Function/Mechanisms Of The Long Non-Coding RNA ElncRNA1 In Promoting Metastasis In ERα-Positive Ovarian Cancer

Ovarian cancer continues to be the leading cause of death from gynaecologic malignancies. Despite advances in surgery and chemotherapy, the five-year survival rate is poor. Such an unfavourable prognosis has been largely correlated with tumour metastasis. To date, the mechanism of ovarian cancer metastasis remains unclear. Emerging data have demonstrated that the involvement of estrogen (E2) has been associated with ovarian cancer metastasis. In fact, the effects of E2 on ovarian cancer metastasis are mainly attributable to the capacity of E2 to regulate target gene expression. Many studies, including ours, have revealed contributions to the ovarian cancer metastasis by multiple E2-regulated protein-coding genes. Despite such findings, our understanding of the exact effects of E2 on ovarian cancer metastasis remains far from complete, and investigating the underlying mechanisms solely in terms of the roles of E2-regulated protein-coding genes would be wholly insufficient.Long non-coding RNAs (lncRNAs) are emerging as new regulators in the cancer paradigm. The misregulation of lncRNAs has been definitively associated with different types of cancers. Although ovarian cancer research has included several small studies of lncRNAs, no published studies have investigated the hormonal regulation of lncRNA expression, to our knowledge. Therefore, in the present study, we determined whether the expression of any lncRNAs is regulated by E2 in ERa-positive ovarian cancer cells and, if so, whether certain E2-regulated IncRNA contributes to ERa-positive EOC progression. Our study may help better understand the mechanism of ovarian cancer metastasis and clinical design of new target therapies based on a perspective of IncRNA.Part 1 Identification of Estrogen-induced long non-coding RNA ElncRNAl in ERa-positive ovarian cancer cellsObjective To identify estrogen-induced long non-coding RNAs through microarray and to preliminary explore the mechanism by which E2 regulates the candidate lncRNA.Methods (1) E2-induced lncRNA profiles in ERα-positive SKOV3 ovarian cancer cells were identified using microarrays. (2) Bioinformatics ERE binding site searching was performed to predict that the E2 regulation of certain lncRNAs is mediated through ERα-ERE pathway. (3) A series of experimental assays (the use of ER inhibitor ICI 182,780 and siRNAs targeting ERα, ChIP assay and Dual luciferase reporter assay) were performed to confirm that E2 regulation of the candidate lncRNA is mediated through ERα-ERE pathway.Results (1) 115 lncRNAs exhibited significant changes in E2-treated SKOV3 cells compared with untreated controls (fold change≥1.5, P< 0.05). (2) E2 regulation of the expression of 19 lncRNAs was bioinformatics predicted to be mediated through ERα-ERE pathway. LncRNA-TC0101441, which was designated as Estrogen-induced long non-coding RNA-1 (ElncRNA1), was chose as a candidate for further study because it was the most significantly altered lncRNA among the 19 lncRNAs. (3) The modulation of ElncRNAl expression by E2 was abrogated by ICI 182,780 and ERα-siRNAs. Additionally, based on the ChIP assay, the enrichment of ERα-associated ElncRNA1 promoter fragments was confirmed by RT-PCR, which was enhanced by E2 treatment. Furthermore, results of the dual luciferase reporter assay revealed that ERa mimic significantly induced luciferase activity of ElncRNAl promoter when compared to negative control, which was enhanced by E2 treatment; whereas mutations in the ERE sequences abrogated the effects of E2-activated ERα on induction of the repoter activity. These findings suggest that E2 regulation of ElncRNA1 is mediated through ERα-ERE pathway.Conclusion Our data demonstrate, for the first time, that E2 can modulate lncRNA expression in ERα-positive EOC cells. Furthermore, our study identifies a novel lncRNA, named as ElncRNA1, and shows that E2 regulation of ElncRNA1 is mediated through ERα-ERE pathway.Part 2 Expression and Clinical Significance of ElncRNA1In ERa-positive Ovarian CancerObjective To investigate the expression pattern and clinical significance of ElncRNAl in ERa-positive ovarian cancer.Methods (1) Expression of ERa was evaluated by immunohistochemistry technique in ovarian cancer tissues. (2) Expression levels of ElncRNAl in ERa-positive, ERa-negative ovarian cancer and normal ovarian surface epithelial tissues were examined by qRT-PCR. (3) The correlation between ElncRNA1 expression and clinicopathological factors as well as prognosis in ER alpha-positive EOC patients was then analyzed.Results (1) In 96 ovarian cancer tissue specimens,74 specimens were ERa-positive, and 22 ERa-negative. (2) ElncRNA1 expression was elevated in ERa-positive ovarian cancer tissues than both ERa-negative ovarian cancer tissues and normal ovarian surface epithelial tissues. (3) High ElncRNAl expression levels were positively correlated with the FIGO stage, the histological grade of the tumor, and the occurrence of lymph node metastasis (P<0.01), but not with other clinicopathological variables such as the age of the patient, the histological type of the tumor, the residual tumor diameter, CA125 levels, and the occurrence of ascites in ERa-positive ovarian cancer. (4) In a univariate analysis, ElncRNA1 expression levels were correlated with both OS and PFS (P<0.01). According to the multivariate Cox regression analysis, ElncRNAl expression levels, in addition to the FIGO stage and the occurrence of lymph node metastasis, were independent predictors of OS and PFS (P<0.05).Conclusion Our results indicate that ElncRNA1 is correlated with tumor aggresiveness and poor clinical outcome and suggestive of a novel prognostic indicator in ERa-positive EOC patients.Part 3 Functional characterization of ElncRNAl in ERa-positive ovarian cancer cell progression in vitroObjective To explore the role of ElncRNAl in cell proliferation, cell cycle/apoptosis, cell migration/invasion using loss-of-function and gain-of-function approaches in ERa-positive ovarian cancer cells.Methods (1) ElncRNA1 expression levels were investigated in a panel of ERa-positive ovarian cancer cells lines (SKOV3、CAOV3、OVCAR3、 HO8910、PEO1 and PEO4) using qRT-PCR. Lentiviral-mediated knockdown/overexpression of ElncRNAl expression was performed in certain cell lines. (2) The cell proliferation changes of lentiviral-mediated knockdown/overexpression of ElncRNAl were determined using the CCK-8 assay. (3) The apoptosis and cell cycle changes caused by lentiviral-mediated knockdown/overexpression of ElncRNA1 were evaluated using the flow cytometry assay. (4) The migration and invasion changes induced by lentiviral-mediated knockdown/overexpression of ElncRNA1 were measured using the wound healing assay and the matrigel transwell assay.Results (1) SKOV3 and CAOV3 cells, with the higher ElncRNA1 expression than other cells, were chose to establish lentiviral-mediated ElncRNA1 knockdown cells; while HO8910, with the lowest ElncRNA1 expression, were chose to establish lentiviral-mediated ElncRNA1 overexpression cells. (2) Results of the CCK-8 assay showed that suppression of ElncRNA1 decreased the proliferation of both SKOV3 and CAOV3 cells, and overexpression of ElncRNAl increased the proliferation of HO8910 cells. (3) Results of the flow cytometry assay revealed that ElncRNAl knockdown induced apoptosis but not cell cycle alteration in both SKOV3 and CAOV3 cells, and overexpression of ElncRNA1 altered cell cycle progression but not apoptosis in HO8910 cells. (4) The wound healing assay and the matrigel transwell assay demonstrated that knockdown of ElncRNAl significantly reduced both the migratory and invasive ability of SKOV3 and CA0V3 cells, and overexpression of ElncRNAl significantly enhanced both the migratory and invasive ability of HO8910 cells.Conclusion Among cell proliferation, cell cycle/apoptosis, cell migration/invasion, ElncRNA1 plays the most important role in migration/invasion in ERa-positive ovarian cancer cells.Part 4 ElncRNA1 silencing inhibits metastasis in ERa-positive ovarian cancer in vivoObjective To construct peritoneal ovarian cancer model in nude mice and to observe pro-metastatic effect of ElncRNA1 in vivo.Methods Stable ElncRNA1-knockdown(LV-ElncRNA1-shRNA-SKOV3) and negative control SKOV3 cells (LV-NC-SKOV3) carrying green fluorescent protein (GFP) were established by selection with puromycin. The intraperitoneal ovarian cancer model was established by i.p. injection of LV-ElncRNA1-shRNA-SKOV3 (the study group) and LV-NC-SKOV3 (the control group) cells in nude mice. The progression of tumors within the abdominal cavity of the animal was monitored by in vivo imaging system every week. The engrafted mice were euthanized four weeks after the intraperitoneal injection of the tumor cells and the signs of tumor distress within the abdominal cavity were inspected. The number and weight of transplanted tumors were compared between the two groups.Results Four weeks after intraperitoneal injection of cancer cells, the diffuse distribution of tumor within the abdominal cavity could be detected by in vivo imaging system. The mice injected with LV-NC-SKOV3 cells (the control group) displayed widespread metastatic lesions; while the mice injected with LV-ElncRNA1-shRNA-SKOV3 cells (the study group) displayed reduced metastatic lesions. After the in vivo imaging assay, all mice were euthanized; significant differences in the patterns of tumor growth in the two groups were observed. The control mice developed numerous large tumors in the abdominopelvic region and numerous tumors studding the mesentery adjacent to the bowel. Moreover, liver metastases could be found. The average number of tumor implants was 51.33 ± 4.59 and the total tumor weight was 0.81 ± 0.05 g. The study mice typically developed significantly fewer implants on the mesentery and in the abdominopelvic region than the controls (9.33 ± 1.86 tumor implants, P<0.05 vs. control). The total tumor weight was also decreased 5.4-fold in the study mice (0.15 ± 0.01 g, P<0.05 vs. control).Conclusion Our results suggest that ElncRNA1 promotes EOC metastasis in vivo.Part 5 Preliminary study on mechanisms of ElncRNA1 in promoting metastasis in ERa-positive ovarian cancerObjective To explore the mechanism of ElncRNA1 in promoting metastasis in ERα-positive ovarian cancer.Methods Metastasis-related gene PCR array and qRT-PCR were employed to investigate the downstream molecular events involving ElncRNA1 and EOC metastasis. A series of experimental assays (western blot, immunohistochemistry and the use of siRNAs) were performed to explore the involved mechanisms.Results Tumor metastasis-related gene expression profiles of LV-ElncRNA1-shRNA-SKOV3 cells were first compared with those of LV-NC-SKOV3 cells using PCR array analysis. The results showed that ten genes were markedly dysregulated (>5-fold) after ElncRNA1 silencing in SKOV3 cells. qRT-PCR was performed to confirm the ten genes in another lentiviral-mediated ElncRNAl overexpressed HO8910 cells. The results showed that five genes, consistent with the results of PCR array, were significantly dysregulated after ElncRNA1 overexpression. Because most of those genes were EMT-related genes, we hypothesized that ElncRNAl may promote EMT. Western blot assays were performed to investigate alterations in EMT markers following ElncRNA1 knockdown. The results showed that the lentiviral-mediated ElncRNA1 knockdown resulted in an increase in the expression of E-cadherin and a decrease in the expression of FN-1, N-cadherin and Snail. In our in vivo model of intraperitoneal metastasis, immunohistochemistry results confirmed that the metastatic tumour nodules originating from LV-ElncRNA1-shRNA-SKOV3 cells had increased expression of E-cadherin and decreased expression of FN-1, N-cadherin and Snail. Additionally, lentiviral-mediated ElncRNAl overexpressed HO8910 cells had decreased expression of E-cadherin and increased expression of FN-1, N-cadherin and Snail. Such modulation of E-cadherin, FN-1 and N-cadherin expression by ElncRNA1 in lentiviral-mediated ElncRNA1 overexpressed HO8910 cells was abrogated by Snail-siRNA, suggesting that ElncRNA1 regulation of certain EMT markers (E-cadherin, FN-1 and N-cadherin) is mediated through Snail.Conclusion The pro-metastatic effects of ElncRNA1 are likely partially mediated by the regulation of EMT through Snail.