Long noncoding RNA LSINCT5 promotes endometrial carcinoma cell proliferation, cycle, and invasion by promoting the Wnt/β-catenin signaling pathway via HMGA2

Background: A review of the evidence has indicated the critical role of long noncoding RNA (lncRNA) LSINCT5 in a large number of human cancers. However, the mechanistic involvement of LSINCT5 in endometrial carcinoma (EC) is still unknown. Here the authors aim to characterize the expression status of LSINCT5 and elucidate its mechanistic relevance to EC. Methods: Relative expression of LSINCT5 and HMGA2 were quantified by a real-time polymerase chain reaction. SiRNAs were employed to specifically knockdown endogenous LSINCT5 in EC cells. Cell proliferation was measured with Cell Count Kit-8 kit (CCK-8, Dojindo, Kumamoto, Japan) and cell growth was assessed by a colony formation assay. The cell cycle was analyzed with propidium iodide (PI) staining. Apoptotic cells were determined by flow cytometry after Annexin V/PI double-staining. Cell migration was evaluated by a wound-healing assay, and cell invasion was assessed using a transwell migration assay. The protein levels of HMGA2, Wnt3a, p-β-catenin, c-myc, β-actin, and GAPDH were determined by western blot. Results: The authors observed positively correlated and aberrantly up-regulated LSINCT5 and HMGA2 in EC. LSINCT5 deficiency significantly inhibited cell proliferation, cell cycle progression, and induced apoptosis. Meanwhile, cell migration and invasion were greatly compromised by the LSINCT5 knockdown. LSINCT5 stabilized HMGA2, which subsequently stimulated activation of Wnt/β-catenin signaling and consequently contributed to the oncogenic properties of LSINCT5 in EC. Conclusions: Our data uncovered the oncogenic activities and highlighted the mechanistic contributions of the LSINCT5-HMGA2-Wnt/β-catenin signaling pathway in EC.


Introduction
Endometrial carcinoma (EC) is one of the most common gynecologic malignancies in women, and ranks as the sixth in morbidity and third in mortality worldwide. In 2012 it was reported that 320,000 new cases were diagnosed and 76,000 deaths resulted from the disease. 1 Epidemiological investigations have revealed that the majority of EC occurs after menopause. Approximately 40% of incidences are associated with obesity, and other risk factors documented include excessive estrogen exposure, high blood pressure, and diabetes. 2 In addition, approximately 2-5% of patients have been identified with specific genomic mutations in the mismatch repair pathway. 3 EC is histologically categorized into endometrioid, serous and clear-cell subtypes. 4 Diagnosis of this disease is frequently achieved by endometrial biopsy or sampling during procedures such as dilation and curettage. Despite the insufficiency in accuracy and reliability, pap smear serves as a typical method for screening purposes. Currently, abdominal hysterectomy is still the leading treatment option for EC. For more advanced disease stages, radiotherapy, chemotherapy, combined with hormone therapy, or not, are also recommended. 5 The overall prognosis of EC is relatively favorable with a five-year survival rate in the USA greater than 80%.
Long noncoding RNA (lncRNA) is defined as a transcript longer than 200 oligonucleotides without protein-coding potential. The documented biological functions of lncRNA include regulation of gene-specific transcription, basal transcription machinery, transcript splicing, translation, and epigenetic modifications. 6 The importance of lncRNA in diverse human diseases, especially cancers, has been increasingly acknowledged. The subject in terms of the pleiotropic functioning of lncRNAs in EC is discussed in several recently published reviews. [7][8][9] Long stress-induced noncoding transcript 5 (LSINCT5) was first identified to be overexpressed in breast and ovarian cancer and affected cellular proliferation. 10 Subsequent investigations have uncovered its critical role in a range of human cancers. For instance, Xu and colleagues suggested that LSINCT5 predicted a negative prognosis and exhibited oncogenic activity in gastric cancer. 11 Zhang and colleagues demonstrated that elevated B-type natriuretic peptide promoted apoptosis in myocardial cells via the B-type natriuretic peptide/LSINCT5/caspase-1/ interleukin 1β signaling pathway. 12 Long and colleagues reported that LSINCT5 promoted ovarian cancer cell proliferation, migration, and invasion by disrupting the CXCL12/CXCR4 signaling axis. 13 In bladder cancer, the study performed by Zhu and colleagues uncovered that LSINCT5 activated Wnt/β-catenin signaling by interacting with NCYM to promote cancer progression. 14 Tian and colleagues indicated that LSINCT5 promoted malignancy in non-small cell lung cancer by stabilizing HMGA2. 15 Qi and colleagues demonstrated that E2F1 induced LSINCT5 transcription and promoted cancer progression via epithelial-mesenchymal transition (EMT) in gastric carcinoma. 16 Of interest, the investigation conducted by Dong and colleagues proposed that circulating CUDR, LSINCT5, and PTEN1 lncRNAs in sera distinguished patients with gastric cancer from healthy individuals. 17 However, to date, the expression status and potential involvement of LSINCT5 in EC is still not known. In this research, the authors' aim is to address this subject with both clinical endometrial tumor tissues and cell culture and attempt to understand the underlying molecular mechanism in cancer biology of this disease.

Patient samples
Tissue specimens were obtained from 30 EC patients and from 30 uninvolved controls from August 2016 to March 2018 at the First Affiliated Hospital, Sun Yat-sen University. Normal endometrial tissue specimens were obtained from women who underwent hysterectomy or endometrial curettage for endometrial-unrelated diseases, including uterine myoma or prolapse and the clinicopathologic characteristics are presented in online supplementary Table 1. All donors gave written informed consent for the collection of tissue specimens. This investigation involving human samples was approved by the Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University (#FAHLC078a). The procedure was performed in strict accordance with protocol.

Real-time polymerase chain reaction
The RNA species were extracted from either tissue samples or cell culture with Trizol reagent (Invitrogen, Waltham, MA, USA). The integrity of RNA was quality-checked by agarose electrophoresis. The RNA concentration was determined by Nanodrop 2000 (ThermoFisher, Waltham, MA, USA). cDNA was prepared from each 1 μg RNA with High-Capacity cDNA Reverse Transcription Kit (ThermoFisher) following the provider's recommendation. Quantitative real-time polymerase chain reaction (PCR) was performed with SYBR Green Mater Mix (Bio-Rad, Hercules, CA, USA) and relative expression of the target gene was calculated by 2 -∆∆Ct method. GAPDH was used for internal reference. The primers were provided as follows:

Colony formation assay
Cell growth was inspected using a colony formation assay. In short, 1000 cells (LSINCT5deficient and scramble control) were seeded into 6-well in triplicate and underwent consecutive culture for up to 2 weeks. The culture medium was replaced every 3 days. The resulting colonies were first fixed with 4% paraformaldehyde and stained with 0.25% crystal violet for 15 min. Colonies were counted under light microscope in three random areas.
Cell cycle analysis A single-cell suspension was prepared from both LSINCT5-deficient and proficient Ishikawa and HEC-1A cells [2 × 10 6 cells/ml in ice-cold phosphate-buffered saline (PBS)], which were subsequently fixed with 70% ethanol at 4°C for 12 h. The fixed cells were then stained with 500 μL PI/ Triton X-100 solution (0.02% RNAse, 0.002% PI, 0.1%, Triton X-100) at 37°C for 15 min. Cell clumps were removed by passing-through a cell strainer (BD BioSciences, CA, USA) and data was acquired using BD Accuri Cytometry (BD BioSciences, Franklin Lakes, NJ, USA).

Apoptosis analysis
Apoptotic cells were detected by Annexin V/PI double-staining in both Ishikawa and HEC-1A cell lines in response to LSINCT5 knockdown. The indicated cells were harvested by trypsin digestion and prepared into single-cell suspension in PBS. Annexin V/PI staining was performed simultaneously in the dark, at room temperature for 15 min. BD Accuri Cytometry (BD Biosciences) was used for cell analysis.

Wound-healing assay
Cell migration capacity was evaluated by wound closure rate. Straight lines were drawn in the monolayer cells using sterile tips. Gap closure was continuously monitored and recorded under a light microscope. The assay was performed three times independently.

Statistical analysis
Data was collected from at least three independent repeats. GraphPad Prism 6.0 software was used for data processing and analysis. SPSS 22.0 was used for statistical analysis. One-or two-way ANOVA followed and the Tukey test was used for statistical comparison. Pearson's correlation coefficient (r) was used for correlation analysis.
The p value was calculated and p < 0.05 was considered as significantly different.

Positively correlated and aberrantly overexpressed LSINCT5 and HMGA2 in EC
To investigate the potential involvement of LSINCT5 in EC, the authors' aim was to analyze the relative expression of this lncRNA in clinical samples (n = 30, Table S1). As shown in Figure  1(a) the amount of LSINCT5 in EC tissues were significantly higher than those in adjacent normal controls. Consistent with the previously reported positive-regulation on HMGA2 expression in lung cancer, 15 the authors noticed that HMGA2 protein expression levels were induced in EC in comparison with control, shown in Figure 1(b). Furthermore, correlation analysis demonstrated significant positivity between LSINCT5 and HMGA2 (Figure 1(c)), which implies the mechanistic relevance and potential regulatory relationship between these two genes in EC.

LSINCT5-deficiency compromised cell proliferation in EC cells
The authors' previous data disclosed the aberrant overexpression of LSINCT5 in vivo. Therefore, the authors investigated the potential oncogenic contribution of LSINCT5 to EC in cell culture. To this end, two EC cell lines journals.sagepub.com/home/tam 5 were used to exclude any bias due to cell contexts and specifically knockdown LSINCT5 with siRNAs. Interference efficiencies were experimentally confirmed by qPCR, where more than 70% knockdown was achieved with two independent siRNAs in both Ishikawa and HEC-1A cells (Figure 2(a) and (b)). Cell proliferation was continuously monitored up to 96 h upon LSINCT5 knockdown. The CCK-8 results demonstrated that cell proliferation in LSINCT5-deficient cells was significantly suppressed in both Ishikawa and HEC-1A cells (Figure 3(a) and (b)). Likewise, the colony formation capacity was greatly decreased in  response to the LSINCT5 knockdown ( Figure  3(c) and (d)). The data suggested that high LSINCT5 was critical for the malignant proliferation in EC cells.

LSINCT5-silencing elicited cell cycle arrest and cell apoptosis
In addition to the impact on cell proliferation, the potential influence of LSINCT5 on cell cycle progression and cell apoptosis was evaluated. As shown in Figure 4(a) and (b), the percentage of cells at the G1 phase was increased, accompanied by a decrease in S phase in LSINCT5-silenced EC cells, in comparison with either parental or scramble control. Quantitative results are summarized in Figure 4(c) and (d). The apoptotic percentage was measured using the Annexin V/PI double-staining method. As shown in Figure 4(e) and (f), apoptosis was greatly induced in LSINCT5-deficient cells. Notably, more intensive effects on apoptosis induction were found in si-LSINCT5-2 cells, which was consistent with the higher knockdown efficiency. Quantitative results are presented in Figure 4(g) and (h). Therefore, the data indicates the importance of LSINCT5 in cell cycle progression and apoptosis inhibition in EC.

LSINCT5-deficiency inhibited cell migration and invasion of EC cells
In addition, the possible alterations with respect to cancer metastatic characteristics upon the LSINCT5 knockdown were assessed. Cell migration behaviors were investigated using a woundhealing assay, and the scratch closure rate was continuously monitored for up to 24 h. Representative images and quantitative results were shown in Figure 5(a)-(d). It was noted that cell migration was significantly compromised in LSINCT5-deficient offspring of both cell lines. Similarly, cell invasive capacity, as indicated by the invaded cells into basement membrane, was greatly reduced upon LSINCT5 knockdown as shown in Figure 5(e)-(h). The data uncovered that LSINCT5-deficiency inhibited cell migration and invasion of EC cells.

LSINCT5 inhibited HMGA2 protein degradation
The previous results disclosed the positive correlation between LSINCT5 and HMGA2 and suggested their possibly regulatory effects in EC tissues. This research tries to characterize the relative expression of HMGA2 in response to LSINCT5 knockdown in vitro. As shown in Figure 6(a) and (b), no noticeable difference in HMGA2 transcripts was observed in both Ishikawa and HEC-1A cells upon LSINCT5 knockdown. However, the protein level of HMGA2 was significantly decreased in LSINCT5-depleted cells, in comparison with either parental cells or scramble control as shown in Figure 6(c) and (d), which indicates a posttranscriptional mechanism underlying the LSINCT5-regulated expression of HMGA2 in EC cells. Densitometric quantitation results are shown in Figure 6(e) and (f). The data suggests that LSINCT5 might suppress HMGA2 degradation, which consequently stabilized HMGA2 protein in EC cells.

Wnt/β-catenin signaling was repressed by HMGA2 downregulation upon LSINCT5 knockdown
In addition, this research aims to clarify whether up-regulated HMGA2 by LSINCT5 impacts the Wnt/β-catenin pathway in EC. The relevant protein factors including Wnt3a, p-β-catenin, β-catenin, and c-myc in response to LSINCT5 knockdown were examined. As shown in Figure  7   both western blot and immunohistochemistry results. Except for HMGA2, all other transcripts were notably up-regulated in EC as well based on real-time PCR results, which was unmistakably in support of the conclusion that LSINCT5 regulated HMGA2 at post-transcriptional level via suppressing protein degradation. Therefore, the importance of these signaling cues in EC was highlighted both in vitro and in vivo.

Discussion
Despite the increasing acknowledgment of the critical roles of LSINCT5 in human malignancies, potential involvement of this lncRNA in EC is still unknown. The authors' aim was to characterize the expression status of LSINCT5 and to attempt to understand its oncogenic properties at the molecular level. First, the authors uncovered aberrant overexpression of LSINCT5 in clinical EC patients. siRNA-mediated LSINCT5 knockdown significantly suppressed proliferation, stimulated cell cycle arrest and apoptosis. Furthermore, the authors uncovered that metastasis-related malignant behaviors, such as migration and invasion, were evidently suppressed by LSINCT5 depletion.
In view of the previously described stabilizing effect of LSINCT5 on HMGA2 in lung cancer, 15 here we analyzed the correlation between LSINCT5 and HMGA2 both in vivo and in vitro. The results disclosed a positive relationship between these two genes in clinical endometrial tumor tissues. More importantly, we characterized that HMGA1 transcripts were not subjected to the influence imposed by LSINCT5 knockdown in EC cells. In contrast, the HMGA2 protein was evidently decreased in LSINCT5-deficient cells, which suggested the post-transcriptional manner in regulating HMGA2 expression by LSINCT5. This was consistent with the stimulatory effects of HMGA2 on Wnt/βcatenin activation, 18 and remarkable decreases in key Wnt/β-catenin effectors including Wnt3a, p-βcatenin, and c-myc were observed. While ectopic introduction of HMGA2 in LSINCT5-depleted cells completely rescued the phenotype with respect to activation of Wnt/β-catenin pathway, which critically highlighted the predominant role of HMGA2 in mediating the LSINCT5 signaling transduction. Therefore, this study demonstrated the oncogenic properties of LSINCT5 in EC via stimulating cell proliferation, inhibiting cell cycle arrest and apoptosis, and promoting migration and invasion. The results also demonstrate that LSINCT5 efficiently stabilized HMGA2 protein, which subsequently activated the Wnt/β-catenin pathway and consequently contributed to EC initiation, progression, and metastasis.
HMGA2 belongs to the non-histone chromosomal high mobility group protein family, which usually functions as a structural component and important factor in facilitating enhanceosome formation on a variety of mammalian promoters. 19 HMGA2 was also suggested as a transcriptional modulator involved in cell cycle control via directly targeting CCNA2, therefore playing a critical role in chromosome compaction in the meiotic spermatocytes. 20 Cumulative evidence has implicated the indispensable roles of HMGA2 in a range of human cancers. For example, Summer and colleagues reported that HMGA2 exhibited dRP/AP site cleavage activity and protected cancer cells from DNA-damage-induced cytotoxicity during chemotherapy. 21 Klemke and colleagues suggested that overexpression of HMGA2 in uterus leiomyomas indicated its universal function in the pathogenesis of this disease. 22 The study carried out by Hristov and colleagues demonstrated that HMGA2 protein expression correlated with lymph node metastasis and increased tumor grade in pancreatic ductal adenocarcinoma. 23 Belge and colleagues proposed that up-regulated HMGA2 could serve as a novel molecular marker to distinguish between benign and malignant follicular neoplasia in thyroid carcinomas. 24 Malek and colleagues indicated HMGA2 as a candidate target for ovarian cancer siRNA therapy exploitation. 25 Langelotz and colleagues demonstrated that the expression of HMGA2 mRNA in the peripheral blood as a potential prognostic biomarker for unfavorable survival in metastatic breast cancer. 26 More recently, Tian and colleagues discovered a key role of HMGA2 in non-small cell lung cancer, which was complexed with and stabilized by lncRNA LSINCT5. 15 Consistent with the oncogenic properties of HMGA2, in this research the authors demonstrated that aberrant overexpression of LSINCT5 in EC led to stabilized HMGA2 protein, which consequently contributed to LSINCT5 pro-tumoral activity. Ectopic introduction of HMGA2 in LSINCT5-negative EC cells was sufficient to activate oncogenic signaling, which underlined the predominant role of HMGA2 in oncogenesis and progression of EC.
The Wnt signaling pathway plays a fundamental role in embryonic development and specifically programmed body axis patterning, cell fate specification, cell proliferation, and migration. Dysregulation in Wnt/β-catenin pathway has been characterized in a wide range of human cancers. 27 For example, Wnt1 was first identified as a protooncogene in a breast cancer mouse model. 28 Increased nuclear presence of β-catenin was reported to correlate with poor prognosis in breast cancer patients. 29 Changes in the β-catenincoding gene CTNNB1 was measured in breast, colorectal, melanoma, prostate, lung, and other cancers. The crucial involvement of HMGA2 in Wnt/β-catenin pathway has been uncovered by multiple investigations. Singh and colleagues reported that HMGA2 was required for canonical Wnt signaling during lung development. 18 Zha and colleagues demonstrated that HMGA2 elicited EMT process through activating the Wnt/βcatenin signaling in gastric cancer. 30 Cui and colleagues suggested that miR-337 regulated the PI3K/AKT and Wnt/β-catenin signaling pathways to inhibit hepatocellular carcinoma progression by targeting HMGA2. 31 In support of the theory that HMGA2 positively modulated Wnt/β-catenin, in this research the authors provide evidence that introducing HMGA2 in LSINCT5-deficient cells significantly restored Wnt/β-catenin activation. Of interest, the molecular mechanisms underlying the stabilizing effect of LSINCT5 on HMGA2 protein in EC still have to be defined. In addition, the mode-of-action of LSINCT5-HMGA2-Wnt/βcatenin unraveled in the in vitro study required further confirmation in vivo.

Conclusion
In summary, this data characterizes aberrant overexpression of LSINCT5 in EC, which exerts its oncogenic function via stimulating cell proliferation, promoting cell cycle, inhibiting cell apoptosis, inducing cell migration, and invasion. The authors have further uncovered the predominant role of HMGA2 downstream of LSINCT5 signaling transduction, which consequently contributes to the oncogenic properties of LSINCT5. This study has highlighted the fundamental role of LSINCT5-HMGA2-Wnt/β-catenin in EC, which may hold significant potential for diagnostic and therapeutic exploitation.

Funding
This research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.