Rationale: Emerging evidences have highlighted the critical roles of lncRNAs in human cancer development. The work sought to assess the biological role and potential underlying mechanisms of lncRNA-CF129 (CF129) which is significantly reduced in pancreatic cancer (PC).Methods: CF129 expression and its association with multiple clinicopathologic characteristics in PC specimens were analyzed. The role of CF129 both in vitro and in vivo was assessed, with RNA pull-down and immunoprecipitation assays being performed to detect the interaction between CF129 and p53 and E3 ligase MKRN1. Chromatin immunoprecipitation and luciferase assays were utilized to identify the interaction between p53 and FOXC2 promoter, HIF-1α/HDAC1 complex and CF129 promoter, FOXC2 and HIF-1α promoter, respectively.Results: CF129 levels were markedly lower in PC compared with paired non-tumor adjacent tissues. Low CF129 expression predicted short overall survival in PC patients. CF129 inhibited invasion and metastasis of PC cells in a FOXC2-dependent manner. In addition, CF129 regulates FOXC2 transcription through association with mutant p53. CF129 directly binds to p53 and E3 ligase MKRN1, and such an interaction leading to p53 protein ubiquitination and degradation. Furthermore, CF129 is a hypoxia-responsive lncRNA, which is transcriptionally downregulated by binding between HIF-1α/HDAC1 complex and CF129 promoter. Finally, it is revealed that HIF-1α is reciprocally regulated by FOXC2 in transcriptional level. Clinically, CF129 downregulation coordinates overexpression of FOXC2.Conclusions: Our study suggests that CF129 inhibits pancreatic cell proliferation and invasion by suppression of FOXC2 transcription, which depends on MKRN1-mediated ubiquitin-dependent p53 degradation. The HIF-1α/CF129/ p53/FOXC2 axis may function as a potential biomarker and therapeutic target. Pancreatic cancer is among the deadliest malignancies, with a mortality rate that ranks among the top four in worldwide. Unfortunately, there is currently no specific marker for early pancreatic cancer diagnosis, and as a result it is often diagnosed in advanced stages when there is already metastatic invasion and no opportunity for radical operation. Nevertheless, pancreatic cancer is resistance to chemotherapy and radiotherapy, which exaggerates the miserable prognosis. Because of complexities of signaling at genetic, epigenetic, and metabolic levels, the therapeutic options and progress in drug development are impeded in pancreatic cancers Long noncoding RNAs (lncRNAs) are RNAs that are over 200 nucleotides long and do not code for protein, even though they play essential roles in many biological contexts including epigenetic regulation, cell cycle control, nuclear import, differentiation, and other pathways in vivo and in vitro. Mechanical experiments further indicated that CF129 promotes an interaction of p53 and E3 ligase MKRN1, which consequently induces the ubiquitination and degradation of p53 protein in pancreatic cancers. We further revealed p53 promoted FOXC2 transcription, thereby, CF129 repressed FOXC2 expression through inducing p53 instability. Specifically, we showed CF129 was inhibited by the complex of HIF-1α and HDAC1 during hypoxia. In the meantime, FOXC2 regulates transcription of HIF-1α, by which CF129 can indirectly inhibit HIF-1α expression. Therefore, the present study demonstrates a lncRNA-interacted feedback of HIF-1α and FOXC2 which is involved in pancreatic cancer progression during hypoxia microenvironment.Many lncRNAs dysregulated in pancreatic cancer have been previously identified through microarray assay Figure ). Moreover, the downregulation of CF129 was further verified in 40 cases of PC tissues relative to paired NP tissues . Compared to immortalized human pancreatic duct cells (HPDC), CF129 was downregulated in most of the cell lines and PANC-1 and BxPC-3 cells were the lowest . Meanwhile, the secondary structure of CF129 is predicted by online database AnnoLnc Figure ). In addition, the protein-coding capacity of CF129 was evaluated by online prediction software, which validated CF129 as a non-coding RNA . Furthermore, results from nuclear/cytoplasmic RNA fractionation revealed that CF129 mainly presents in the nucleus . This result was further confirmed via fluorescence in situ hybridization (FISH) using a CF129 probe . We next evaluated the relationship between CF129 expression and the clinicopathologic characteristics of PC patients. The correlation analysis showed that low expression of CF129 was associated with large tumor size, poor tumor differentiation, lymphatic invasion, distant metastasis (Supplementary Table 1), and shorter overall survival . This suggested CF129 might be involved in PC development.Differential lncRNA expression between pancreatic cancer (PC) and paired noncancerous peritumoral (NP) tissues determined via microarray, revealing lncRNA-CF129 (CF129) to be among the most significantly decreased lncRNAs in PC samples , the PANC-1/BxPC-3 proliferation and colony formation were remarkably suppressed . Additionally, both the wound-healing and transwell assays demonstrated suppression of migration and invasion of PC cells due to CF129 overexpression .Since CF129 was downregulated in PC tissues, we explored its biology by overexpression of CF129. After transfection with CF129-containing vector (CF129-OE) . In addition, the number of mice with liver metastases and the visible metastasis loci in the LV-CF129 group was apparently less than those in the control group .To validate these findings, PANC-1 cells were transfected stably using lentivirus containing negative control (LV-NC) or CF129 sequences (LV-CF129) and further implanted into nude mice to construct xenograft tumor model. Relative to LV-NC mice, the tumors in LV-CF129 mice were smaller and weighed less . Contrary to CF129 overexpression, downregulation of CF129 remarkedly promoted proliferation, colony formation of PANC-1/BxPC-3 cells . In addition, migration and invasion were also significantly increased following transfection with siCF129 .To further elaborate impacts of CF129 on pancreatic cancer cells, three siRNAs targeting CF129 were designed and siCF129-1 and siCF129-2 were used for experiments , we further explore whether CF129 could regulate expression of FOXF1, FOXC2 or FOXL1. Interestingly, CF129 overexpression or knockdown dramatically inhibited or increased expression of FOXC2 mRNA, but not expression of FOXF1 and FOXL1 mRNA . Moreover, the luciferase reporter assay showed that siCF129 obviously increased the luciferase intensity of PANC-1 cells which were transfected with vector containing FOXC2 promoter . Coincidently, expression level of FOXC2 protein was significantly increased by siCF129 but decreased with CF129 overexpression . As that of CF129 overexpression, FOXC2 knockdown with siFOXC2 dramatically suppressed proliferation and invasion of PANC-1 and BxPC-3 cells . Furthermore, the co-transfection with FOXC2-OE effectively rescued FOXC2 expression, proliferation, and invasion of PANC-1 cells which were inhibited by CF129 overexpression . On the contrary, siFOXC2 dramatically depleted FOXC2 expression, proliferation and invasion of PANC-1 cells which were induced by siCF129 . These results intensively suggest that CF129 exerts function in pancreatic cancer cells by repressing FOXC2 expression in transcriptional level.Researches had demonstrated that lncRNAs regulate expression of neighborhood gene in cis. Since CF129 is adjacent to forkhead family of transcription factors including FOXF1, FOXC2 and FOXL1 . Moreover, the results of Jaspar revealed 3 putative binding sits for p53 in the FOXC2 promoter region . Meanwhile, knockdown of p53 dramatically inhibited FOXC2 expression both in mRNA and protein level . Therefore, we assumed that p53 may act as transcription factor to mediate the regulation of CF129 on FOXC2 expression. To further identify the association of CF129 and p53, we performed RNA pulldown and RNA immunoprecipitation (RIP) assays. The western blot results showed that p53 protein was accumulated by CF129 but not by antisense transcript . The qPCR results confirmed that CF129 was significantly enriched by anti-p53 antibody but not IgG . In addition, deletion-mapping analyses identified that the 195nt region at the 5' end of CF129 is required for its association with p53 . In addition, the ChIP assay verified the association of p53 and the chromatin fragments corresponding to the site P2/P3, rather than the site P1 within the FOXC2 promoter region . Furthermore, we used luciferase reporter assays to validate whether p53 activating transcription of FOXC2 promoter. FOXC2 promoter sequence containing wild-type (WT) or mutant-type P2/P3 (MUT) was inserted in the luciferase reporter plasmid promoter. We found that luciferase activity from the WT, but not the MUT, was markedly decreased by p53 knockdown .lncRNAs have recently been found to exert effects in part via interacting with transcription factors. To predict the putative transcription factor involved in regulation of CF129 on FOXC2, an intersection analysis of online database catRAPID and Jaspar was applied. The catRAPID analysis showed high affinity between CF129 transcript and p53 protein . Coincidently, the luciferase activity of WT was decreased by CF129 overexpression, but increased by CF129 downregulation . Coincidently, the siCF129- induced upregulation of FOXC2 was inhibited by the knockdown of p53 in BxPC-3 cells . Importantly, the enhanced proliferation and invasion abilities of CF129-knockdown BxPC-3 cells were reversed by co-transfection with sip53 . Since both BxPC-3 and PANC-1 cell lines are p53 mutant phenotype Moreover, the CF129 overexpression markedly decreased, while the CF129 knockdown increased the enrichment of p53 on FOXC2 promoter , while CF129 knockdown enforced expression of p53 protein . Nevertheless, neither siCF129 nor CF129-OE demonstrated obvious effects on mRNA expression of p53 . To further reveal whether CF129 regulated p53 stability, p53 protein was measured in cycloheximide (CHX) treated cells, to prevent protein synthesis. The results showed that p53 protein in pancreatic cancer cells was destabilized with CF129 overexpression , while knockdown of CF129 increased the stability of p53 protein .Researches had reported that lncRNA regulate protein degradation and thereby plays a critical role in tumorigenicity Figure ) or downregulation on p53 expression . Given that p53 is regulated by the ubiquitin-proteasome pathway Figure ), while knockdown of CF129 decreased ubiquitination of p53 , respectively. Together, these data suggest that CF129 inhibits p53 protein expression in post- transcriptional level by promoting ubiquitin- dependent degradation.Moreover, the proteasome inhibitor MG132 could abolish the regulation of CF129 overexpression . It has been reported that MKRN1 induces the ubiquitination and proteasomal degradation of p53 Figure ). A significant upregulation of p53 protein upon MKRN1 knockdown was detected in both PANC-1 and BxPC-3 cells . Based on the findings, we presumed that MKRN1 might be involved in the process about CF129- induced degradation of p53. RNA pulldown assays using lysates from PANC-1 cells confirmed the association between CF129 and MKRN1 . The association of CF129 and MKRN1 was further confirmed by RIP analysis with anti-MKRN1 antibody . Deletion-mapping analyses identified that the segment (5' 246-377nt 3') of CF129 were required for the association with MKRN1 . Coincidently, MKRN1 knockdown abrogated the decrease of p53 protein caused by the CF129 overexpression in PANC-1 and BxPC-3 cells . Vice versa, knockdown of CF129-induced p53 expression was rescued by overexpression of MKRN1 . We then further evaluated whether CF129 modulated the association between MKRN1 and p53. Co-IP assays revealed that overexpression of CF129 promoted the interactions between MKRN1 and p53 . On the contrary, the interaction between MKRN1 and p53 was reduced upon the CF129 knockdown . In addition, MKRN1 knockdown dramatically decreased p53 ubiquitination, which was increased after co-transfected with CF129 . In contrast, MKRN1-enhanced ubiquitination of p53 was reduced by CF129 knockdown . Interestingly, the knockdown of MKRN1 did not affect the interaction between CF129 and p53, whereas the knockdown of p53 also did not affect the association between CF129 and MKRN1 .We further explore the precise mechanism about how CF129 regulates the ubiquitin-dependent degradation of p53 protein. Interestingly, depending on the online prediction from catRAPID, Makorin Ring Finger Protein 1 (MKRN1), a E3 ligase, might be associated with CF129 , we next explored whether CF129 was transcriptionally regulated by HIF-1α during hypoxia. Coincidently, CF129 were significantly downregulated with treatment of hypoxia  or COCl2  in a time-dependent manner. Moreover, both qRT-PCR and FISH assays demonstrated that HIF-1α knockdown obviously increased CF129 expression which was repressed by the hypoxia  or COCl2 treatment . Coincidently, the ChIP assays validated an association between CF129 promoter and HIF-1α was enhanced by hypoxia or COCl2 but suppressed after transfection with siHIF-1α . Additionally, luciferase reporter plasmid containing CF129 promoter  or HRE-mutant CF129 promoter  was transfected into PANC-1 and BxPC-3 cells. The results verified the siHIF-1α enhanced luciferase intensity which was inhibited by hypoxia  or COCl2 treatment .Hypoxia is a common feature of solid tumors and plays key roles in tumor development. Recent results had shown lncRNAs were aberrantly expressed during hypoxia Figure ) or COCl2 . Since CF129 is a hypoxia-responsive lncRNA, we further identified the effects of CF129 on growth of PC cells during different oxygen concentration. Similar to 5% O2 , the growth of PANC-1 cells were significantly promoted by CF129 depletion but inhibited by CF129 overexpression during both 1% O2 and 0.5% O2 . Therefore, these data intensively indicate CF129 is a hypoxia-responsive lncRNA in pancreatic cancer.The expression of p53 and FOXC2 was further evaluated to validate the critical roles of CF129 during hypoxia. The western blot results showed that both siHIF-1α and CF129 overexpression could reverse p53 and FOXC2 expression which was induced by hypoxia . PANC-1/BxPC-3 CF129 expression during hypoxia was obviously upregulated by treatment of HDAC1 knockdown or deacetylase inhibitor trichostatin A (TSA) . Meantime, the ChIP assay displayed the hypoxia-enhanced binding of HDAC1 with HRE of CF129 promoter was significantly downregulated by siHDAC1 but not TSA . Nevertheless, the hypoxia-inhibited luciferase density of WT reporter was upregulated by siHDAC1 or TSA . Coincidently, we also revealed that hypoxia-induced p53 and FOXC2 was markedly repressed by siHDAC1 or TSA . Therefore, these results indicate that the downregulation of CF129 is regulated via the HIF-1α/HDAC1 complex in hypoxic contexts.Research has revealed that HDACs are involved in downregulation of noncoding RNA including lncRNA and microRNA under hypoxic condition Figure ), which implied HIF-1α might be regulated by FOXC2 in transcriptional level. As expected, both HIF-1α mRNA and protein expression was significantly reduced after knockdown of FOXC2  but increased by overexpression of FOXC2 . Coincidently, the ChIP assay demonstrated FOXC2 antibody induced obvious accumulation of HIF-1α promoter . To further validate whether FOXC2 binding on the HIF-1α promoter was functional, HIF-1α promoter containing wild-type (WT) or mutant (MUT) FOXC2-binding sequence was inserted into the luciferase reporter plasmid. Results showed the luciferase density of the WT reporter, but not the MUT reporter, was markedly induced by FOXC2 overexpression .Based on that FOXC2 is a transcriptional factor, DNA sequence analysis showed a putative HRE (-1649 to -1639nt) in the HIF-1α promoter region . Moreover, the ChIP and luciferase reporter assay demonstrated that CF129 overexpression significantly inhibited the accumulation and activation of CF129 on FOXC2 promoter . Meantime, the hypoxia- induced HIF-1α was dramatically attenuated by CF129 but rescued by the FOXC2 overexpression . Vice versa, the siCF129 markedly compensated the HIF-1α expression which was inhibited by siFOXC2 during hypoxia .Since FOXC2 is repressed by CF129, we further presumed whether HIF-1α can be reciprocally regulated by CF129. Results showed CF129 knockdown increased, while CF129 overexpression decreased both mRNA and protein level of HIF-1α in PANC-1/BxPC-3 cells . Moreover, the publicly available databases for pancreatic cancer  also displayed that FOXC2 was significantly higher in human PC tissues than that of NP tissues . Furthermore, a significant inverse correlation was observed between CF129 and FOXC2 transcript levels in 40 pancreatic cancer specimens  . Moreover, the online database (ChIPbase) demonstrated an obvious correlation between HIF-1α and FOXC2 mRNA expression in pancreatic adenocarcinoma (PDDA) . Kaplan-Meier and log-rank test analyses showed that pancreatic cancer patients with high FOXC2 expression had a shorter overall survival time than those patients with low FOXC2 expression . Based on our results, we depicted a schematic diagram to depict that CF129 played an important role in regulation of HIF-1α/FOXC2 pathway during hypoxia stress .To further assess the regulation of CF129 on FOXC2 expression, we measured FOXC2 mRNA level in the same cohort of the 40 pancreatic cancer tissues. The FOXC2 level was significantly higher in PC tissues than that in (NP) tissues  including FOXF1, FOXC2 and FOXL1, we further investigated whether CF129 exerts its function by regulating these FOX proteins expression in cis. Interestingly, knockdown or overexpression of CF129 obviously decreased or increased FOXC2 expression, without effects on FOXF1 and FOXL1 expression. FOXC2 has been reported to be overexpressed in numerous cancers and function as a central regulator for epithelia-mesenchymal transition (EMT) and metastasis, including breast cancer, lung cancer, colorectal cancer, et alMany studies have found that lncRNAs can exert their effects in part via interactions with specific proteins. Therefore, we further applied bioinformation analysis to define the putative protein which might be involved in the regulation of CF129-FOXC2 pathway. The intersecting analysis of catRAPID and ChIPBase demonstrated that p53 protein might be recruited by both CF129 transcript and FOXC2 promoter. Coincidently, the RIP and RNA-pull down assays revealed that CF129 can bind to p53. Moreover, the ChIP and luciferase reporter assays verified the accumulation and activation of p53 on FOXC2 promoter, which was impeded by CF129 overexpression. In addition, p53 knockdown obviously reduced FOXC2 expression which was inhibited by CF129. Therefore, these results indicate that p53 is required for the regulation of CF129 on FOXC2. The accumulation of mutant p53 protein inactivates its anti-proliferative properties but promote tumorigenesis and invasion through a gain-of-function activity The stability of p53 is carefully regulated via the ubiquitin-proteasome system, allowing levels of this protein to rise and fall in response to stressful conditions. lncRNAs are also known to play a role in regulating p53 Base on bioinformatic prediction from online database catRAPID, we revealed an association between CF129 and Makorin Ring Finger Protein 1 (MKRN1). MKRN1 is an E3 ligase for hTERT Hypoxia is common in solid tumors including those in pancreatic cancer, and it can modulate tumor progression as well as therapeutic responses Given the HIF-1α promoter containing potential binding sites for FOXC2, we further assumed whether HIF-1α can be reciprocally regulated by FOXC2. In agreement, our data showed that siFOXC2 dramatically inhibited HIF-1α expression in both mRNA and protein level. Moreover, the recruitment of FOXC2 on HIF-1α HREs was further identified by ChIP and luciferase reporter assays. Furthermore, CF129 dramatically inhibited HIF-1α expression in hypoxic conditions, and FOXC2 overexpression reversed this. In contrast, CF129 knockdown significantly increased HIF-1α expression which is inhibited by siFOXC2 during hypoxia. This suggests that FOXC2 mediates a reciprocal regulation of CF129 and HIF-1α. A result from Fan et al. reported that hypoxia/HIF-1α-induced lincRNA-p21 can disrupt the VHL-HIF-1α interaction and reciprocally enhances HIF-1α accumulation, by which promoting glycolysis under hypoxia In summary, we have shown that CF129 inhibits PC cell proliferation and invasion by suppression of FOXC2 transcription, which depends on MKRN1- mediated ubiquitination and degradation of mutant p53. Our findings suggest that the CF129/MRKN1/ p53/FOXC2 axis may be a promising target for PC therapy.In this study, 40 pancreatic cancer and corresponding non-tumor tissue samples were obtained from individuals undergoing operations at the Pancreatic Surgery Center, Union Hospital  from October 2015 to June 2016. If patients had previously undergone radiotherapy, chemotherapy or immunotherapy, they were not eligible for this study. All pancreatic cancer tissues were confirmed by the histopathological reports. Tumor staging was based on the revised international system. These clinical samples were used after obtaining informed consent from participants and their parents. All protocols were approved by the Union Hospital ethics committee.2, 1% O2, 5% O2, 5% CO2 and corresponding 94.9% N2 , 94% N2, 90% N2 ,respectively or treated with CoCl2 (400 μmol/L). Total RNA and protein were collected after 0h, 6h, 12h, 24h and 48h.The PANC-1 and BxPC-3 cell lines came from American Type Culture Collection . These cell lines were cultivated in RPMI-1640 (HyClone) tinting with 10% FBS (ScienCell) as well as penicillin/streptomycin in a standard 37℃ incubator. To induce hypoxia, cells were cultured with 0.1% OSupplementary Table The siRNA targeting CF129, FOXC2, p53, MKRN1, HIF-1α, HDAC1 and appropriate negative controls were obtained . The plasmids containing CF129, FOXC2 and MKRN1 and the matched negative control plasmid were synthesized from GeneChem Co. . Transfections were performed with lipofectamine 2000 (Invitrogen) based on provided instructions. The siRNAs were transfected at a final concentration of 50nM. For plasmids, 1.6μg was transfected into samples in 12-well plate, while 0.2μg was used for 96 well plate. Protein and total RNA were extracted at 48 h post-transfection for downstream analyses.The siRNA sequences used in this study are all shown in Supplementary Table An RNAiso Plus kit (TAKARA) was used for total RNA extraction based on provided protocols. The PrimeScript® RT Master Mix Perfect Real Time kit (TAKARA) was used to synthesize DNA. PCR amplifications were performed with the SYBR Premix Ex Taq II (TAKARA). The expression levels of LncRNA-CF129, FOXC2, p53, MKRN1, HIF-1α and HDAC1 were normalized to GAPDH as reference genes.Primer sequences used in this study are all shown in An MTT assay was employed to assess the proliferation of target cells. In 96-well plate, stable transfected cells (2000/well) with eight vices well for each sample. The proliferation was observed for 1 to 5 days. At certain hour every day,20μl MTT (5 mg/mL) was added to appropriate wells, and after 4h it was replaced with 150μl DMSO (Sigma). Finally, ELISA reader was used to determine the absorbance at 570nm.PANC-1 and BxPC-3 migratory abilities were measured using a wound healing assay. Stable transfected cells were allowed to form a confluent monolayer in 12-well plates. We observed the healing of the wound for 0h, 24h and 48h after would scratching with microscope. To invasion assay, the Matrigel Invasion Chamber of pore size 8μm  coated with Matrigel  was used. 5×10 cells in serum-free media were placed in the upper chamber with 250μL serum-free medium, while 700μL medium with 30% FBS was added to the lower chamber. After 24h, the cells were fixed and stained with removed in upper chamber. Finally, cells on the lower chamber were counted via microscopic assessment of five fields.Stable transfected cells in 6-well plates (1000/well) were cultured for 8 days. Wash cells with PBS and Fix cells with % polyformaldehyde for 20 minutes, and followed 1% Crystal Violet for 20 minutes after discarding solution. Finally, we count the number of cells in each well under the microscope.lncRNA CF129 was assessed via fluorescence in situ hybridization, a kit from named FISH Tag™ RNA Multicolor Kit  was used. The prepared cell samples were fixed with 5% formaldehyde for 25 minutes. Samples were then combined overnight at 42°C with the CF129 probe. Cells were then washed, nuclei were stained using DAPI, and stained cells were imaged via an LSM 5 Pascal Laser Scanning Microscope (Zeiss).The Pierce™ Magnetic RNA-Protein Pull-Down  and MAXIscript®  kits were used for this assay based on provided protocols. Briefly, PANC-1 nuclear extracts were combined with biotinylated CF129 or antisense- CF129, after which streptavidin beads were added to the samples. Pulled down RNA was then isolated by washing these beads, boiling them in SDS buffer, and subsequent western immunoblotting.A Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  was used based on provided protocols. Briefly, 500uL lysis buffer supplemented with protease and RNase inhibitors was used to lyse cells in 6-well plates. Lysates were spin at 16,000 xg for 10 minutes, and supernatants were used for precipitation with antibodies against p53, MRKN1, and anti-mouse IgG or anti-rabbit IgG for 3 h at 4°C with gentle agitation. Beads were then washed three times in a wash buffer, twice in PBS, and RNA was isolated using the RNAiso Plus (TAKARA), for qRT-PCR as described above.Protein samples were electrophoretically separated on 10% polyacrylamide gels, followed by transfer to PVDF membranes (Millipore). Blots were then probed using the following primary antibodies: p53 , FOXC2 , MKRN1 , HIF-1α , HDAC-1 , GAPDH .For co-IP analysis, anti-MKRN1, anti-p53 or normal mouse/rabbit IgG were used as the primary antibody, and then the antibody-protein complex was following incubated with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). The agarose-antibody- protein complex was collected and then analyzed by Western blot.The EZ-ChIPTM Chromatin Immunoprecipitation Kit  was used based on provided instructions. All the procedures were performed according to the manufacturer's instructions. Rabbit anti-HIF-1α/HDAC-1 was used and corresponding rabbit-IgG as controls. PCR was used to amplify bound DNA, which was then assessed via electrophoretic separation on a 2% agarose gel.To investigate whether FOXC2 is transcriptionally regulated by p53, PANC-1 cells were transfected with FOXC2 promoter (P2/P3-WT or P2/P3-MUT) reporter constructs, and further co-transfected with sip53. After a 48-hour culture, the Dual-Luciferase reporter assay system was used to assess promoter activity, with Renilla luciferase used for normalization.To determine how HIF-1α and HDAC1 affect the activity of HRE on the CF129 promoter under hypoxia condition. PANC-1 and BxPC-1 cells expressing pGL3-based construct containing the HRE of CF129 were transfected with siNC and siHIF-1α, and further treated with and without TSA. Twenty-four hours later, cells were cultured with or without CoCl2 for an additional 24h. The reporter activity was measured as above.PANC-1 cells that had been lentivirally transduced to express negative control (LV-NC) or CF129 sequences (LV-CF129) were injected into the dorsal flanks of 4-week-old male nude BALB/c mice. . (n=7 per group). Tumor growth in these animals was monitored every 3 days, and mice were euthanized 21 days after implant at which time tumor weights were determined.In other experiments, mice instead received tail vein injections of stably transfected PANC-1 cells, and upon sacrifice the frequency and number of liver metastases in these mice was determined. The total RNA of tumors was extracted and analyzed by qRT-PCR. Studies on animals were conducted with approval from the Animal Research Committee of the Academic Medical Center of Huazhong University of Science and Technology.SPSS v13.0 and GraphPad Prism were utilized for all data analyses. Data are given as Mean ± SD. Comparisons between 2 groups were made with Student's t-tests, while survival was assessed via Kaplan-Meier approach with a Log-rank test. The Wilcoxon signed-rank test was used to assess significant differences of CF129, p53 and FOXC2 between samples. χ2 tests were utilized to analyze the relationships between lncRNA-CF129 levels and clinical characteristics. P<0.05 was the threshold of significance.Supplementary figures and tables.Click here for additional data file.


While we observed evidence suggesting that L1 expression has a weak positive effect on A3B and A3G expression and A3B promoter activity, neither efficient siRNA-mediated knockdown nor overexpression of functional L1 elements affected catalytic activity of A3 proteins consistently. However, L1 knockdown diminished proliferation of a UC cell line exhibiting robust endogenous L1 expression, but had little impact on a cell line with low L1 expression levels. Our results indicate that UC cells express A3B at levels exceeding A3A levels by far, making A3B the prime candidate for causing genomic mutations. Our data provide evidence that L1 activation constitutes only a minor and negligible factor involved in induction or upregulation of endogenous A3 expression in UC.The most common mutational signature in urothelial carcinoma (UC), the most common type of urinary bladder cancer is assumed to be caused by the misdirected activity of APOBEC3 (A3) cytidine deaminases, especially A3A or A3B, which are known to normally restrict the propagation of exogenous viruses and endogenous retroelements such as LINE-1 (L1). The involvement of A3 proteins in urothelial carcinogenesis is unexpected because, to date, UC is thought to be caused by chemical carcinogens rather than viral activity. Therefore, we explored the relationship between A3 expression and L1 activity, which is generally upregulated in UC. We found that UC cell lines highly express A3B and in some cases A3G, but not A3A, and exhibit corresponding cytidine deamination activity  They re viruses . Importa viruses  and inhi viruses . A3 prot viruses . Recentl viruses . Many tunfection . Indeed,nfection . Additionfection . In contnfection  in urothnfection .A3 locus in primates , and non-LTR retrotransposons such as long interspersed nuclear element-1  and short interspersed nuclear elements (SINEs), have been the original targets of A3 activity and have provided the evolutionary pressure necessary for the continuous expansion of the primates . Mobilizprimates . For insprimates . Importaprimates   used in this study  were cultured in DMEM GlutaMax , supplemented with 10% fetal calf serum . BC61 ceTo minimize DNA contamination, total RNA was extracted by acid phenol extraction followed by column purification. Synthesis of complementary DNA was performed using the QuantiTect Reverse Transcription Kit , according to the manufacturer’s instructions, including an extra DNA removal step by DNase as recommended by the supplier. Briefly, 1 μg of total RNA was subjected to genomic DNA elimination reaction in a 14 μl volume, comprised of 2 μl of a 7x gDNA-Wipeout-Buffer, RNA, and water. The reaction mixture was incubated at 42°C for 2 min and then kept on ice. One microliter of the reaction mixture was taken and mixed with 14.38 μl of water in a new tube , which served as mock RT template for RT-qPCR assay. With the remaining 13 μl reaction mixture, cDNA synthesis was performed , 1 μl RT primer mix, 1 μl water, and 13 μl DNAse treated RNA) by incubating the RT reaction components for 30 min at 42°C and then inactivating the RT enzyme by boiling for 95°C for 3 min (according to the manufacturer’s instruction). In order to minimize potential inhibitory effects of the RT buffer system on qPCR, a 1:10 dilution of the cDNA product was generated prior to the PCR reaction quantifying FL-L1 transcripts. The final nucleic acids concentration of the RNA suspension (used for the Mock RT-qPCR) and the cDNA suspension were both adjusted to 4.64 ng/μl prior to qPCR.Supplementary Table 1. Ct values obtained from mock-RT experiments were found to be comparable with those obtained from blank control (water). The qPCR conditions were as follows: initial denaturation step at 95°C for 15 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 15 s, annealing at 55°C for 20 s and extension at 72°C for 30 s, using the primers presented in the following method section and Supplementary Table 2.The efficiency of DNAse treatment was assessed by qPCR on RNA samples that were not incubated with any Reverse Transcriptase before. Data  obtained from cell lines 5637, VM-CUB1, 639-V, SD, BC61, RT4 are provided in Supplementary Table 2) were employed using the following PCR conditions: initial denaturation at 95°C for 15 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 15 s, annealing at the appropriate temperature for 20 s and extension at 72°C for 30 s. Assay specificity was controlled for by using UCSC In-Silico PCR and melting curve profiles. All measurements were performed at least in duplicates; assay variance was <10%. Relative expression was calculated by the modified ΔΔCt method using TATA-box binding protein (TBP) mRNA levels as a reference gene transcript , UMUC3 , PC3 , 5637 (A3G), and VM-CUB1 (FL-L1), respectively.RT-qPCR was performed on a 7500 Fast Real-Time PCR System  or Roche LightCycler 96  using the QuantiTect SYBR Green PCR Kit  with cDNA (1:10 diluted) from DNAse-treated RNA samples  as described previously . To quananscript . To asceSupplementary Table 2). RT-qPCRs with these primers were performed as previously described ], washed in 1xPBS-T, and incubated overnight with the respective primary antibody at 4°C.Twenty micrograms of each protein lysate were boiled in 3x SDS sample buffer , loaded on 4–12% Bis/Tris gels (Invitrogen), subjected to SDS-PAGE, and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in 10% non-fat milk powder in 1xPBS-T . Lysates were clarified by centrifugation for 20 min at 14,800 rpm at 4°C. For immunoblot analyses, samples were boiled at 95°C for 5 min in Roti load reducing buffer , separated on an SDS-PAGE gel followed by transfer to a PVDF membrane. Membranes were blocked in TBST containing 5% skimmed milk powder and probed with the respective primary antibody. A rabbit anti-A3G antibody  that is cross-reactive against A3A and A3B was used to detect A3B and A3G proteins (HEK293T cells were transfected with plasmids expressing hemagglutinin (HA)-tagged A3B or A3G using Lipofectamine LTX (Thermo Fisher Scientific). 5637, UMUC3 and VM-CUB1 UCCs were transfected with the pJM101/L1proteins . Mouse aIn vitro deamination reactions were performed as described , respectively, using a Wallac 1420 VICTOR2TM plate reader . Cell proliferation was measured by an EdU incorporation assay , according to the manufacturer’s instructions. The SA-β-galactosidase assay was performed as described .A3H haplotype I encodes an active cytosine deaminase described to reside in the nucleus and linked to cancer mutagenesis  the unpaired Student’s t-test with Graphpad Prism . Data were represented as the mean ± standard deviation (SD). Significant differences (p < 0.05) are marked by asterisks. Correlation coefficients and significance were calculated by non-parametric Spearman’s rank correlation (Spearman’s rho) and were corrected for multiple testing using the Bonferroni method.A3A, A3B, A3C, A3D, A3F, A3G, and A3H) in 19 UCCs and five independent primary cultures of normal urothelial cells (UPs) by RT-qPCR. A3A was only detectable in the urothelial cell culture UP86, the UCC BFTC905  and in activated PBMCs serving as positive control for A3A expression (data not shown). A3A expression was not detectable in all remaining UCCs and UPs. In contrast, A3B expression was barely detectable in all analyzed urothelial cell cultures, but was high in all analyzed UCCs except for the 647-V line harboring no detectable A3B mRNA at all . A3C, D, and F transcripts were detectable at moderate levels in all tested UPs and the majority of UCCs, but a few UCCs expressed A3C and A3D at extremely low or undetectable levels . A3G expression was robust in UPs but low in most UCCs, especially in those originating from muscle-invasive urothelial cancers . A3H transcripts were below detection level in the majority of muscle-invasive UCCs and in two UPs, while the remaining cell lines and UP cultures displayed moderate or robust transcript levels. In the papillary UCC BC61, A3H expression was exceptionally upregulated .As a first step to investigate if the expression of specific members of the A3 protein family of cytidine deaminases is a response to the upregulation of endogenous L1 expression, we profiled mRNA levels of all seven members of the A3 protein family . If L1 upregulation contributed to activation of A3 enzymes, mRNA expression of L1 and A3 would be expected to correlate. We therefore calculated whether any correlation between FL-L1 mRNA expression levels and mRNA levels of the various A3 genes could be detected in UCCs using Spearmen’s rho correlation , which was lost after Bonferroni correction for multiple testing.Quantification of FL-L1 transcripts revealed moderate L1 expression in all UPs and robust transcriptional L1 upregulation in almost all analyzed UCCs with the exception of the UCCs 253J and 5637 , a specific allele of A3H, has been implicated in breast and lung carcinogenesis . Approximately two thirds of the tested UCCs harbor the G105/K121 allele associated with the A3H-I haplotype in a homozygous (6/18) or heterozygous (7/18) manner. However, expression of A3H was not detectable in selected UCCs irrespective of the A3H genotype . This finding implies A3H as a possible but unlikely source of A3 mutations in several but not all UCCs.Because ogenesis , we addiFigure 1) which is based on the L1.3 reference sequence , elevated amounts of L1 ORF1p were detected in the UCC lines BFTC905, RT-112, VM-CUB1, and SD . More moderate but still detectable L1 ORF1p levels were present in J82, SW-I710, UMUC6, 253J, 5637, 639-V, 647-V, HT-1376, T24, and UMUC3 cells . The relatively modest amounts of L1 ORF1p in BC61 and RT4 cells do not seem consistent with the transcriptional L1 upregulation in these cells , but could be explained by the fact that a subset of FL-L1 elements transcribed in these cell lines does not encode intact L1 proteins. Of note, L1 ORF1p expression was not detectable in the normal urothelial cell culture UP239 and the TERT-NHUC immortalized normal urothelial cell line .Consistent with their previously assessed relative FL-L1 mRNA levels   or high (VM-CUB1 and SD) L1 mRNA and ORF1p expression levels, respectively. Expression of full-length L1Hs elements was downregulated in the four UCCs by transfecting either siRNA#1  or siRNAFigures 2B,D), 5637 , and SD UCCs. L1 mRNA and ORF1p expression was barely detectable in 639-V and remained unchanged after L1 siRNA treatment . Interestingly, RT-qPCR using primer combinations specific for the 5′end of the L1 5′-UTR  revealed that overall FL-L1 transcript levels were reduced only by at most 50% . Since the partial mRNA knockdown observed in these cell lines nevertheless resulted in a highly efficient L1 ORF1p depletion , this observation indicates that the siRNAs target most if not all intact protein-coding L1 elements harboring an intact ORF1 efficiently. In contrast, non-functional FL-L1 elements with mutations in ORF1 that differ from the L1.3 reference sequence were targeted less efficiently.Transfection of each of the two L1-specific siRNAs efficiently knocked down L1 ORF1p expression in VM-CUB1 . A3H expression was only detectable in SD cells among these four UCCs  and not altered by the knockdown of L1 expression in the examined UCCs , whereas L1 knockdown exerted variable effects on the expression of A3B to A3G in the tested UCCs.Following transfection with L1-specific siRNAs, Figures 2E–H), and was associated with a drop of A3B expression by 25% (5637) to 55% (VM-CUB1). A3G expression was diminished by 14, 47 and 36% in VM-CUB1, 5637, and SD cells, respectively, and remained unaffected in 639-V cells. However, only minor, negligible effects on A3C, A3D and A3F transcript levels were observed in VM-CUB1, 5637, and SD cells. In 639-V cells, A3C expression was unchanged, whereas A3D and A3F transcript levels were increased by 56% and 38%, respectively.Transfection of L1_siRNA#1 diminished overall FL-L1 transcript levels in the analyzed UCC lines by 8% (639-V) to 42% (5637) . The knockdown of functional L1 elements was associated with an increase of A3F transcript levels in 639-V cells by 44%, but had no meaningful effect on A3F expression in the remaining cell lines. L1_siRNA#2-mediated knockdown of ORF1p-expressing endogenous L1 elements was associated with a strong increase of A3G transcript levels in three UCCs ranging from 50% in the SD line to fourfold in VM-CUB1 cells but had no consequences for A3F expression in 5637 cells, and no noteworthy effect on 5637 cells .Following transfection with L1_siRNA#2, A3C, D, F, and G transcript levels in three out of four analyzed UCCs, while L1_siRNA#1-mediated knockdown was associated with a comparable major increase in A3C, A3D, and A3F transcript levels only in 639-V cells and a minor increase in VM-CUB1 cells. A decrease of A3 transcript levels was observed only after L1_siRNA#1-mediated knockdown in 5637 and SD UCCs and was minor, ranging from 10 to 50% among the A3 genes. Taken together, no consistent correlation between the knockdown of functional endogenous L1 elements and expression of A3 genes could be observed, but knockdown of intact L1s was more often associated with increases rather than decreases of A3 gene expression. In sum, these findings are not consistent with the hypothesis that expression of A3 genes is upregulated in UCCs as a consequence of functional L1s activation. Furthermore, it is currently unclear why the effects of functional L1 knockdown on A3 gene expression differed between L1_siRNA#1- and L1_siRNA#2-mediated knockdowns.In summary, knockdown of functional L1 elements with L1_siRNA#2 resulted in a general increase of A3B and A3G transcript levels in UCC lines, we next transfected either of the L1 expression plasmids pJM101/L1RP and pAJG101/L1RP  into the cell lines VM-CUB1 and 5637, which are characterized by relatively high and low endogenous L1 transcript levels, respectively . Following transfection, FL-L1 RNA levels increased 2.5- to 3-fold in VM-CUB1 cells and by 23% to 46% in 5637 cells, as demonstrated by the L1_5′-UTR-specific RT-qPCR assay . To analyze whether ectopic L1 overexpression affects endogenous A3 expression, we quantified A3A, A3B, and A3G mRNA levels in the transfected UCCs. We found that expression of A3B and A3G was slightly increased in VM-CUB1 UCC after transfection with pAJG101/L1RP, but only A3B expression changes reached the level of significance . In 5637 UCC, A3B and A3G expression was not altered significantly. Of note, A3A expression remained undetectable in both cell lines (data not shown).To investigate the consequences of ectopic overexpression of retrotransposition-competent L1 elements on endogenous A3B promoter activity, we co-transfected VM-CUB1 and 5637 cells with either of the two A3B promoter luciferase reporter constructs pA3B-120 or pA3B-1200  together with the L1 expression plasmids pJM101/L1RP and pAJG101/L1RP or the empty pCEP4 vector as negative control. Activity of the A3B promoter encoded by pA3B-1200 increased by ∼36% – 42% and 64% – 80%, respectively, after co-transfection of the luciferase reporter construct with pJM101/L1RP or pAJG101/L1RP into VM-CUB1 and 5637 cells relative to the effect of the control vector . This increase in promoter activity is consistent with the increase in endogenous A3B mRNA levels by ∼27% after transfection of plasmid pAJG101/L1RP in VM-CUB1 cells . Taken together, induction of L1 activity has only minor effects on A3B expression as well as promoter activity and significant effects are limited to VM-CUB1 UCC with high L1 expression.To study whether ectopic expression of functional L1 elements can induce A3 mRNA expression levels. We chose specifically (i) the UCC 5637 exhibiting major transcriptional upregulation of A3B and A3G, (ii) UMUC3 showing robust transcript levels of most A3 genes (with A3B expression being the highest), and (iii) VM-CUB1 with very low transcript levels of most A3 family members except for A3B . Distinguishing A3B from A3G by standard immunoblotting techniques is challenging due to the high amino-acid sequence homology between both proteins and their comparable molecular masses . Similarly, A3G expression levels dropped after transfection with A3G-specific siRNA to below 10% in all three UCCs . Simultaneous treatment with A3B- and A3G-specific siRNAs resulted in diminished A3B and A3G mRNA levels in all examined UCCs comparable to those observed after single siRNA treatment .We investigated expression and deaminase activity of A3 enzymes suspected to cause mutations during bladder carcinogenesis in selected UCCs with different r masses . TherefoFigure 4B) and their mRNA expression pattern in 5637 cells . The intensity of the 45 kDa band was slightly diminished after transfection of the cells with A3B-specific siRNA, but the band disappeared almost entirely after transfection with A3G-specific siRNA or a combination of both siRNAs . Expression of the 45-kDa protein was not affected by transfection of control siRNA.Immunoblot analyses of cell extracts isolated from the differently transfected 5637 cells with an anti-A3G antibody (ApoC17)  reportedA3G-specific siRNA strongly depleted the amounts of the 45-kDa protein in both UMUC3 and VM-CUB1 cells, whereas the A3B-specific siRNA had only a minor effect on the 45-kDa protein levels in UMUC3 cells and did not affect its expression at all in VM-CUB1 cells . These findings suggest that the majority of the 45-kDa proteins detected with the anti-A3G antibody represents A3G. A note of caution on the detection of endogenous A3B: Since a more specific antibody capable of selectively detecting endogenous A3B enzyme in UC cell lines is currently not available, we cannot formally exclude that A3B protein is not depleted by A3B-specific siRNA, despite downregulation of A3B mRNA. Of note, A3A expression was not considered because there was no evidence for the presence of A3A mRNA in the analyzed UCC lines , and consistently, immunoblot analysis with anti-A3A antibodies did not provide any evidence for the presence of A3A proteins (data not shown).Transfection of A3B- and A3G-siRNAs as controls . To measure deaminase activity, we applied a qualitative PCR-based in vitro DNA deamination assay to identify C→U conversion in an 80-nt single-stranded DNA substrate harboring the isozyme-specific motif TTCA or CCCA, specifically recognized by A3B or A3G, respectively . Of note, whereas the substrate TTCA (YTCA) was reported as a statistically favorable target for A3A over A3B in cancer tissues . A3G deaminase activity was robust in 5637 cells, but lower in UMUC3 cells and barely detectable in VM-CUB1 cells. Importantly, as expected from the CCCA substrate specificity of A3G . Using the CCCA substrate, A3B downregulation slightly reduced product formation, whereas simultaneous knockdown of A3B and A3G abolished detectable deaminase activity. Conversely, using the TTCA substrate, A3B knockdown, but not A3G knockdown resulted in complete loss of detectable deaminase activity . Taken together, these data confirm that A3G favors the CCCA sequence motif and A3B prefers the TTCA motif, but also indicate that A3B might mutate CCCA sequences on ssDNA substrates with a low frequency. More importantly, combining the deamination assay data  with the A3 expression data presented in Figures 1 and 4A leads to the conclusion that in vitro A3B is the predominantly active member of the A3 family in the tested UCCs, whereas A3G-specific deaminase activity is comparably low.In order to investigate if A3B and/or A3G are enzymatically active in UCC lines, DNA deamination activity assays were performed using cells lysates from the different UCCs transfected with ectively . Catalyt tissues , TTC wasc assays . The assy of A3G , siRNA-mRP in UCCs. Lysates from transfected and untransfected UCCs were then either treated with RNase A to eliminate potential inhibitory RNA molecules, or left untreated, before they were assayed for A3B- or A3G-specific deaminase activity .Next, we investigated the effect of the expression of functional L1 elements on the deamination activity of A3 proteins by ectopically overexpressing transfected functional L1 elements encoded by pAJG101/L1activity . EctopicFigure 5A). The number of viable 5637 UCCs was less severely depleted to 68% and 46% after treatment with L1_siRNA#1 and L1_siRNA#2, respectively . Caspase 3/7 activity, measured as an indicator of apoptosis, decreased to 43% and 8% in VM-CUB1 cells after L1_siRNA#1 and L1_siRNA#2 treatment, respectively . In 5637 UCCs, caspase activity was increased after treatment with L1_siRNA#1, but not with L1_siRNA#2 . According to flow cytometry data, the fraction of VM-CUB1 cells in G2/M phase was increased by roughly 8% in cells treated with either L1 siRNA . In VM-CUB1 cells treated with L1_siRNA#2, the fraction of subG1 cells also decreased. S-phase fraction was unchanged  and accordingly, incorporation of EdU was only slightly diminished, especially following L1_siRNA#1 treatment . Only minor changes in cell cycle distribution and, accordingly, EdU incorporation were seen in 5637 UCCs . Thus, the decrease in viable cells following L1 knockdown does not reflect apoptosis.While our results do not indicate that L1 expression affects A3B (or other A3) expression consistently, L1 silencing by siRNA impaired cell proliferation. In VM-CUB1 cells expressing L1 more strongly, the number of viable cells decreased to 47% and 7% after L1 knockdown using L1_siRNA#1 and L1_siRNA#2, respectively, compared to control siRNA-transfected cells . Moreover, some VM-CUB1 cells showed morphological changes typical of senescent cells and stained positive for senescence-associated (SA)-β-galactosidase after treatment with either L1 siRNA, but very rarely after treatment with control siRNA . Unexpectedly, in 5637 cells, clonogenicity reproducibly increased after L1 knockdown using L1_siRNA#2, whereas L1_siRNA#1 exerted no significant effects on clonogenicity . Accordingly, no indications of senescence were detected in 5637 cells after treatment with L1 siRNAs or control siRNA (data not shown). Thus, L1 knockdown affected VM-CUB1 UCCs with high L1 expression levels more severely than 5637 cells with low L1 expression.In keeping with its effects on cell viability in short-term experiments, FL-L1 knockdown strongly diminished the clonogenicity of VM-CUB1 cells , whereas A3A was essentially undetectable in almost all (16/17) analyzed UCCs. Obviously, expression and enzymatic activity of A3 family members may vary during urothelial carcinogenesis and may not be fully reflected in the pattern seen in UCCs. However, A3 mutational activity was shown to be involved in both early and late mutation events that occurred during urothelial carcinogenesis arguing rather for continuous A3 mutational activity   in UCCs 5637, UMUC3 and VM-CUB1, this is very likely to be the case for A3B too. Moreover, cytidine deamination assays coupled with knockdown experiments convincingly revealed the expected substrate-specific activity levels for both A3B and A3G. Of note, the general DNA motif reported to be recognized by APOBEC proteins to introduce somatic mutations in cancer is “TC”  . Further(NMIBCs) , or may CC lines . However is “TC”  . In addition, endogenous FL-L1 and A3 expression levels did not correlate with each other across the tested panel of cell lines. Here, future investigations are required to unambiguously elucidate any role of L1 expression and/or retrotransposition activity in the activation of A3 proteins in tumor cells.To address the general question of what triggers A3 activation in urothelial cancer cells, we pursued the hypothesis that A3 activation may be elicited by endogenous retroelement activity rather than the presence of exogenous viruses. Expression of functional endogenous L1 elements seems a plausible cause for A3 activation, because in urothelial cancer cells, L1 promoter sequences are frequently hypomethylated, and FL-L1 expression is increased even more than in other cancer types . In comp in UCCs . HoweverORFeus-Hs . This could be explained by the fact that the L1 5′UTR-specific primers used for the RT-qPCR assay also detect transcripts from FL-L1 elements with non-functional ORF1 sequences, which are not or less efficiently targeted by the siRNAs.Knocking down the expression of endogenous FL-L1 elements with two different siRNAs targeting the intact ORF1 coding region resulted in the efficient depletion of endogenous L1 ORF1p. This observation indicates that the majority of transcripts from active L1Hs elements harboring intact ORF1 sequences were removed from the tested cell lines. However, these siRNAs did not decrease the overall FL-L1 transcript levels as measured by RT-qPCR to the same degree , but had less effect on UCCs exhibiting lower L1 expression levels (such as 5637 cells). These results are in good agreement with previous reports that L1 knockdown causes a loss of proliferative ability in tumor cells independent from apoptosis , ultimatFinally, if there is no evidence that A3 activation in UC is elicited by either exogenous virus infection or endogenous L1 retrotransposon activation, what causes it? Several alternative hypotheses deserve investigation. For instance, A3B is induced by several cytokines in normal liver  and throMost advanced muscle-invasive UCs contain mutations inactivating p53, which are rare in non-muscle invasive UC . The p53Moreover, recent results suggest that A3B may target ssDNA accumulating as a result of replication stress  or transThus, we conclude that several factors may cooperate to activate A3 in urothelial carcinogenesis. This work largely excludes the pervasive activation of L1 retroelements as one potential factor. Moreover, in line with some, but not other previous reports, detailed analysis of UCCs suggests A3B rather than A3A as the predominantly active enzyme.The major limitations of our study concern the detection of A3 proteins and the high L1 copy number in the human genome. With respect to A3 proteins, we could not obtain antibodies that are sufficiently sensitive and specific to detect endogenous expression of each isoenzyme. A reliable array of such antibodies would be very helpful to characterize the expression pattern of A3s in UC cell lines and tissues more precisely. Also the highly repetitive character of endogenous L1 retroelements causes major complications for our studies. To fully understand the impact of L1 activity in UC cells and tissues, a complete characterization of the repertoire of transcripts from retrotransposition-competent L1 elements and, ideally, from non-functional L1 elements will be required. Third generation techniques currently under development will hopefully enable this investigation.RP, and participated in drafting the manuscript. UK and AK performed some experiments. WG, WS, AAJV, DH, and CM analyzed the data. WS, GS, and CM contributed reagents and tools. WG, WS, AAJV, GS, and CM wrote the paper.WG, WS, AAJV, and CM conceived and designed the experiments. AV and WG performed most of the experiments. GS performed immunoblot analyses, generated the L1 reporter plasmid pAJG101/L1The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a new carbon-based nanomaterial, graphene has exhibited unique advantages in significantly improving the combination properties of traditional polymer hydrogels. The specific properties of graphene, such as high electrical conductivity, high thermal conductivity and excellent mechanical properties, have made graphene not only a gelator to self-assemble into the graphene-based hydrogels (GBH) with extraordinary electromechanical performance, but also a filler to blend with small molecules and macromolecules for the preparation of multifunctional GBH. It fully exploits the practical applications of traditional hydrogels. This review summarizes the preparation methods, properties, and the applications of GBH. Further developments and challenges of GBH are also prospected. Graphene is a new nanomaterial with strict two-dimensional layers structure Geim, . With exThis review aims to demystify the preparation methods, performance aspects, and the applications of GBH. Further developments and challenges of GBH are also prospected.Self-assembly method means that the basic structure of graphene oxide (GO) is spontaneously translated into a stable 3D graphene structure under the interaction of non-covalent π-π bond. It is considered an effective approach for fabricating GBH. Various self-assembly methods such as hydrothermal process  was greater several orders than the traditional hydrogels. The 3D multifunctional GBH, which was self-assembled by combining DNA and GO sheets, possessed good mechanical property, big dye-adsorption capacity, and excellent self-cure capability  / poly (PVA) hydrogel, which was made by SG and PVA mixed solution, showed good mechanical property and intelligent adsorption property for cationic dyes compared to ordinary pure PVA hydrogel  composite hydrogel was synthesized via cross-linking reaction of PAA at low temperature , poly (PDMAA), etc.), GBH are attracted much attention in tissue engineering. Jing et al. studied the mussel-inspired GO/CS composite hydrogel, which was prepared by incorporating protein polydopamine (PDA)  after 42 h. However, only 51% vitamin B12 molecules can be released in the acidic medium (pH = 1.7) at the same time. Li et al. successfully fabricated an NIR-responsive GBH as an active cell scaffold by pNIPAAm derivative solution and arginine-glycine-aspartic acid (RGD). The NIR-responsive GBH could efficiently capture cells, but also could release the cells on the stimulus of NIR light  (pNIPAAm), etc.) has shown attractive prospects in drug controlled release system on account of the huge specific area of GO or graphene. The graphene/Ag (mass ratio of 1:5) composite hydrogel, which was made through the cross-linking reaction of graphene with acrylic acid and methylene bisacrylamide, exhibited good biocompatibility, and high swelling ratio , good cycling performance and high rate capability  hydrogel exhibited excellent mechanical property, high Photo-Fenton activity and big adsorption capacity. The hydrogel was achieved a 90% degradation of Rhodamine B  and a 72.7% degradation of chemical oxygen demand  in wastewater for 1 h under visible light  and L-ascorbic acid (LA). The hydrogel possessed high selectivity to oil-water separating by pre-soaking (Hu et al., The specific properties of graphene, such as high electrical conductivity, high thermal conductivity, and excellent mechanical properties, have made graphene not only a gelator to self-assemble into the GBH with extraordinary electromechanical performance, but also a filler to blend with small molecules and macromolecules for the preparation of multifunctional GBH. It fully exploits the practical applications of traditional hydrogels. In view of the developing trend of hydrogel in recent years, theoretical researches are relatively high. Researchers are very interested in the application prospect of hydrogel for biomedical, tissue engineering, supercapacitor, water treatment, dye absorption, catalyst carrier, and intelligent response for microfluidic system. However compared to the practical applications, the actual operation research is very weak. The formation mechanism of graphene/GO hydrogel in the aqueous solution and the influencing function are not apparent yet. Design and fabrication of GBH with new structures and functions will be a significant challenge in the future.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) is an immune checkpoint (ICP) overexpressed in various types of tumors; thus, it has been considered as an important target for cancer therapy. To determine important residues for ligand binding, we applied molecular docking studies to PD-1/PD-L1 complex inhibitors against the PD-L1 protein. Our data revealed that the residues Tyr56, Asp122, and Lys124 play critical roles in ligand binding to the PD-L1 protein and they could be used to design ligands that are active against the PD-1/PD-L1 complex. The formation of H-bonds with Arg125 of the PD-L1 protein may enhance the potency of the PD-1/PD-L1 binding. Immune checkpoints (ICPs) are paramount regulators of the immune system, and they can differentiate between the healthy and foreign cells and prevent activation of immune cells ,2,3. Can+ and CD8+ T cells, natural killer T (NKT) cells, B cells, activated monocytes, and dendritic cells (DCs) [PD-1 is a type I transmembrane immune-inhibitory protein that is expressed on activated CD4ls (DCs) ,19. PD-1ls (DCs) . PD-1 hals (DCs) . In normls (DCs) ,23,24. Ils (DCs) ,26. The ls (DCs) ,28. PD-Lls (DCs) ,32,33,34ls (DCs) ,36,37. THowever, the occurrence of immune-mediated adverse effects has been observed in some patients receiving the immune checkpoint inhibition (ICI) by mAbs. The side effects can be colitis, autoimmune hepatitis, endocrine or neurological disorders . A studyTherefore, a small molecule inhibitor that binds to PD-L1 only would spare the function of PD-1 and may be an alternative therapeutic approach that may minimize the immune-related adverse effects. Unfortunately, the development of small molecules targeting the PD-1/PD-L1 axis lags far behind the mAb development targeting the same pathway. This is due, in part, to insufficient structural information of the PD-1/PD-L1 complex with small molecule inhibitors. The crystal structure of the fully human PD-1/PD-L1 complex  that was determined in this crystal structure suggests that there are several binding sites on the PD-1 and PD-L1 that can be targeted to develop small molecule inhibitors . The cryHere, we report the structural basis for PD-L1 interactions with the reported Bristol-Myers Squibb (BMS) inhibitors . The docPRED) of PD-1/PD-L1 complex inhibitors. The comparisons of predicted docking scores to the experimental free energy of bindings, converted from the IC50s, show that the docking scores of both models 5NIU and 5N2F are in good agreement with the experimentally observed data, with mean errors of 1.07 and 0.91 kcal/mol, and the root-mean-square errors of 1.66 and 1.51 kcal/mol for model proteins 5NIU and 5N2F, respectively. The low standard deviations of 1.29 and 1.22 for model proteins 5NIU and 5N2F, respectively, further confirm the validity of the glide docking method and confirm the consistency of the docking studies in our PD-L1 system. The more negative the docking score, the more favorable the interaction of the complex. To determine the protonation state of amino groups in the compounds outlined in 8), which has a high pKa of 10. This suggests that the nitrogen atom on most ligands should not be protonated and thus remains neutral, whereas BMS-1220 (8) was protonated.To identify amino acids that are essential for small molecule binding, we carried out docking studies of 29 experimentally verified inhibitors of the PD-1/PD-L1 complex . These 2In addition to binding affinity, ligand binding can also be evaluated by binding free energy (ΔG). Herein we use a knowledge-based moveable-type (MT)-based approach  to estim1  ,55,56. TAfter the validity of the glide dock method was confirmed by the aforementioned methods, we can confidently use the docked poses to identify PD-L1/inhibitor interactions. To design new molecules with the desired potency, it is important for the designed molecules to maintain the proper interactions with essential residues in the binding pocket. Thus, it is very important to identify critical binding residues for effective PD-L1 binding.1)), the 2, 3-dihydro-1, 4-benzodioxine fragment creates π–π stacking interaction with Tyr56, and the (2R)-2- amino-3-hydroxypropanoic acid moiety formed H-bonds with the carbonyl of Asp122, Tyr123, Lys124, and the main chain carbonyl oxygen of Phe19 . No missing residues were observed in these two crystal structures except a few residues with missing parts of side chains. The missing side chains were regenerated during the protein preparation step in MOE to correct the missing side chains, to optimize the hydrogen bonding network, to allow protonation to be assigned to charged residues, and to allow the flipping of the side chains of Asn, Gln, and His in MOE to maximize H-bond interactions [The X-ray crystal structures of the human wild type PD-L1/BMS-1001   and the ractions . Due to ractions  in MOE, ractions  to allow8) has a pKa of 10.73, which is greater than 10 and thus should be protonated. The pKa values of nitrogen atoms of all 29 inhibitors are listed in We built 29 PD-1/PD-L1 complex inhibitors  from difTo calculate the enrichment factor in order to evaluate the effectiveness of the docking program, we downloaded a database of 260,071 ligands from the National Cancer Institute (NCI)  and thisPRED. The output ΔGPRED was then related to the experimental ΔGEXP, which calculated from the experimental IC50 (nM) using the following equation:The compounds were then subjected to energy minimization using the MacroModel module in Maestro . We usedFurthermore, we created an electrostatic map for the binding site of PD-L1 to estimate the electrostatically favored locations of H-bond donors, H-bond acceptors, and hydrophobic interactions. The electrostatic map was made using the MOE program . The recAfter 29 inhibitors were docked to the 5NIU protein, the model protein was saved in PDB format and the 29 ligands were each saved separately in mol2 file format. The saved protein and ligand files were fed as input files for the movable-type (MT) free energy calculation method, an in-house program developed by Prof. Kenneth Merz Jr. at Michigan State University and which was generously given to us for complimentary use. The output data of the MT-based method is the absolute free energy of binding; these data were used to compare with those experimentally observed values and are listed in 9 (The prevalence of the PD-1/PD-L1 complex in several human cancer cells has made it an attractive target for anticancer drug discovery. The positive results of mAb targeting the PD-1/PD-L1 complex in cancer treatment are boosting and inspiring the design and development of small molecules targeting this pathway. Our studies on the protein/ligand dockings and structural analysis on the docked complexes have suggested that the glide dock approach and the MT-based free energy calculation method are very dependable in terms of their predictability, with low error compared to those observed values. The low RMS deviations of the docked pose to the native conformation and the very good enrichment factor further confirm the effectiveness of the Glide Dock program. The analyses of the protein/ligand interactions reveal that PD-L1 residues Tyr56, Asp122, and Lys124 may be very important for inhibitor design and that two aromatic rings may be expected in new PD-L1 inhibitors. From a drug design point of view, Phe19 and Asp122 can interact with the amino moiety of ligands and Lys124 and Arg125 provide interactions the carboxylate groups of ligands whereas Tyr56 from chains C and D, forming π–π interactions with two different aromatic rings as shown in compound 9 . All the


The lubrication performance of graphene used as additive in 4-n-pentyl-4′-cyanobiphyl liquid crystal (5CB) for steel/steel contacts was studied on a ball-on-plate tribotester. The friction test results show that when the graphene content in the 5CB was 0.15 wt.%, and the lubricant and friction pairs were heated to 44–46 °C before friction tests, the lubrication performance of the 5CB was most improved. Compared with pure 5CB, 5CB+0.15 wt.% graphene suspension reduced the friction coefficient and wear scar diameter by up to 70.6% and 41.3%, respectively. The lubrication mechanisms have been tentatively proposed according to the test results. We speculate that the excellent lubrication performance of graphene/5CB suspensions may be attributed to the low shear resistance adsorption layer formed by graphene and 5CB molecules on the sliding surfaces. As the protective layer, it not only prevents direct contact between the rough sliding surfaces but also is easy to slide. Friction and wear between mechanical parts widely exist in mechanical systems. Friction consumes a lot of energy, and wear will lead to premature failure of parts . In ordeLiquid crystals exhibit mesophase between liquid and crystal phase, which have both crystal anisotropy and liquid fluidity. Liquid crystals can be classified in two categories: (1) monomer liquid crystals, irrespective of whether they are able to polymerize, and (2) polymer liquid crystals . 5CB belRecently, nanoparticles have been introduced into industry as a new lubricant additive ,8,9. The2 stripping of graphite. The antifriction and antiwear properties of the multilayer graphene as an additive of polyalphaolefin-2 (PAO2) oil were studied by using four-ball test method. They pointed out that the PAO2 with 0.05 wt.% multilayer graphene showed the best lubrication performance. By analyzing the worn surfaces, it was found that the excellent lubrication performance of the multilayer graphene additives can be attributed to its small sheet diameter and layered structure, which make the multilayer graphene easily enter the friction interfaces and form a protective layer, thus preventing the friction surfaces from directly contacting.Multilayer graphene can be well dispersed in some specific lubricants, and shows its antifriction and antiwear properties. For instance, Fan et al.  reportedBesides multilayer graphene, few-layer graphene also has been reported as lubricant additive. For instance, Eswaraiah et al.  evaluateTo exert the lubricating properties of graphene in a lubricant, the dispersibility of graphene in the lubricant must be considered. The dispersity of graphene in most lubricants is very poor . ChemicaIn addition to using chemical modification to improve the dispersion of graphene, it has also been reported to improve dispersion by changing the shape of graphene to prevent adsorption and agglomeration between sheets. Inspired by the fact that crumpled paper sheets are not easily adhered to each other, Dou et al.  used capThe dispersion effect of graphene in base oil will directly affect its lubrication performance. However, using chemical modification to improve the dispersion of graphene will affect its chemical properties. At the same time, changing the morphology of graphene through external force to improve its dispersion will destroy the physical structure of graphene.In order to achieve the good dispersion of graphene in lubricating oil without changing its physical and chemical properties, we chose 5CB as the basic lubricant in this study. Since the rod-shaped molecules of 5CB tend to be arranged along the surface of the nanostructure that graphene provides ,31,32, tIn this study, the adsorption between the 5CB molecules and graphene was fully utilized. Considering the fact that the higher the temperature, the more intense the thermal movement of the liquid molecules and the more fierce the Brownian movement, we have achieved good dispersion of graphene in 5CB by heating the graphene/5CB suspension. The lubrication properties of the suspension were tested on a tribometer with a ball-on-plate configuration. Transmission electron microscopy (TEM), optical microscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Raman spectroscopy were used to characterize graphene and study the wear surfaces. Based on the above tests and analysis results, we deduced the possible antifriction and antiwear mechanism of graphene as additive in 5CB.The 5CB was supplied by the Shijiazhuang Huarui Scientific and Technological Co. . This liquid crystal exhibits a nematic phase at 22–35 °C . The graThe preparation procedures of Graphene/5CB suspension are as follows: Firstly, 5 mg of graphene was added in 10 ml of anhydrous ethanol with ultrasonic dispersion for 1 h to ensure that graphene was uniformly dispersed the solution. Secondly, 10 g of 5CB was added to graphene/anhydrous ethanol solution in 40–50 °C water bath with ultrasonication for 1 h to obtain a homogeneous suspension. After that, the suspension was put in vacuum at 50 °C for 12 h to completely evaporate the anhydrous ethanol. Finally the suspension of 0.05 wt.% graphene in 5CB (5CB+0.05 wt.% G suspension) was obtained. According to the above method, 5CB+0.15 wt.% G suspension, 5CB+0.25 wt.% G suspension and 5CB+0.35 wt.% G suspension were prepared respectively. No additional dispersion or surfactant was used to improve the dispersion of graphene in 5CB.The friction tests were carried out on a high temperature tribometer  with a ball-on-plate configuration, in indoor air conditions . The tribometer is shown in −1. The normal load applied to the steel ball was 3 N. The duration for each sliding friction test was 1 h.Before conducting the friction tests, the steel balls and steel plates were ultrasonically washed with acetone, washed with anhydrous ethanol and dried. During the tests, the steel plate rotated at a speed of 200 rpm and the friction track radius was 18 mm, which was equivalent to sliding at a speed of 0.38 msIt is well known that the concentration of additives in basic lubricants plays an important role in reducing friction and wear . In ordeFor the influence of temperature on lubrication performance of the graphene/5CB suspension, three different temperature conditions were investigated. The first temperature condition: the steel plate was heated to 44–46 °C while the graphene/5CB suspension lubricant was stirred at 44–46 °C for 120 s, to achieve good dispersion of the graphene in the 5CB, then the graphene/5CB suspension lubricant was applied to the plate and immediately stopping heating at the beginning of the friction test. The heating was stopped at the beginning of the friction test to make the 5CB return to the nematic phase, thus restoring the lubrication properties of the 5CB as a liquid crystal lubricant. After the friction test started, the shear force caused the graphene to be uniformly distributed on the surface of the friction region. Therefore, it is not necessary to rely on continuous heating to maintain a good dispersion state of graphene. The second temperature condition was at room temperature (24–26 °C). The third temperature condition was at 44–46 °C.Before the friction tests, the lubricant was dripped onto the steel plate with a dropper. Then the wear track surface was fully covered with a lubricating layer formed by the lubricant droplets. Each test condition was repeated at least three times, and the final results of friction coefficient and wear scar diameter were the average values.Before analyzing the friction surfaces, the steel balls and steel plates were washed with anhydrous ethanol to remove contaminants. An optical microscope  was used to observe wear profile and measure diameter of wear scar. Scanning electron microscopy  was used to further analyze the surface textures of steel ball wear scar. Energy dispersive spectroscopy (EDS) was used to analyze the content of various elements on the wear surfaces. Raman microscope  was used to study Raman spectra of the graphene and wear scar. The transmission electron microscope  was used to analyze the structure and morphology of graphene before and after the friction tests.In order to achieve good dispersion of graphene in the 5CB and make sure the 5CB exhibits a nematic phase during the rubbing process, the friction tests were performed in the first temperature condition. The friction coefficient and the wear scar diameter at different graphene concentrations were studied.The relationship between the concentration of graphene in 5CB and the average friction coefficient is shown in The relationship between the friction coefficient and the time is shown in When the addition amount of graphene is less than 0.15 wt.%, the coefficient of friction of the lubricant is relatively stable, this may be attributed to the absence of excess graphene on the friction surfaces and all graphene is adsorbed on the friction surfaces to fill surface pits or grooves to make the friction surfaces smooth ,37. WhenGraphene, as a sheet-like nano material, is easy to enter the friction region . In addiWhen addition amount of graphene is less than 0.15 wt.%, the average diameter of steel ball wear scar decreases with the increase of concentration. This phenomenon further proves that when the concentration is less than 0.15 wt.%, there is not enough graphene to form an antifriction and antiwear protective layer covering the wear surface . The conHowever, when the graphene concentration is higher than 0.15 wt.%, the average wear scar diameter starts to increase. This phenomenon further proves that graphene tends to agglomerate into large bodies at higher concentrations, and it is not easy to form a continuous and uniform antifriction layer on the friction region ,42. In aThe friction coefficient and wear scar diameter of the 5CB+0.15 wt.% G suspension were tested under the three temperature conditions set. The variation of the friction coefficient with the friction test time is shown in The lubrication mechanism of 5CB+0.15 wt.% G suspension were emphatically analyzed with 5CB as the benchmark. All the test data and images analyzed were obtained under the first temperature condition.To determine how graphene influences lubrication performance, the topography of wear surfaces after tests were investigated. As shown in In order to prove that graphene exists on the friction contact surfaces and plays an important role in the friction tests process, the wear surfaces of the balls lubricated by 5CB and 5CB+0.15 wt.% G suspension were analyzed by SEM and EDS, respectively. The test results are shown in Obviously, the friction surface of the ball for 5CB had irregular and deep furrows, and the severe scratch was attributed to the relatively poor antiwear property of 5CB. The corresponding EDS analysis showed that the content of C element on the worn surface was only 1.44 wt.%, which may be due to the basal component of the steel ball and carbonizations of the 5CB, as shown in By contrast, some very regular and shallow furrows appeared on the wear surface of the ball for 5CB+0.15 wt.% G suspension. Many literatures show that friction coefficient is closely related to surface morphology ,48. In a−1), D peak (1350 cm−1) and 2D peak (2712 cm−1) which are the typical Raman signals of graphene [Because Raman spectroscopy is a most suitable characterization technique for graphene materials , the Ramgraphene . This di2D/IG increased after the friction tests, which indicates that the thickness of the graphene is reduced [D/IG ratio to 0.89, as compared with 0.75 in the pristine graphene powder. This indicates that the defect density of graphene increases after friction tests [Comparing  reduced ,50,51. Ion tests ,53. ThisGraphene protects the friction surfaces and reduces the friction coefficient by forming a protective layer on the friction surfaces during the friction process. Graphene has strong mechanical strength, so the protective layer can greatly protect the friction surfaces from abrasion and corrosion. In addition, graphene sheets with weak van der Waals force are easy to slide between each other, which helps to reduce shear resistance during friction ,24. In oIn this work, the lubrication performances of graphene as additive of 5CB for steel/steel contacts were studied by using a tribometer with a ball-on-plate configuration. Based on the analysis of the test results, the conclusions are as follows.The concentration of graphene with the best antifriction and antiwear properties in the graphene/5CB suspension was 0.15 wt.%. Compared with pure 5CB, 5CB+0.15 wt.% G suspension reduced the friction coefficient and wear scar diameter by up to 70.6% and 41.3%, respectively. This outstanding lubricity is due to the low shear resistance and chemically stable protective layer formed by graphene and 5CB on the friction surfaces. Graphene with a sheet structure easily enters the friction interfaces and forms a protective layer on the friction surfaces, which makes the friction surfaces inert and prevents the two friction surfaces from direct contact, thereby reducing wear. In addition, graphene sheets are easy to slide, which is beneficial to reducing frictional resistance. Therefore, the addition of appropriate amount of graphene into 5CB can significantly reduce the friction and wear.