnei isolates were obtained from 2002 to 2006, whereas 300 isolates (88.2%) were obtained in the following 5 years in Jiangsu Province, which was a rapidly developing area in China. The noticeable increase of S. sonnei has also been observed in other fast-developing countries [23], and in some areas such as Beijing and Vietnam [6,24]. S. sonnei has even replaced S. flexneri to become the predominant subgroup. The pattern shift might due to improvements in the economic situation, environmental conditions and hygiene habits in these regions. Another finding was that only two S. sonnei isolates were collected in 2003 during the severe acute respiratory syndrome (SARS) epidemic. Since the main transmission mode of Shigella is person-to-person contact or consumption of contaminated food [25], fewer people getting out and eating out in order to avoid SARS infections may have resulted in the obvious decrease of S. sonnei isolates.
Antimicrobial resistance of Shigella has become one of the most serious global public health concerns [14]. Over the past few decades, Shigella have become resistant to most of the widely used antimicrobials [26,27]. In this study, the resistance patterns of S. sonnei were analysed from three different angles: changing resistance categorised by year; changing resistance categorised by geographical region; and MDR analysis.   In this study during the period 2006-2011, more than one-half of S. sonnei isolates were resistant to ampicillin (67.7%), nalidixic acid (75.2%), tetracycline (73.7%) and SXT (68.7%). Resistance to these antimicrobials is commonly reported and the frequency of resistance differs by country. In Belgium, an 18-year research showed that resistance rates to these four antimicrobials were 19.2%, 3.8%, 59.3% and 85.9%, respectively [12]. In the USA during 2000-2010 [28], resistance rates to the common drugs nalidixic acid (1%), tetracycline (18%) and SXT (33%) were dramatically lower than those observed in the current study, whilst ampicillin had a similar resistance rate of 74%. In addition, in Eastern China high levels of resistance to ampicillin (70.8%), nalidixic acid (69.3%), tetracycline (74.8%) and SXT (73.8%) were reported [9]. The widespread nature of nalidixic acid and fluoroquinolone resistance has been documented in many countries [29,30]. In the current study, resistance to nalidixic acid was found to be very high, with a peak at 95.8% in 2007, whilst the third-generation fluoroquinolone norfloxacin showed a lower resistance rate of 21.0% on average. Cefotaxime also had a lower frequency of resistance compared with cefalotin. Thus, the third-generation fluoroquinolones and cephalosporins are recommended as first-line drugs to treat infections caused by S. sonnei.
This study further analysed the resistance of S. sonnei in different regions of Jiangsu Province. Resistance rates of isolates from the South, Centre and North differed significantly for eight of nine antimicrobials tested. North Jiangsu exhibited low resistance rates compared with the Centre and the South. The great geographical variation found in resistance patterns may be ascribed to the various prescribing practises of doctors in different regions. This calls for strong monitoring of the local resistance patterns in order to provide effective empirical treatment regimens.
Together with the increased single-drug resistance, the frequency of MDR isolates during 6 years remained at a high level, slowly rising from 78.9% in 2006 to 87.5% in 2011. The same trend had already been revealed a few years ago in a long-term surveillance in Belgium, where the MDR rate of S. sonnei increased from 55.9% in 1990 to 80.2% in 2007, reaching a peak of 89.0% during 2004-2005 [12]. Geographical distribution of multidrug resistance indicated that South Jiangsu represented the predominant MDR region (77.3%), followed by the Centre (76.9%) and the North (63.1%).
The declining susceptibility to commonly used antimicrobials and the emergence of MDR S. sonnei could be highly related to inappropriate prescription and easy access to antimicrobials among outpatients. It is therefore important to strictly control antimicrobial application and to monitor local resistance in order to facilitate the rational use of antimicrobial agents.
This high frequency of MDR S. sonnei strains has led to tremendous interest in molecular typing and determination of resistance mechanisms. Bacterial molecular typing is frequently applied for outbreak surveillance and phylogenetic investigation. Strain-specific fingerprints generated are used to facilitate the identification of disease transmission routes and sources [7][8][9][10][11]. To date, various genotyping methods including biotyping, multilocus variable-number tandem-repeat analysis (MLVA) and PFGE have been used for the epidemiological investigation of S. sonnei [31,32]. Biotyping is a relatively earlier phenotypic method with insufficient discrimination.
Although MLVA has a higher discriminatory power for distinguishing epidemiological relationships, it is not a universal method and is not 100% reproducible, and it has no wellestablished protocol for surveillance networks [5]. On the other hand, PFGE, as the acceptable gold-standard technique for molecular typing, has the standardised PulseNet PFGE protocol for interlaboratory comparison of results [18] and has been successfully used for subtyping sporadic or epidemic isolates of Shigella [11,24]. PFGE is an electrophoresis-based method that can directly or indirectly reflect pathogen variation, in other words, the changing in DNA sequence.
In this study, PFGE was used to characterise the homology of all 340 S. sonnei isolates. The results revealed that many PFGE clusters contained strains from multiple geographical regions; some clusters even contained strains from six to seven cities throughout the province. Large-scale transmission of different clones was found in different years. In 2009, cluster SC6 containing 33 isolates spread between Changzhou and Nantong; in 2008, cluster SD5 containing 35 isolates was prevalent in Wuxi, Zhenjiang, Suzhou, Lianyungang, Nanjing and Xuzhou; and in 2007, cluster SE1 containing 35 isolates was present in Xuzhou, Lianyungang, Changzhou, Taizhou, Suzhou, Yancheng and Huai'an, a total of 7 cities. Similarly, the clonal spread of S. sonnei has also been described by other studies in Malaysia during 1997-2009 [32] and on a global scale [33]. These observations emphasise the necessity of monitoring the molecular subtypes of S. sonnei from endemic regions and controlling outbreaks of S. sonnei.
The most commonly applied method of integron detection is to use the specific primer of various integrons for single PCR and to examine them separately, which is time consuming and complicated. This study used degenerate primers combined with restriction enzyme digestion to detect class 1, 2 and 3 integrons concurrently. A total of 55.9% of S. sonnei isolates carried class 1 integrons in Jiangsu Province, perceptibly higher than that in Bangladesh (2.5%) [5], South Korea (14.9%) [16] and Zhejiang Province of China (12.9%) [21]. This difference may due to geographical variation or the increased prevalence of class 1 integrons in recent years. Class 2 integrons were detected in 80.3% of S. sonnei isolates, which was in concordance with previous reports (100.0% in Gabon [13], 100.0% in South Korea [16], 81.0% in Uzbekistan [34] and 80.6% in Zhejiang Province [21]). Class 3 integrons were not detected in any isolates. Furthermore, an atypical class 1 integron, ever present in Africa, Europe and America [35], was also detected with a positive rate of 49.4%. Overall, the current study searched for four classes of integron (classes 1, 2 and 3 and atypical class 1 integrons) in all isolates obtained. The results showed that class 2 integrons remained the preponderant integron among S. sonnei strains but it was not as dominant as reported previously. It is well known that classes 1 and 2 and atypical class 1 integrons play an important role in increased antimicrobial resistance. This was supported by the fact that the resistance rates of the class 1 integron-positive group were all statistically higher than the class 1 integron-negative group for all nine antimicrobials, thus integrons were associated with resistance of S. sonnei to multiple antimicrobials at the phenotype level.
Further analysis of the variable region sequencing showed the close correlation between drug resistance and the presence of integrons at the genotype level. Four of five amplicons carried one or two gene cassettes; only one amplicon (0.15 kb) carried the skeletal structure of 5 0 CS and 3 0 CS. The class 1 integron containing 0.15-kb and 2.2-kb amplicons was the most common type in this study, and aacA4-cmlA1 was the most prevalent gene cassette, conferring resistance to aminoglycoside and chloramphenicol, respectively. The gene cassette array dfrA1-sat1-aadA in class 2 integrons conferred resistance to trimethoprim, streptothricin and streptomycin [16,36]. The gene cassette bla OXA-30 -aadA1, conferring resistance to b-lactams and aminoglycosides, was detected in an atypical class 1 integron. The decrease of AMC resistance is probably due to the decrease of the number of strains harbouring an atypical class 1 integron, which contains bla OXA-30 encoding an enzyme conferring resistance to AMC. So far, a similar atypical class 1 integron has been reported in China, India and France [21,[37][38][39].
In conclusion, this study systematically analysed the subtype shift, antimicrobial resistance patterns, genetic homology and molecular characteristics of integrons in 340 S. sonnei collected from 2002 to 2011 in Jiangsu Province, China. The multicentre, large-sample research, globally unusual, is of great significance for the clinical therapy, prevention and control of S. sonnei infection in Jiangsu Province, China.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

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Knockdown of Long Non-Coding RNA KCNQ1OT1 Restrained Glioma Cells’ Malignancy by Activating miR-370/CCNE2 Axis

Accumulating evidence has highlighted the potential role of long non-coding RNAs (lncRNAs) as biomarkers and therapeutic targets in solid tumors. Here, we elucidated the function and possible molecular mechanisms of lncRNA KCNQ1OT1 in human glioma U87 and U251 cells. Quantitative Real-Time polymerase chain reaction (qRT-PCR) demonstrated that KCNQ1OT1 expression was up-regulated in glioma tissues and cells. Knockdown of KCNQ1OT1 exerted tumor-suppressive function in glioma cells. Moreover, a binding region was confirmed between KCNQ1OT1 and miR-370 by dual-luciferase assays. qRT-PCR showed that miR-370 was down-regulated in human glioma tissue and cells. In addition, restoration of miR-370 exerted tumor-suppressive function via inhibiting cell proliferation, migration and invasion, while promoting the apoptosis of human glioma cells. Knockdown of KCNQ1OT1 decreased the expression level of Cyclin E2 (CCNE2) by binding to miR-370. Further, miR-370 bound to CCNE2 3′UTR region and decreased the expression of CCNE2. These results provided a comprehensive analysis of KCNQ1OT1-miR-370-CCNE2 axis in human glioma cells and might provide a novel strategy for glioma treatment.

INTRODUCTION
Gliomas are one of the most life-threatening and common primary human brain malignancies with an annual incidence of five cases per 100,000 people. Despite advancements in surgery and adjuvant therapy, the prognosis remains very poor, with a median survival of less than 15 months (Xu et al., 2010;Li et al., 2014).
Long non-coding RNAs (lncRNAs) have been confirmed to act as key molecules in cancer development and progression (Cloutier et al., 2016;Liu et al., 2016). Dysregulations of lncRNAs are discovered in various tumor tissues and cancer cells where they serve as oncogenes or tumor suppressors (Ellinger et al., 2015;Jiang et al., 2016). KCNQ1OT1, an imprinted antisense lncRNA in the human KCNQ1 locus on chromosome 11p15.5, is involved in cis-limited silencing within an imprinted KCNQ1 cluster Sunamura et al., 2016). Earlier studies showed that KCNQ1OT1 was up-regulated in breast cancer and conferred to breast cancer malignancy (Zhang et al., 2016a). Similarly, over-expressed KCNQ1OT1 contributed to the carcinogenesis of hepatocellular carcinoma (Wan et al., 2013). However, the expression and function of KCNQ1OT1 in glioma remain unclear.
MicroRNAs (miRNAs) play crucial roles in post-transcriptional gene regulation and serve as negative gene regulators by targeting 3 -UTR of downstream genes (Karsy et al., 2012). MiRNAs have emerged as key regulators in cancer cell biology including cell proliferation, migration, invasion and apoptosis (Zhao et al., 2015). Aberrant expression of miR-370 has been reported in various cancers. In most cases, miR-370 serves as a tumor-suppressive gene in tumors such as liver cancer and osteosarcoma (Duan et al., 2015;Sun G. et al., 2016). More importantly, miR-370 has been confirmed to be down-regulated in glioma and reduces glioma cells growth . But the detailed function of miR-370 still needs to be explored.
Cyclin E2 (CCNE2) is a member of cyclin E family, and one of its major function is making transition from G0/G1 to S phase through binding with CDK2 and phosphorylation of Rb protein (Bizama et al., 2014;Ng et al., 2014;Cong et al., 2015). CCNE2 expression was found to be dysregulated in many cancers such as breast cancer and thyroid cancer Liang et al., 2015). However, the characterization of CCNE2 in glioma has not been investigated.
In the present study, we investigated the expression and function of KCNQ1OT1, miR-370 and CCNE2 in glioma tissues and cells. We also studied the underlying interactions among them. There was a specific binding site between KCNQ1OT1 and miR-370. KCNQ1OT1 and miR-370 negatively regulated their respective expression. MiR-370 targeted CCNE2 3 -UTR and impaired its expression. We hypothesized KCNQ1OT1/miR-370/CCNE2 might exert crucial function in glioma cells progression and might be a novel therapeutic target.

Clinical Tumors
All human glioma tumors and normal brain tumors (NBTs) were collected from the Department of Neurosurgery, Shengjing Hospital of China Medical University, from 2013 to 2016. Glioma samples were divided into four groups: Grade I, Grade II, Grade III and Grade IV, according to the World Health Organization (WHO) classification. Informed consent was obtained from all patients and the study procedure was approved by Research Ethics Board at the Shengjing Hospital of China Medical University. The tumor specimens were immediately frozen and preserved in liquid nitrogen until used in our research.

Cell Culture
Human glioma cell lines (U87, U251) and human embryonic kidney (HEK) 293T cells were acquired from Shanghai Institutes for Biological Sciences Cell Resource Center. They were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). Primary normal human astrocytes (NHA) were obtained from Sciencell Research Laboratories (Carlsbad, CA, USA) and cultured in astrocyte medium (Carlsbad, CA, USA). All cells were incubated at 37 • C a humidified incubator with 5% CO 2 .

RNA Extraction and Quantitative
Real-Time PCR (qRT-PCR) We used trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA ) extracted total RNA from NBTs, glioma tissues, NHA, U87 and U251 cell lines. The primers for KCNQ1OT1 and GAPDH were synthesized from Takara Bio (Japan). The primers for miR-370 and U6 were synthesized from the Applied Biosystems. RNA concentration and quality were measured by the 260/280 nm ratio using a Nanodrop Spectrophotometer (ND-100, and we used One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time; Takara Bio, Inc., Japan) to test the expression of KCNQ1OT1 in NBTs, glioma tissues, NHA, U87 and U251 cells. KCNQ1OT1: forward 5 -TGCAGAAGACAGGACACTGG-3 , reverse 5 -CTTTGGTGGGAAAGGACAGA-3 ; GAPDH: forward 5 -TGCACCACCAACTGCTTAGC-3 , reverse 5 -GGCATGCACTGTGGTCATGAG-3 . GAPDH was used as endogenous controls for genes expressions. High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) and Taqman Universal Master Mix II (Life Technologies Corporation, Carlsbad, CA, USA) were used to examine the expression levels of miR-370 and U6 (Applied Biosystems, Foster City, CA, USA) levels. Fold change was calculated as 2 −∆∆Ct in gene expression.

Cell Proliferation Assay
After knockdown efficacy was confirmed, cell counting Kit-8 (CCK-8, Dojin, Japan) assay was used for cell proliferation. Twothousand cells were seeded per well in 96-well plates at a same density, and 10 µL of Cell Counting Kit-8 was added into each well after 48 h. Then incubated at 37 • C for 2 h and absorbance was measured at 450 nm.

Apoptosis Detection
Apoptosis was evaluated using Annexin V-PE/7-AAD staining apoptosis detection kit (Southern Biotech, Birmingham, AL, USA) according to the manufacturer's instructions. After the cells washed two times with 4 • C PBS, cells were collected and stained with Annexin V-PE and 7-AAD. Then acquired the cells by flow cytometry (FACScan, BD Biosciences, San Jose, CA, USA) and analyzed by CELL Quest 3.0 software.

Cell Migration and Invasion Assay
The abilities of cell migration and invasion were tested using the 24-well transwell chambers with 8 µm pore size polycarbonate membrane (Corning, NY, USA). For cell invasion assay, the filter was pre-coated with 500 ng/ml matrigel solution (BD, Franklin Lakes, NJ, USA) and incubated at 37 • C for 4 h before the invasion assay started; placed 500 µL of 10% FBS medium in the lower chamber, and placed 100 µL serum-free medium in the upper chamber. After incubation at 37 • C for 24 h, the cells on the upper membrane surface were scraped off. Surface were fixed with methanol and glacial acetic acid 30 min and then stained with 10% Giemsa solution. Number of cells was counted, and five randomly fields were counted randomly in each well. The photographs were taken at 200× magnification.

RNA Immunoprecipitation
RNA immunoprecipitation was performed as previously reported . Briefly, to explore whether KCNQ1OT1 and miR-370 were associated with the RISC, we performed RNA immunoprecipitation. Glioma cells were lysed in complete RNA lysis buffer with protease inhibitor and RNase inhibitor from an EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA). Whole cell lysates from the control groups and miR-370 groups were incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute 2 antibody (Millipore, Billerica, MA, USA) and the negative control (normal mouse IgG; Millipore, Billerica, MA, USA). Samples were incubated with Proteinase K buffer, and then the immunoprecipitated RNA was isolated. Purified RNA was obtained and qRT-PCR was performed with the primers mentioned above to illustrate the presence of the binding targets.

Western Blot Analysis
Total protein of cells were lysed using ice-cold RIPA (Beyotime Institute of Biotechnology) buffer supplemented with protease inhibitors and centrifuged at 17,000× g 4 • C for 40 min. Equal amount of total cell lysate protein samples (40 µg) went electrophoresis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then Equal amount of total cell lysate protein samples (40 µg) went electrophoresis in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were incubated in 5% nonfat milk dissolved in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 2 h at room temperature. After blocking of non-specific bindings, the protein went immunoblotting with primary antibodies against CCNE2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), YAP1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-YAP (1:2000, CST, USA) and GAPDH (1:1000, Proteintech, Chicago, IL, USA). Followed by incubation with appropriate correlated HRP-conjugated secondary antibody, then the membranes were incubated with secondary antibodies (goat anti-rabbit or goat anti-mouse, 1:5000 respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and scanned using ChemImager 5500 V2.03 software. The integrated density values (IDV) were calculated using FluorChem 2.0 software.

Subcutaneous and Orthotopic Xenografts in Nude Mice
The stably transfected human glioma tissues were used in the in vivo study. Four-week-old male nude mice were purchased from the National Laboratory Animal Center (Beijing, China). All mice were free to autoclaved food and water during the experiment, and all experiments with nude mice were performed strictly in accordance with a protocol approved by the Administrative Panel on Laboratory Animal Care of the China Medical University. Lentivirus encoding miR-370 was generated using pLenti6.3/V5eDEST Gateway Vector Kit (Life Technologies Corporation, Carlsbad, CA, USA). The miR-370 and short-hairpin RNA targeting human KCNQ1OT1 were ligated into the pLenti6.3/V5eDEST vector and LV3-CMV-GFP-Puro vector (GenePharma, Shanghai, China), respectively, and then pLenti6.3/V5eDEST-miR-370 and LV3-CMV-GFPPuro-sh-KCNQ1OT1 vectors were generated. The ViraPower Packaging Mix was used to generate Lentivirus in 293FT cells. After infection, the stable expressing cells of miR-370 and sh-KCNQ1OT1 were obtained. The lentiviruses of miR-370 were transduced in sh-KCNQ1OT1 stably transfected cells to generate miR-370 + sh-KCNQ1OT1 cells. The nude mice were divided into four groups: control group (only U87 or U251), sh-KCNQ1OT1 group (sh-KCNQ1OT1 stable expression U87 or U251 cells), miR-370 group (miR-370 stable over-expression U87 or U251 cells) and sh-KCNQ1OT1 + miR-370 group (KCNQ1OT1 inhibition and miR-370 over-expression stable U87 and U251 cells). 3 × 10 5 cells were subcutaneously injected in the right flanks of the mice. Tumor volume was measured every 4 days when the tumors were obviously identified and the volume was calculated by the formula: volume (mm 3 ) = length × width 2 /2. Fourty-four days after injection, mice were sacrificed and tumors were isolated. As previously described (Rubin et al., 2003;Zhou et al., 2015), for survival analysis in orthotopic inoculations, 3 × 10 5 cells were stereotactically implanted into the right striatum of the mice in the position of 2 mm posterior to the bregma, 2 mm laterally, and 2 mm deep to the dura. The number of survived nude mice was marked and survival analysis was investigated using Kaplane-Meier survival curve.

Statistical Analysis
Data are presented as mean ± standard deviation (SD). SPSS 18.0 statistical software with the Student's t-test or one-way analysis of variance (ANOVA) were used to evaluate all statistical analyses. Multiple comparison between the groups was performed using S-N-K method. Differences were considered to be significant when P < 0.05.

KCNQ1OT1 was Up-Regulated in Glioma Tissues and Cell Lines, KCNQ1OT1 Inhibition Impeded Glioma Cells Malignant Progression
The latest studies confirmed aberrant KCNQ1OT1 expression was ubiquitous in various tumors. We first investigated KCNQ1OT1 expression in glioma tissues and cells. As Figures 1A,B showed, the expression of KCNQ1OT1 in glioma tissues and cell lines was robustly up-regulated in glioma tissues (P < 0.01), U87 and U251 cells (P < 0.05), and was positively correlated with the histopathological grades of gliomas. Therefore, we hypothesized KCNQ1OT1 might contribute to glioma cells' malignancy. U87 and U251 cells with stable inhibition of KCNQ1OT1 were established. We then examined the knockdown efficiency ( Figure 1C; P < 0.01). As expected, cell proliferation was reduced in sh-KCNQ1OT1 group compared with sh-NC group (Figure 1D; P < 0.05). Similarly, flow cytometry results showed that down-regulation of KCNO1OT1 induced glioma cells apoptosis compared with sh-NC group ( Figure 1E; P < 0.05). Further, as shown in Figure 1F, U87 and U251 cells' migration and invasion ability were weaker in experiment group compared with sh-NC group (P < 0.05). These results indicate KCNQ1OT1 acts as an oncogene in glioma cells.
Over-Expression of miR-370 Impaired Cell Proliferation, Migration and Invasion, While Promoted Apoptosis of Glioma Cells An earlier study has shown miR-370 was down-regulated in glioma tissues and cells . In addition, the restoration of miR-370 reduced glioma cells proliferation. We further explored miR-370's detailed function in glioma cells. Consistent with previously reported, CCK-8 assay indicated over-expression of miR-370 restrained the proliferation of U87 and U251 cells compared with pre-NC group at different times (Figure 2A; P < 0.05). Meanwhile, over-expression of miR-370 led to an increased ratio in apoptosis compared with pre-NC group ( Figure 2B; P < 0.05). Transwell assays were conducted to assess the effects of miR-370 on the migratory and invasive abilities of glioma cells. As Figure 2C showed, the number of cells was reduced in pre-miR-370 group compared with pre-NC group (P < 0.05). Therefore miR-370 might act as a tumor suppressor in glioma cells.

MiR-370 Mediated the Tumor-Suppressive Effect of KCNQ1OT1's Inhibition on Glioma Cells
Multiple studies have confirmed lncRNAs harbor miRNAs binding sites (Su et al., 2016). LncRNAs can sponge miRNAs in a sequence-specific manner (Ballantyne et al., 2016). In most cases, lncRNAs and miRNAs regulate expression respectively in an Argonaute2-dependent manner (Kobayashi and Tomari, 2016). We used a bioinformatics database (Starbase) to infer the potential targets of miR-370. KCNQ1OT1 was identified as a potential target and harbored one putative binding site of miR-370. We first examined the expression of miR-370 in KCNQ1OT1 inhibition glioma cells. As shown in Figure 3A, miR-370 expression was increased in sh-KCNQ1OT1 group compared with sh-NC group (P < 0.01). Further, as shown in Figure 3A, we found KCNQ1OT1 expression was negatively correlated with miR-370 expression in glioma cells (P < 0.01). Luciferase assays were used to confirm the functional binding site between KCNQ1OT1 and miR-370. The binding site and the designed mutated site are shown in Figure 3B. As shown in Figure 3C, luciferase activity was significantly reduced in KCNQ1OT1-WT + miR-370 group compared with KCNQ1OT1-WT + miR-370-NC group (P < 0.05). We further performed RNA-binding protein immunoprecipitation (RIP) experiment to ascertain whether KCNQ1OT1 and miR-370 were in a RNA-induced silencing complex (RISC). As shown in Figure 3D, KCNQ1OT1 and miR-370 were both detected in anti-Ago2 group (P < 0.01). These results demonstrated an  insight into the mechanism of KCNQ1OT1 negatively regulating miR-370.
To determine whether miR-370 conferred to the tumorsuppressive effect of KCNQ1OT1's inhibition, we transfected over-expression of miR-370 or miR-370 inhibitors into the stable sh-KCNQ1OT1 cells prior to the assessment of cell proliferation, apoptosis, migration and invasion. As shown in Figure 3E, sh-KCNQ1OT1 cells co-transfected with over-expression of miR-370 had the strongest inhibitory effect on cell proliferation compared with sh-NC + pre-NC group at different times (P < 0.05). Flow cytometry results showed that glioma cells treated with sh-KCNQ1OT1 + pre-miR-370 exhibited increased apoptotic ratio compared with sh-NC + pre-NC group (Figure 3F; P < 0.05). Similarly, knockdown of KCNQ1OT1 combined with restoration of miR-370 robustly decreased migrating and invading glioma cells (Figure 3G; P < 0.05).

CCNE2 was Involved in KCNQ1OT1/miR-370-Mediated Glioma Cells Malignant Progression
CCNE2 exerts oncogenetic function in various tumors such as breast cancer and bladder cancer (Gupta et al., 2016). We first detected the expression of CCNE2 in glioma tissues using western blot. As shown in Figure 4A, CCNE2 expression were obviously up-regulated in high-and low-grade glioma tissues compared with normal brain tissues (NBTs; P < 0.05). CCNE2 was identified as one of the downstream genes of miR-370 by Bioinformatics database (Targetscan, miRanda and Starbase). Luciferase reporter assay was performed to verify the putative binding site between miR-370 and CCNE2. The binding site and the designed mutated site are shown in Figure 4B. As shown in Figure 4C, luciferase activity was significantly reduced in CCNE2-Wt + miR-370 group compared with CCNE2-Wt + miR-370-NC group (P < 0.05). Further, we examined the expression of CCNE2 protein in cells treated with KCNQ1OT1 or miR-370. As shown in Figure 4D, CCNE2 protein level was decreased in sh-KCNQ1OT1 group compared with sh-NC group (P < 0.05). CCNE2 protein levels were also examined in overexpressed or inhibited miR-370 cells. As expected, CCNE2 protein level was attenuated in pre-miR-370 group, while it was up-regulated in anti-miR-370 group (Figure 4E; P < 0.05). Having confirmed both KCNQ1OT1 and miR-370 affected glioma cells' malignant biological behaviors, we further investigated the underlying molecular mechanisms. Figure 4F showed that CCNE2 protein expression was robustly decreased in sh-KCNQ1OT1 + pre-miR-370 group, while anti-miR-370 rescued sh-KCNQ1OT1 induced down-regulation of CCNE2 protein expression (P < 0.05).

CCNE2 Promoted Cell Proliferation, Migration and Invasion, While Inhibited Apoptosis of Glioma Cells
Having confirmed CCNE2 was up-regulated in glioma cells and participated in KCNQ1OT1/miR-370 regulating glioma cells' malignancy, we examined the effect of CCNE2 on glioma cells' biological behaviors. CCK-8 assay indicated over-expression of CCNE2 (CCNE2 (+)) promoted the proliferation of glioma U87 and U251 cells compared with CCNE2 (+)-NC group (Figure 5A; P < 0.05). Meanwhile, over-expression of CCNE2 (CCNE2 (+)) led to a decreased apoptotic ratio of glioma cells compared with CCNE2 (+)-NC group (Figure 5B; P < 0.05). Transwell assays were conducted to assess the effects of CCNE2 (+) on the migratory and invasive ability of glioma cells. As expected, the migratory and invasive ability was stronger in CCNE2 (+) group compared with CCNE2 (+)-NC group (Figure 5C; P < 0.05).
A latest study showed CCNE2 promoted breast cancer cells' malignant progression via activating Hippo pathway (Pegoraro et al., 2015). Therefore, we detected p-YAP and YES-associated protein (YAP) expression in cells treated with KCNQ1OT1 and miR-370. As shown in Figure 7D, p-YAP expression was up-regulated in sh-KCNQ1OT1 group and pre-miR-370 group compared with control group (P < 0.05). Besides, p-YAP expression was robustly increased in sh-KCNQ1OT1 + pre-miR-370 group.

KCNQ1OT1 Inhibition Combined with miR-370 Over-Expression Significantly Reduced Tumor Growth In Vivo
As shown in Figures 8A,B, knockdown of KCNQ1OT1 or over-expression of miR-370 reduced tumor growth. Further, knockdown of KCNQ1OT1 combined with over-expression of miR-370 exhibited the smallest tumor compared with each group in the experiment (P < 0.05). Similarly, the survival analysis demonstrated both KCNQ1OT1 inhibition and miR-370 restoration prolonged survival period compared with control group, and as expected, KCNQ1OT1 inhibition combined with miR-370 reintroduction contributed to the longest survival period ( Figure 8C).

DISCUSSION
In this study, we demonstrated KCNQ1OT1 was up-regulated in glioma tissues and cells. Knockdown of KCNQ1OT1 suppressed FIGURE 4 | Cyclin E2 (CCNE2) was up-regulated in glioma tissues. CCNE2 was a target of miR-370, both KCNQ1OT1 and miR-370 could modulate CCNE2 expression. (A) CCNE2 protein expression levels in normal brain tissues and glioma tissues using GAPDH as an endogenous control. Representative protein expression and their integrated light density values (IDVs) of CCNE2 in normal brain tissues, low-grade glioma tissues ([WHO] I-II), and high-grade glioma tissues ([WHO] III-IV) are shown. Data are presented as the mean ± SD (n = 5, each group). * P < 0.05 vs. NBTs group. (B) CCNE2 harbored a putative miR-370 binding site, and designed mutant sequences were indicated. (C) Dual-luciferase reporter assay of HEK 293T cells co-transfected with CCNE2-Wt and miR-370-NC or CCNE2-Wt and miR-370, and CCNE2-Mut and miR-370-NC or CCNE2-Mut and miR-370. Data are presented as the mean ± SD (n = 5, each group). * P < 0.05 vs. CCNE2-Wt + miR-370-NC group. (D) Western blot analysis for CCNE2 in KCNQ1OT1 knockdown of human glioma cells using GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 5, each group). * P < 0.05 vs. sh-NC group. (E) Western blot analysis for CCNE2 in miR-370 over-expression and miR-370 inhibition of human glioma cells using GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 5, each group). * P < 0.05 vs. pre-NC group; # P < 0.05 vs. anti-NC group. (F) Western blot analysis for CCNE2 in co-transfected KCNQ1OT1 inhibition with miR-370 over-expression or miR-370 inhibition of human glioma cells using GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 5, each group). * P < 0.05 vs. sh-NC + pre-NC group. the malignant behaviors of human glioma U87 and U251 cells. In addition, the KCNQ1OT1 knockdown up-regulated miR-370, which was lowly expressed in glioma tissues and cells. CCNE2 is a direct target of miR-370. We found that CCNE2 was up-regulated in glioma tissues. We further discovered that miR-370 played a tumor-suppressive role by down-regulating CCNE2 in glioma cells. The in vivo study demonstrated knockdown of KCNQ1OT1 combined with miR-370 over-expression produced the smallest tumor and the longest survival period in nude mice. Taken together, the KCNQ1OT1/miR-370/CCNE2 axis might exert an important role in human glioma tumorigenesis and malignant progression, which provided a novel promising therapeutic target.
Many research indicate that lncRNAs are abnormally expressed in multiple tumors, and play important roles in the biological processes of tumor cells (Bartonicek et al., 2016;Serghiou et al., 2016). LncRNA MALAT1 is up-regulated in pancreatic cancer, epigenetically inducing MALAT1 promotes tumor cell growth and invasion via regulating autophagy . LncRNA TUG1 is highly expressed in hepatoblastoma tissues and cells, silencing TUG1 impairs cell proliferation, migration and invasion through increasing miR-34a-5p expression (Dong et al., 2016). KCNQ1OT1 encodes a paternally expressed long non-coding RNA, which has been shown to regulate the imprinting of several genes present at 11p15.5 locus in cis (Bliek et al., 2001;Higashimoto et al., 2006;Fedoriw et al., 2012;Schultz et al., 2015). KCNQ1OT1 aberrant expression is ubiquitous in various cancers. KCNQ1OT1 is up-regulated in Wilms' tumor cells. Knockdown of KCNQ1OT1 promotes apoptosis of Wilms' tumor cells (Yoshizawa et al., 2015). Consistent with previously reported, we proved that KCNQ1OT1 was highly expressed in glioma tissues and cells. Also, epigenetically silencing KCNQ1OT1 restrained proliferation, migration and invasion, while promoting apoptosis of glioma cells. Therefore, KCNQ1OT1 might be an oncogene in glioma and promote glioma cells' malignant progression.
MiRNAs have various expression patterns and different biological functions in tumors. MiR-29a is highly expressed in breast cancer, and over-expressed miR-29a predicts shorter survival in patients with breast cancer. Inhibition of miR-29a impairs cell proliferation and weakens cell migratory abilities via up-regulating TET1 (Pei et al., 2016). MiR-497-5p exerts tumorsuppressive function in human angiosarcoma, reintroduction of miR-497-5p reduces KCa3.1 expression by targeting its mRNA 3 -UTR . MiR-370 is first identified as a tumor-suppressor in human cholangiocytes, and is found to be methylated by Interleukin-6 (Meng et al., 2008). MiR-370 exerts different function in various tumors. In most cases, miR-370 is lowly expressed and exerts tumor-suppressive role in tumor cells (Lo et al., 2012;Chen et al., 2014). A latest Representative images and accompanying statistical plots were presented. Data are presented as the mean ± SD (n = 5, each group). * # P < 0.05 vs. miR-370 + CCNE2 group. Scale bars represent 20 µm. (D) Western blot analysis of the YES-associated protein (YAP) pathways regulated by CCNE2 in human glioma cells. Data are presented as the mean ± SD (n = 5, each group). * # P < 0.05 vs. Sh-KCNQ1OT1 group and pre-miR-370 group. study shows that miR-370 is down-regulated in human glioma tissues cells . In addition, it has been confirmed that miR-370 inhibits glioma cells' proliferation via targeting cyclin D1 and c-myc. Therefore, we further investigated the potential detailed function miR-370 might exert. As expected, our results showed that overexpression FIGURE 8 | In Vivo study. (A) The stable expressing cells were used for the in vivo study. The nude mice carrying tumors from respective groups were shown. The sample tumor from respective group was shown. (B) Tumor volume was calculated every 4 days after injection, and the tumor was excised after 44 days. * P < 0.05 vs. Control group; # P < 0.05 vs. pre-miR370 group; P < 0.05 vs. sh-KCNQ1OT1 group. (C) The survival curves of nude mice injected into the right striatum (n = 15). P < 0.05 (pre-miR-370 or sh-KCNQ1OT1 group vs. Control group); P < 0.01 (pre-miR-370 + sh-KCNQ1OT1 group vs. Control group).
of miR-370 restrained glioma cells' malignant progression, including cell proliferation, migration and invasion, while promoting apoptosis.
Recent studies have showed that lncRNAs can function as endogenous miRNA sponges by sponging miRNAs in a sequence-specific manner and impairing their function in a RISC manner. LncRNA NEAT1 promotes non-small cell lung cancer (NSCLC) cell via decreasing miR-377 expression. Mechanically, miR-377 targets NEAT1, and NEAT1 and miR-377 are in a RISC complex . Similarly, lncRNA MIR31HG harbors two miR-193b binding sites. MIR31HG and miR-193b respectively regulates each expression in a RISC manner . Our results showed KCNQ1OT1 exerted oncogenic role while miR-370 exerted the opposite function in glioma cells. Bioinformatics software (Starbase) indicated KCNQ1OT1 might harbor a binding site of miR-370. Luciferase reporter confirmed the binding site of miR-370 in KCNQ1OT1, and RIP assays supported the hypothesis that KCNQ1OT1 and miR-370 were in a RISC complex. Further, KCNQ1OT1 negatively regulated miR-370, and miR-370 inversely modulated KCNQ1OT1 expression. Knockdown of KCNQ1OT1 combined with miR-370 significantly restrained malignant behaviors of glioma cells in vitro and reduced tumor growth in vivo. Furthermore, KCNQ1OT1 inhibition combined with miR-370 over-expression significantly decreased CCNE2 and increased p-YAP expression. These results supported our assumption that KCNQ1OT1 promoted glioma cells progression via decreasing miR-370 expression. However, the underlying mechanism between miR-370 and CCNE2 remained unclear.
MiRNAs regulate cellular events by binding to the 3 -UTRs of downstream genes. For example, miR-208a-3p promotes gastric cancer cell proliferation via targeting PDCD4 3 -UTR (Yin et al., 2016). Meanwhile, miR-504 exerts tumor-suppressive function via targeting FOXP1 3 -UTR in glioma (Cui et al., 2016). In our study, three kinds of bioinformatics softwares indicated CCNE2 was a target of miR-370. Further, luciferase reporter results confirmed the binding site between miR-370 and CCNE2. CCNE2 belongs to the G(1) cyclin family and exerts important role in G1/S transition of the cell cycle . Dysregulation of CCNE2 has been reported in various tumors, such as breast cancer and NSCLC (Matsushita et al., 2015;Ye et al., 2016). In human prostate cancer, CCNE2 expression is elevated in tumor tissues (Wu et al., 2009, and when PTEN, which is conventionally lowly expressed in prostate cancer cells, is reintroduced, CCNE2 is blocked and results in cycle G(1) arrest (Macdonald et al., 2014). Moreover, miR-30a expression is negatively correlated with CCNE2 in prostate cancer pathological specimens (Zhang et al., 2016b). Similarly, CCNE2 expression is increased in NSCLC, over-expressed CCNE2 facilitates NSCLC cell growth and the effect can be abolished by reintroduction of miR-30d-5p . Consistent with previous studies, our results showed that CCNE2 expression was elevated in glioma tissues. Inhibition of CCNE2 hindered glioma cells progression. Also, CCNE2 inversed miR-370-induced suppression on glioma cells malignant behavior. Besides, a latest study shows CCNE2 is involved in regulation of Hippo pathway in breast cancer (Pegoraro et al., 2015). Hippo pathway is characterized as a tumor-suppressive axis, and plays important roles in tumor cellular process including proliferation, migration, invasion, and apoptosis (Alam et al., 2016;Atkins et al., 2016;Kemppainen et al., 2016;Mao et al., 2016). YAP is an important transcriptional activator and can be phosphorylated by Hippo pathway (Bouvier et al., 2016). Phosphorylation of YAP induces YAP functional nucleus localization transit to nonfunctional cytoplasmic cell distribution (LaQuaglia et al., 2016). We have demonstrated that KCNQ1OT1 regulated CCNE2 expression via decreasing miR-370 expression. Therefore, phosphorylated YAP expression were detected in cells treated with KCNQ1OT1 inhibition, miR-370 restoration and co-transfected KCNQ1OT1 inhibition and miR-370 restoration glioma cells. As expected, phosphorylated YAP expression was predominantly increased in cells co-transfected with KCNQ1OT1 inhibition and miR-370 restoration. This may partially declare the underlying mechanism of KCNQ1OT1/miR-370 regulation on glioma cells. The mechanism underlying tumorgenesis of human glioma cell lines by KCNQ1OT1 is schematically presented in Figure 9.
In conclusion, our study demonstrated that knockdown of KCNQ1OT1 inhibited glioma cells malignancy by up-regulating miR-370. Moreover, miR-370 suppressed glioma cells' malignant biological behaviors by targeting CCNE2, and further activated Hippo pathway. Thus, therapy targeting KCNQ1OT1/miR-370/CCNE2 axis may be a promising option for the treatment of human gliomas.


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Malignant pericytes expressing GT198 give rise to tumor cells through angiogenesis

Angiogenesis promotes tumor development. Understanding the crucial factors regulating tumor angiogenesis may reveal new therapeutic targets. Human GT198 (PSMC3IP or Hop2) is an oncoprotein encoded by a DNA repair gene that is overexpressed in tumor stromal vasculature to stimulate the expression of angiogenic factors. Here we show that pericytes expressing GT198 give rise to tumor cells through angiogenesis. GT198+ pericytes and perivascular cells are commonly present in the stromal compartment of various human solid tumors and rodent xenograft tumor models. In human oral cancer, GT198+ pericytes proliferate into GT198+ tumor cells, which migrate into lymph nodes. Increased GT198 expression is associated with increased lymph node metastasis and decreased progression-free survival in oral cancer patients. In rat brain U-251 glioblastoma xenografts, GT198+ pericytes of human tumor origin encase endothelial cells of rat origin to form mosaic angiogenic blood vessels, and differentiate into pericyte-derived tumor cells. The net effect is continued production of glioblastoma tumor cells from malignant pericytes via angiogenesis. In addition, activation of GT198 induces the expression of VEGF and promotes tube formation in cultured U251 cells. Furthermore, vaccination using GT198 protein as an antigen in mouse xenograft of GL261 glioma delayed tumor growth and prolonged mouse survival. Together, these findings suggest that GT198-expressing malignant pericytes can give rise to tumor cells through angiogenesis, and serve as a potential source of cells for distant metastasis. Hence, the oncoprotein GT198 has the potential to be a new target in anti-angiogenic therapies in human cancer.

INTRODUCTION
Angiogenesis has long been recognized as a hallmark of cancer [1,2], and is essential for the development of many, if not all, human solid tumors [3][4][5]. Clinical anti-angiogenesis therapies such as bevacizumab treatment have shown only transient benefits in cancer patient survival [6]. The challenge remains to reconcile conflicting observations in angiogenesis research, and to identify additional angiogenic factors with better therapeutic potential.
Angiogenesis with the formation of new blood vessels is a normal biological process during embryonic development and in adult tissue repair [7][8][9]. This process is hijacked in tumor development. In normal angiogenesis, under stimulation by angiogenic factors, www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 31), pp: 51591-51607 Research Paper endothelial cells first sprout from existing capillaries to provide a monolayer luminal structure. Pericytes are then recruited to enclose and stabilize these endothelial cells to form microvessels [8,10]. Further remodeling and maturation of microvessels is facilitated by a complex molecular signaling network that establishes a stable new vasculature [7,11]. Tumor angiogenesis appears to mirror normal angiogenesis with the exception of having defects characterized by continued microvessel formation and constant reconstruction of the vasculature [10]. Several interconnected hypotheses of tumor angiogenesis have been proposed [12][13][14][15]. These include: sprouting angiogenesis [16], the sprouting of endothelial cells from existing capillaries; vessel co-option [5], the adoption of existing vessels by tumor cells; intussusception [17], the splitting of existing vessels to form new ones; vascular mimicry [18][19][20], de novo formation of new vessels by tumor cells; vasculogenesis [21], recruiting bone-marrow derived progenitors to form new vessels; and the theory of cancer stem cells [22][23][24], the differentiation of tumorderived stem cells into new vessels, and importantly, into pericytes [25,26]. In this study, we provide evidence to support existing angiogenesis hypotheses [27], and suggest that the presence of oncoprotein-stimulated malignant pericytes is a shared defect in tumor angiogenesis.
Glioblastoma xenografts closely mimic primary human glioblastoma, a common and aggressive type of brain tumor with poor prognosis [28]. The U-251 glioblastoma xenograft is characterized by extensive angiogenesis with hypertrophic vascular proliferation [29]. Despite genetic instability [30], this glioblastoma cell line is an excellent model for studies on angiogenesis, in part because of the presence of CD133 + stem cells which transdifferentiate into vascular lineages [22,24,31]. The plastic and angiogenic nature of tumor stem/progenitors is crucial for tumor development since these cells can recapitulate new tumor vasculature. The mouse GL261 glioma also possesses similar characteristics to human glioblastoma, and has been extensively investigated as a mouse tumor model for testing immunotherapy [32,33]. Although angiogenic stimuli or risk factors in human cancers versus rodent models may be distinct, the fundamental principles of tumor angiogenesis are likely shared.
The hypotheses regarding tumor angiogenesis are also in accordance with the theory of epithelial-tomesenchymal transition (EMT), where mesenchymal stromal stem cells have been shown to give rise to angiogenic malignant pericytes [26]. Epithelial-tomesenchymal transition is a process by which tumor cells become stromal stem cells, which can then differentiate into a variety of cell types and gain mobility and invasiveness during metastasis [34]. When differentiate into pericytes, they stimulate tumor angiogenesis by forming tumor vasculature with poor pericyte coverage.
Clinical data have previously shown that low pericyte coverage in vasculature is correlated with decreased patient survival [35,36]. These malignant pericytes may further acquire properties promoting their mobility and invasiveness during tumor metastasis. Thus, malignant pericytes may be of central importance for both tumor angiogenesis and tumor metastasis.
Here, we investigate malignant pericytes using the oncoprotein GT198 as a marker. The human GT198 gene (PSMC3IP; encodes GT198, also known as Hop2 or TBPIP) is a DNA repair gene and also a breast and ovarian cancer gene located within the BRCA1 locus at chromosome 17q21. Germline mutations in GT198 have been identified in familial breast and ovarian cancer [37,38]. Somatic mutations in GT198 are prevalent in tumor stroma in sporadic breast, ovarian, and fallopian tube cancers [39][40][41]. GT198 mutations induce a truncated active mutant form containing the GT198 DNA-binding domain, resulting in cytoplasmic GT198 expression [39,40]. Tumor-specific cytoplasmic expression of GT198 has previously been demonstrated in angiogenic pericytes located within the mutant breast tumor stroma [41]. GT198 is a transcriptional coactivator stimulating a variety of target genes including adipogenic and angiogenic factors such as VEGF [41,42]. GT198 directly stimulates the human VEGF promoter [41]. In addition, extensive analyses have shown that GT198 stimulates DNA strand exchange and regulates DNA recombination in both DNA repair and meiosis [43][44][45][46]. While the broad functions of GT198 in transcription and DNA repair may rely on its DNA-binding capacity [39,43,45], GT198 is also expressed in embryoid body stem cells [39], as well as in pericyte progenitors [41]. It is therefore conceivable that both transcription and DNA repair may be affected in stem/progenitors producing malignant pericytes, thereby impacting tumor angiogenesis and in turn cancer.
We have previously shown that cytoplasmic GT198 is expressed in mutant breast tumor stromal cells including in angiogenic pericytes [41]. In this study, we show that GT198 + pericytes are a common feature among various human solid tumors as well as rodent xenograft tumor models. Specifically, we show that GT198 + pericytes have malignant potential and give rise to tumor cells in both human oral cancer and in rat glioblastoma xenografts. We further show that GT198 vaccination in mouse glioma containing GT198 + pericytes resulted in suppressed tumor growth and prolonged mouse survival. Consistent with the notion that tumor progenitors actually evolve into pericytes [25,26], we propose that malignant pericytes also can differentiate into tumor cells through the process of angiogenesis. The presence of GT198 + pericytes provides a rationale for the integration of existing hypotheses in tumor angiogenesis, and implies the potential of oncoprotein GT198 as a new target in antiangiogenesis therapy. www.impactjournals.com/oncotarget

GT198 expression in pericytes is a common feature in tumor stroma of human solid tumors and xenograft mouse tumors
Angiogenic pericytes expressing cytoplasmic GT198 have been previously identified in mutant breast cancer stroma [41]. Here, we further analyzed multiple human solid tumors for GT198 + pericytes by immunohistochemistry. GT198 + pericytes in capillaries, and perivascular cells in small vessels, were found in angiogenic tumor stroma of human primary cancers in breast, ovary, uterus, fallopian tube, prostate, bladder, testis, lung, brain, melanoma, kidney, oral cavity, thyroid, and colon ( Figure 1A). Positive blood vessels were often not sporadically located but clustered in angiogenic tumor stroma where GT198 + fibroblasts were also present. In contrast, GT198pericytes in vessels were often located in non-angiogenic areas of tumor stroma (Supplementary Figure 1). The highest positive rates were found in brain astrocytoma (83%) and oral cancer (82%), which were mostly angiogenic ( Figure 1B). Although GT198 mutations have not been analyzed in every types of cancer, GT198 somatic mutations that cause cytoplasmic GT198 overexpression have been previously identified in breast, ovarian, uterus, and fallopian tube cancer [37,[39][40][41]. The current results suggest that angiogenic GT198 + pericytes are frequently present in tumor stroma of multiple types of human solid tumors.
In addition, we analyzed xenograft mouse tumors produced by six human cancer cell lines including breast cancer BT474, ovarian cancer OVCAR-3, glioblastoma T98G, lung cancer Calu-6, prostate cancer DU145, and colon cancer SW480. We found GT198 + capillaries were present in all cases, particularly in angiogenic tumor stroma adjacent to tumor ( Figure 1C). Thus, GT198 expression in pericytes is also a common feature in mouse transplantation tumor models.

Normal expression of GT198 is mostly nuclear and decreases during development
GT198 expression in normal tissues is essential to assess its abnormal patterns in cancer, and is indispensible to predict therapeutic side effects, should GT198 be a drug target. Here, we systematically characterized GT198 expression using normal mouse tissues. GT198 was previously shown to increase in primitive ectoderm during embryonic stem cell differentiation [39]. During mouse embryonic development, GT198 mRNA revealed by in situ hybridization showed a marked increase in brain at embryonic stages from E8.5 to E10.5 (Figure 2A), with a peak in protein expression at E12.5 in all three germ layers ( Figure 2B). This pattern is consistent with the previous Northern blot analysis [42]. GT198 expression was rapidly downregulated in most tissues and major organs from E13.5 to E18.5, except in testis where spermatocytes were continuously positive ( Figure 2C-2D). In adult mice, other than testis, low level of GT198 expression was also detected in the ovary, thymus, bone marrow, and certain types of neurons in brain ( Figure 2E). Blood vessels in the heart and lung were negative in expression ( Figure  2D-2E). Except for normal ovarian corpus luteum and certain neural cells, GT198 protein expression was mostly nuclear in normal tissues. This was in sharp contrast to the cytoplasmic expression observed in human cancer ( Figure  1A). The overall expression pattern of GT198 resembles a class of proteins called cancer-testis antigens with the expression in cancer, embryo, testis, but not in most adult tissues. GT198 expression is also remarkably similar to that of BRCA1 and BRCA2 expression in mice [47]. The developmental decrease of GT198 reflects its functional involvement in stem or progenitor cell differentiation. Its re-expression in cancer infers a dedifferentiation status.

GT198 + pericytes give rise to tumor cells in human oral cancer
Using GT198 cytoplasmic expression as a marker, we analyzed 40 cases of human oral cancers with associated clinical information (Supplementary Table  1). In angiogenic tissues adjacent to tumor, pericytes in capillaries were thickened, and overexpressed cytoplasmic GT198 ( Figure 3A-3B). These GT198 + pericytes further proliferated into small tumor nodules and detached from blood vessels ( Figure 3C-3D). In advanced tumor, the GT198 + pericytes in nodules continued to differentiate into tumor cells ( Figure 3E-3F). Pericyte-enclosed nodules at the early stage were still functional vessels with red blood cells in their cavities (Figure 4), which is consistent with the observations from others that tumor produces its own vessels [27]. In lymph node metastasis, pericyte-derived GT198 + nodules were found in the lymph nodes ( Figures  3G-3H and 4). These observations suggest that pericytes overexpressing GT198 have malignant potential and give rise to tumor cells which are capable to migrate into lymph nodes.
GT198 expression in pericytes or in their derived tumor cells was found in all stages of oral tumors analyzed in 40 patients ( Figure 3I). However, the expression scores were higher in tumor adjacent tissues where angiogenic blood vessels were abundant ( Figure 3J). GT198 expression levels in tumor were higher in smokers versus non-smokers ( Figure 3K), and were positively associated with lymph node metastasis ( Figure 3L). When patients were divided into two groups by GT198 expression scores, higher level of GT198 expression was significantly associated with decreased progression-free survival in patients (HR = 6.127, 95% CI) ( Figure 3M), suggesting that the presence of GT198 + pericytes promotes tumor progression and is a worse prognostic indicator in oral cancer patients.
To further confirm that GT198 + vessels give rise to tumor cells in human oral cancer, we co-stained GT198 together with CD31 for endothelial cells or α-SMA for pericytes or perivascular cells using adjacent tissue sections ( Figure 5). In angiogenic tumor stroma, GT198 was co-stained with α-SMA + pericytes enclosing a thin layer of CD31 + endothelium ( Figure 5 left panels). Within the tumor, CD31 + endothelium was still intact and was surrounded by a thickened layer of GT198 + α-SMA + perivacular cells ( Figure 5 middle panels). At this stage, its vessel cavity had already infiltrated with tumor cells. When tumor progressed, the vessel structure disintegrated with a gradual loss of CD31 + endothelial layer and proliferation of perivascular cell-derived GT198 + tumor cells (Supplementary Figure 2). In contrast, GT198 expression in pericytes was mostly negative in normal oral tissues in the absence of angiogenesis ( Figure 5 right panels). These results indicate that GT198 + pericytes in angiogenic blood vessels are capable to give rise tumor cells in human oral cancer.

GT198 + pericytes give rise to tumor cells in U-251 glioblastoma xenografts
To investigate malignant pericytes in brain tumor, we examined U-251 human gliobastoma intracranial xenografts in rat brain which are characterized by extensive microvascular proliferation in tumor angiogenesis. We found high level of GT198 expression  tissues were analyzed for pericytes. Immunohistochemical staining of GT198 is shown in the top panels counter-stained with hematoxylin. Fluorescent double staining of GT198 in red, CD31 and α-SMA in green, and DAPI in blue, is shown in the middle and bottom panels, respectively. Each enlarged boxed area is shown at the right. In tumor stroma, GT198 + α-SMA + pericytes enclose a thin layer of CD31 + endothelial cells. In tumor, CD31 + endothelial layer is enclosed by GT198 + pericytes, which further proliferate into tumor cells (Supplementary Figure 2). In normal vessels, GT198 expression is largely negative. Arrows indicate pericytes. Scale bars = 100 μm. associated with angiogenic pericytes in tumor but not with quiescent pericytes in normal vessels ( Figure 6A-6C). In particular, GT198 expression is cytoplasmic consistent with an active GT198 status ( Figure 6B) [39]. Compared to the surrounding GT198normal vessels in rat brain ( Figure 6C), GT198 + pericytes were specific to angiogenic vessels at the periphery of the developing tumor ( Figure 6A). The GT198 + vessels also expressed VEGF, vWF ( Figure 6D), as well as pericyte marker α-SMA (Supplementary Figure 3). Importantly, the stem cell marker CD133 was co-expressed with GT198 in cells surrounding the vessels ( Figure 6E). It appeared that GT198 + pericytes are able to proliferate as well as differentiate to produce larger vessels out of smaller capillaries ( Figure 6D). The larger vessels further merge into a tumor mass leaving original vessel lumens in the middle of the tumor ( Figure 6E). Tumor cells surrounding vessel lumens were undifferentiated expressing CD133, while cells distal to the lumens were more differentiated lacking CD133 ( Figure 6E). These observations suggested that GT198 + pericytes are derived from tumor stem cells or progenitors and are able to proliferate and differentiate back into tumor cells, utilizing vessel formation during the process, so that continued cycles expand tumor size. This conclusion is in part consistent with the observations from others on vascular mimicry of tumor-derived vessels. Our data indicate that GT198 + pericytes are both angiogenic and tumorigenic.

Vascular co-option between human GT198 + pericytes and rat endothelial cells
The above observation raised a question about the relationship between GT198 + pericytes and endothelial cells in this xenograft tumor model. To examine the origins of both cell types, we performed FISH analysis using dual-colored human and rodent FISH probes to distinguish cell origins. Human and rodent chromosome painting probes were first validated and found specific to human or rodent cells ( Figure 7A and Supplementary Figure 4A). Subsequent FISH analysis showed that the rat cell-derived endothelial layer in red was surrounded by human tumor-derived cells, with smaller vessels at the tumor periphery and large fused vessels at the center of the tumor ( Figure 7B-7C). In larger vessels, the endothelial layer was disorganized ( Figure 7C and Supplementary Figure 5). Although the human FISH probe in green was weak, it was clear that these human cells lacked red signal. Red signal was also found in normal rat brain tissue adjacent to tumor (Supplementary Figure 4B). The human origin of pericytes or tumor cells was also confirmed by immunohistochemical staining using a human-specific antibody against the human leukocyte antigen HLA-A, in which human HLA-A + pericytes captured rat HLA-Aendothelial cells ( Figure 7D). Using adjacent sections, we also confirmed that GT198 + pericytes were both VEGF + and HLA-A + , enclosing a thin layer of rat endothelium stained in red by rat FISH probe ( Figure 7E). Human tumor cells stained in green were able to infiltrate into rat tissues stained in red (Supplementary Figure 4C). Together, these data suggest that the angiogenic vessels in U-251 xenograft tumors are mosaic, containing human tumor-derived pericytes and rat host-derived endothelial cells. Therefore, vessel co-option and vascular mimicry may result from a single process where tumorderived GT198 + pericytes adopt the host's endothelial layer to differentiate into tumor cells via the process of angiogenesis.

GT198 activation stimulates VEGF and promotes tube formation in U-251 cells
The U-251 cell-derived GT198 + pericytes are clearly not quiescent. To test if cytoplasmic GT198 expression activates the pericytes, we utilized a previously characterized siRNA that suppresses wild type GT198 but stimulates its active splice variants [39]. We have previously shown that GT198 alternative splice variants induce cytoplasmic translocation of the wild type [40], and stimulate target genes including VEGF [41]. In U-251 cells, while GFP-wild type GT198 (1-217 aa) was nuclear and GFP-mutant GT198 (126-217 aa) was cytoplasmic, the siRNA induced cytoplasmic translocation of the wild type ( Figure 8A). We then tested this siRNA by transfection of U-251 cells to evaluate marker gene expression during GT198 activation. We found that the siRNA induced a switch in alternative splicing balance by diminishing wild type GT198 and increasing its active splice variants ( Figure 8B), and also induced cytosolic VEGF and vWF isoforms expression ( Figure 8C). As controls, nuclear VEGF expression detected by the same anti-VEGF antibody was unchanged which may represent a less angiogenic VEGF isoforms in nucleus [48]. Wild type GT198 was downregulated by its siRNA as expected ( Figure 8C), the expression of cytosolic GT198 was too low to be detected by Western blot although can be detected by fluorescent imaging ( Figure 8A). In addition, using a human VEGF-luciferase reporter [41], we found that GT198 siRNA stimulated the VEGF promoter in U-251 cells ( Figure 8D). Furthermore, GT198 siRNA Immunohistochemistry and FISH analyses using adjacent sections. GT198 + pericytes (black arrows) are HLA-A + and VEGF + , and enclose rat endothelial cells (white arrows). Scale bars = 100 μm. www.impactjournals.com/oncotarget increased U-251 cell survival when tested under increasing concentrations of a cytotoxic drug paclitaxel in an MTT assay ( Figure 8E). In siRNA-transfected U-251 cells, both efficacy and sensitivity of paclitaxel cytotoxicity were reduced ( Figure 8E). Together, these results indicate that activation of GT198 induced angiogenic factors including VEGF and promoted cell survival in U-251 cells.
Hypoxia is a well-characterized condition to induce angiogenesis. Using FACS analysis, we surprisingly found that GT198 protein was detectable on living U-251 cell surface, and was markedly increased in hypoxia ( Figure  9A). Thus, GT198 is capable to translocate into cytoplasm as well as to cell surface, given that translocation among cellular compartments is a well-observed phenomenon in many oncoproteins such as p53 [49], Rb [50], BRCA1 [51], and EWS [52]. Taken the advantage of surface GT198 in FACS analysis, we found that hypoxia induced an increase of pericyte marker CD146, in contrast to CD31 or CD133, in GT198 + cell population ( Figure 9B). This result supports the expression of GT198 in pericytes in U-251 cells. In addition, when U251 cells were transfected with wild type GT198, mutant GT198, or siRNA, only siRNA induced detectable tube formation on Matrigel in serum-free medium ( Figure 9C). Mutant-transfected cells were apoptotic [39], and thus did not induce tube formation. The activity in siRNA-transfected cells was potentially through the stimulation of VEGF ( Figure 8C).
The isolated GT198 + population from siRNA-transfected cells also showed increased tube formation than GT198population ( Figure 9D). Taken together, our results support that activation of GT198 promotes vasculogenic activity in U-251 cells.

GT198 vaccination suppresses GL261 glioma growth in mouse tumor model
The cell surface expression of GT198 may permit targeted immunotherapy against angiogenesis, and we tested this potential in a mouse brain tumor model implanted with syngeneic GL261 glioma. GT198 was strongly expressed in a proportion of tumor cells and vessel pericytes of GL261 tumor implanted in C57BL/6 mouse brain ( Figure 10A). Mice were subcutaneously vaccined with repeat doses of denatured recombinant GT198 protein as antigen, or PBS as control, starting at six weeks before tumor implantation ( Figure 10B). Anti-GT198 antibody titer measured from each bleed showed that the antibody peaked around the time of tumor implantation but with marked individual variability in the vaccined mice, while the PBS control mice lacked anti-GT198 antibody ( Figure 10B). The control group (n=4) died of tumor between 21-23 days after tumor implantation, whereas the vaccine group (n=4) died between 28-39 days, with significantly prolonged survival time ( Figure 10C). The V4 vaccined mouse had highest antibody titer and survived longest. Optical imaging of tumor sizes suggested a reciprocal correlation between anti-GT198 antibody titers and the tumor sizes ( Figure  10D-10E), particularly at the early stage of tumor development at week 2. Our results suggested that anti-GT198 antibody, if successfully produced by vaccination, may have protective effect against GL261 tumor growth and prolong mouse survival.
We also evaluated GT198 vaccination using primed dendritic cells in a mouse tumor model implanted with 4T1 breast tumor containing GT198 + pericytes and GT198 + stromal cells (Supplementary Figure 6A). GT198 protein-primed matured dendritic cells inhibited tumor growth without metastasis, while the control mice showed metastasis (Supplementary Figure 6B-6D). Although more in vivo stimuli may be present during angiogenesis, the above data together support that cytoplasmic GT198 is a marker for activated pericytes which have malignant potential to recapitulate tumor.

DISCUSSION
Solid tumor growth is often dependent on angiogenesis. Despite intensive studies on the cellular and molecular basis of angiogenesis [4], transient clinical benefit and drug resistance to anti-angiogenic therapies in patients indicate the need of new knowledge [6,53,54]. Using the oncoprotein GT198 as a marker, we report here that malignant pericytes expressing GT198 give rise to tumor cells through the process of angiogenesis. The finding of GT198 + pericytes provides a strengthen link among existing angiogenesis models. In a possible scenario, sprouting angiogenesis may be initiated by surrounding malignant pericytes, which produce higher than normal levels of VEGF ( Figure 7E and 8C) to stimulate the sprouting of endothelial tip cells growing toward the pericytes. Next, malignant pericytes capture, adopt, and enclose incoming endothelial cells as vessel co-option ( Figure 7B-7D). Then, intussusception is promoted by the abundance of non-quiescent malignant pericytes ( Figure 6B), due to the lack of quiescent normal pericytes to maintain vessel stability. Thereafter, proliferation and differentiation of tumor cells derived from pericytes result in thick-walled large vessels ( Figure  6D), which have previously been described as vascular mimicry. Furthermore, CD133 + cancer stem cells can differentiate into malignant pericytes in tumor vessels [25], since tumor cells and pericytes are interchangeable ( Figure 6E). Finally, it is possible that bone marrowderived myeloid stem/progenitor cells may differentiate into pericyte progenitors. Evidence from others also supports the incorporation of bone marrow cells into perivascular locations rather than into vessel lumens [21,55]. Our study does not exclude the possibility of the existence of tumor-derived endothelial cells [20,24], or non-endothelial lined vessels [18,19], but emphasizes that tumor-derived pericytes are more evident, at least in the models studied here. Specifically, we provide strong evidence to show that the endothelial cell layer is derived from the host in U-251 xenografts ( Figure 7B-7C and Supplementary Figure 5). The host-derived endothelial layer is gradually lost into disintegration and is ultimately overgrown by tumor cells (Figure 7B-7C). Together, these studies suggest that previous hypotheses in the field are not mutually exclusive; rather, they are multiple steps in a linked process mediated by malignant pericytes during the process of angiogenesis. An integrated hypothetical model is presented in Figure 11. First, malignant GT198 + pericyte progenitors capture sprouting capillaries to form smaller vessels. Continued differentiation from pericytes into tumor cells yields larger tumor-enclosed vessels that are still functional. The fusion of these vessels produces tumor masses containing GT198 + tumor nodules, in which pericyte progenitors continue the cycle. In support of this hypothetic model, tumor-derived pericytes have been previously demonstrated [25,26]. Tumor cells with migratory pericyte behavior have been described in details [56]. Pericyte-derived tumor cells were also observed in reports of vascular mimicry or tumor-do-it-yourself vessels [27], or even as the product of reversible epithelial to mesenchymal transition [57]. Here we extend these previous observations using GT198 as a marker to confirm that these two processes are actually interconnected in concurrent cycles during tumor angiogenesis. When tumor cells are already present, such as in metastatic tumors or in xenografts, malignant pericytes can be tumor-derived. In contrast, in early human primary cancers before tumor growth has occurred, malignant GT198 + pericytes could be derived from normal pericytes, such as through acquisition of somatic mutations [41]. Thus, distinct initiating stimuli would lead to the development of malignant pericytes as a common step in tumor angiogenesis. Our observations on migrating GT198 + pericytes in human oral cancer (Figure 4), are consistent with this hypothesis. These findings also provide direct evidence to support the notion that during the process of epithelial-to-mesenchymal transition, mesenchymal stromal cells become malignant pericytes [26], and are thereby able to migrate.
The existence of malignant pericytes may have implications for the effectiveness of current anti-cancer strategies. For example, the development of radiation resistance in tumors, may be explained by the survival of GT198 + malignant pericytes; since activation of GT198 stimulates DNA repair [37,39], which could support the survival of resistant cells. Similarly, the transient effect of anti-VEGF therapy is often due to the development of drug resistance. The VEGF target may not be specific enough to completely suppress the tumor vasculature [53]. While VEGF + pericytes may be inhibited, VEGF -GT198 + pericytes would be predicted to escape anti-VEGF therapy, particularly in advanced or metastatic tumors [58]. Based on our preliminary evidence (Figure 10), anti-GT198 in combination with anti-VEGF therapy may be one practical approach for overcoming such resistance.
In summary, our study shows that overexpression of cytoplasmic GT198 in malignant pericytes is a common feature of the angiogenic tumor vasculature among various human primary cancers and rodent tumor models. By analyzing human oral cancers and glioblastoma xenografts, we suggest that malignant pericytes expressing GT198 give rise to tumor cells. These observations imply that existing hypotheses in tumor angiogenesis are interconnected and reconcilable when malignant pericytes are incorporated. GT198 protein has the potential to be a new target in anti-angiogenic therapy.

Human cancer sections
Institutional Review Board approval was obtained following institutional guidelines using de-identified human cancer formalin-fixed paraffin-embedded (FFPE) sections from Augusta University, Augusta, GA. Human oral tumor microarray FFPE sections containing 40 cases of triplicate cores at 1.0 mm and associated clinical information were originally prepared by the Head and Neck Cancer SPORE at University of Pittsburgh Cancer Institute, Pittsburgh, PA. Additional common human tumor FFPE microarrays at 2.0 mm in diameter were from Imgenex Corporation, San Diego, CA. Pathology diagnosis was verified through histological examination before immunohistochemistry analysis.

Xenograft mouse tumors
FFPE sections of xenograft tumors were originally prepared using human cancer cell lines xenografted into nude mice. The six human cancer cell lines include breast cancer BT474, ovarian cancer OVCAR-3, glioblastoma T98G, lung cancer Calu-6, prostate cancer DU145, and colon cancer SW480.

Whole mount in situ hybridization
Mouse embryos (FVB/N) at stages E8.5, E9.5, and E10.5 were fixed overnight in 4% paraformaldehyde in PBS with 0.1% Tween at 4°C and dehydrated through serial methanol at 25%, 50%, 75% and 100%. The dehydrated embryos were treated with RNase-free DNase I (50 U/ml) before hybridization with denatured riboprobe. The antisense and sense riboprobes were produced by in vitro transcription in the presence of Digoxigenin-UTP (Roche Diagnostics) using full-length GT198 cDNA in the pcDNA3 vector. Stained embryos were fixed and photographed.

U-251 glioblastoma xenograft in rat brain
The rat xenograft tumor model of U-251 glioblastoma was previously described [59]. The human U-251 cell line was authenticated with the following short tandem repeat and Amelogenin profiles matched: HT01, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, and TPOX. Animal protocols were approved by the IACUC at Augusta University. NIH-RNU nude rats (Charles River Laboratories, Frederick, MD) at five weeks of age were anesthetized and injected with 5 μl of U-251 cells (4x10 5 ). Briefly, the surgical zone was swabbed before incision, and a 3 mm hole in the skull was made by a dental drill. U-251 cells were stepwise injected at a rate of 0.5 μL per 30 second. The surgical hole was sealed with bone wax after the procedure. Rats were sacrificed on day 22. Tumors were harvested to prepare FFPE sections for immunohistochemistry, immunofluorescence, and fluorescent in situ hybridization (FISH) analyses.

Fluorescent in situ hybridization (FISH)
FISH analysis was carried out using FFPE sections of U-251 glioblastoma xenograft tumors in rat brain. Immunohistochemical staining of GT198, VEGF and HLA-A using adjacent sections was carried out for comparison. Paraffin-embedded rat brain tissue sections were deparaffinized through xylene and ethanol series, followed by antigen retrieval as described above. The sections were further digested by 5% pepsin in 0.01 N HCl at 37°C for 10 min before use. Gene-specific or locus-specific FISH probe signals were found weak due to higher background signals in FFPE sections, so that only chromosome painting FISH probes were used in this study. The rodent-specific chromosome painting probe was a Cy3-labeled mouse painting probe in red (1200-11MCy3-02, StarFISH, Cambridge, UK), which detected both mouse and rat but not human nuclei. The humanspecific chromosome painting probe was prepared by labeling purified human placenta genomic DNA in green using SpectrumGreen dUTP (Nick Translation Kit, Vysis) following the manufacturer's protocol. Prior to FISH analysis, probe specificity was validated using mixed human U-251 and rat GH3 cells, or human HeLa and www.impactjournals.com/oncotarget mouse P19 cells. Subsequently, rodent and human probes were applied simultaneously on rat brain tumor sections for dual-color visualization. Sections were hybridized using Vysis reagents and counterstained with DAPI II before visualization using fluorescence microscopy.

Western blot and reverse-transcript PCR
U-251 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 0.1 μg/μl streptomycin, and incubated in 5% CO2 at 37°C. Cells were transfected in 6-well plates by Lipofectin (Invitrogen, Carlsbad, CA) using 0-100 nM GT198 siRNA as indicated. After 16 hours, the cytosolic fraction was isolated using lysis buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 μg/ml protease inhibitors) with the addition of 1% Triton X-100 for 15 min on ice, and was probed by rabbit anti-VEGF (RB-9031) and anti-vWF. For the nuclear extract fraction, cells were lysed in the above lysis buffer with 0.05% Triton X-100 for 15 min on ice. The nuclei were then collected by centrifugation and further lysed in the above buffer with 420 mM NaCl for 30 min. Nuclear extracts were probed by anti-VEGF, anti-GT198, and anti-β-actin (Sigma, St Louis, MO). Western blots were detected using the ECL system (GE Healthcare, Piscataway, NJ). For reverse-transcription PCR, total RNA from siRNA-transfected U-251 cells was isolated by Trizol and analyzed using one-step RT-PCR kit (Qiagen, Valencia, CA). GT198 primers used are at Exon 1 and 5: 5'-CTTCCCCTTCAGCCAATCAC-3' and 5'-GG TAGCTGCTTTAATGTTCTTCAA-3'.

VEGF promoter luciferase assay and MTT assay
Human VEGF promoter luciferase reporter (-1970 to +19) was previously described [41]. U-251 cells were transfected in triplicate in 24-well plates using 0.1 μg/well VEGF-luciferase reporter together with GT198 siRNA (0-100 nM), as indicated. Cells were harvested 16 hours after transfection, and relative luciferase activities were measured by a Dynex luminometer. Data are presented as means of triplicate transfections ± standard errors. For MTT assay, U-251 cells were transfected in duplicate in 48-well plates using 0 nM or 100 nM GT198 siRNA, and incubated for 72 hrs using increasing concentrations of paclitaxel (0, 0. 27

FACS analysis and tube formation assay
FACS analysis and data acquisition were performed on a flow cytometer (Accuri C6, BD Biosciences). Hypoxia U-251 cells were generated in DMEM with 10% fetal bovine serum, in 1% oxygen and 5% CO2 at 37°C overnight. Living normoxia and hypoxia U-251 cells were stained in PBS with 1% BSA on ice using anti-GT198 and anti-rabbit Alexa 448; or co-stained with PE-conjugated anti-CD31 or CD146 (342004, BioLegend, San Diego, CA), or CD133 (Miltenyi Biotech, Germany). For fixed cells, cells were incubated in 1% paraformaldehyde for 10 min and methanol for 20 min on ice before antibody staining. A minimum of 10,000 cells within the gated region were analyzed.
In tube formation assay, U-251 cells were lipofectintransfected for 4 hours in 6-well plates using wild type GT198 (1-217), mutant GT198 (126-217), GT198 siRNA (100 nM), or pcDNA3 vector as a control. After overnight culture, transfected cells were seeded at 2x10 4 per well in duplicate in a 96-well plate containing 50 μl Matrigel in serum-free medium, and incubated for overnight before photograph and quantification. To isolate GT198 + cell population, siRNA-transfected U-251 cells (2x10 5 ) were incubated with anti-GT198 antibody (1:100, 1 ml) and protein A agarose beads (30 μl). Bound cells were eluted by His-tagged GT198 protein (3 μg/ml, 200 μl). Unbound cells (GT198 -) and eluted cells (GT198 + , 28% by cell count) were washed and subjected to the tube formation assay. www.impactjournals.com/oncotarget GT198 vaccination in GL261 and 4T1 mouse tumor models For GL261 glioma model, denatured GT198 antigen for vaccination was produced from insoluble inclusion body of recombinant GST-GT198 protein isolated from E. coli BL21(DE3), since insoluble antigen has greater efficacy in vaccination. Briefly, the isolated inclusion body containing 95% pure GT198 protein was repeatedly washed by sonication in PBS and sterilized by 70% ethanol. Incomplete Freund's adjuvant (IFA) was mixed with PBS at 1:1 ratio together with GT198 protein pellet, and was sonicated using a sterilized probe to produce GT198 antigen at 1 mg/ml for each subcutaneous injection at 100 μg in 100 μl (on days 0, 14, 28, 46, 63). GST was too soluble to yield inclusion body so that PBS alone was served as control. Mouse tail blood was collected at each vaccine time point to produce serum (5-10 μl). The antibody titers were measured at the end of experiment using His-tagged GT198-coated 96-well white plate (100 ng GT198 and 5 μg BSA/well), which was incubated with 200 μl of 1:1000 diluted mouse sera in duplicate wells, and detected by HRP-conjugated anti-mouse antibody with ECL detection reagents. Antibody titers were counted by a Dynex luminometer. Tumor implantations in mice brain were carried out in eight C57BL/6 mice weighing 20-22 g using luciferase-positive GL261 glioma cells (5x10 4 in 3 μl) six weeks after the initial vaccine (n=4) or PBS (n=4) injection. For optical imaging, the living mice were intraperitoneal injected with luciferin at 150 mg/kg and optical bioluminescent imaged at weeks 2 and 3 after tumor implantation using an AMI-x optical imager.
For 4T1 breast tumor model, immunocompetent Balb/c mice were implanted with luciferase-positive 4T1 breast cancer cells (5x10 4 ) two weeks after two doses of immunizations at 2 months intervals using GT198-primed matured dendritic cells. Purified soluble recombinant GST-GT198 and control GST proteins (200 μg/mouse) were used as antigens to prime dentritic cells. The living mice were intraperitoneal injected with luciferin at 150 mg/kg for bioluminescent imaging every week using an AMI-x optical imager. Photon intensity reflecting tumor growth was graphed using GraphPad Prism software.

Statistical analysis
Statistical analyses were carried out using GraphPad Prism software. Scattergrams with means and Kaplan-Meier curves are presented using immunohistochemical staining scores in human oral cancer. P values in scattergrams or in bar graphs were calculated using unpaired two-tailed t test. * P<0.05, ** P<0.01, *** P <0.001; NS, not significant. P value in Kaplan-Meier curves was calculated by log-rank test. P value in luciferase assay was calculated by linear progression. IC50 values in MTT assay were calculated by non-linear progression sigmoidal curve fit. A P value of less than 0.05 is considered statistically significant.