Junjun Qiu

Overexpression of long non-coding RNA HOTAIR predicts poor patient prognosis and promotes tumor metastasis in epithelial ovarian cancer

OBJECTIVES Although long non-coding RNAs (lncRNAs) are emerging as new regulators in the cancer paradigm, the involvement of lncRNAs in epithelial ovarian cancer (EOC) is just beginning to be studied. In this study, we focused on lncRNA HOX transcript antisense RNA (HOTAIR) and investigated its expression pattern, clinical significance, and biological function in EOC. METHODS HOTAIR expression in EOC tissues was examined and its correlation with clinicopathological factors and patient prognosis was analyzed. A series of in vitro and in vivo assays were performed to understand the role of HOTAIR in EOC metastasis. RESULTS HOTAIR expression was elevated in EOC tissues, and HOTAIR levels were highly positively correlated with the FIGO stage, the histological grade of the tumor, lymph node metastasis, and reduced overall survival (OS) and disease-free survival (DFS). A multivariate analysis showed that HOTAIR expression is an independent prognostic factor of OS and DFS in patients with EOC. Additionally, the results of in vitro assays showed that the suppression of HOTAIR expression in the three highly metastatic EOC cell lines (SKOV3.ip1, HO8910-PM, and HEY-A8) significantly reduced cell migration/invasion. The results of in vivo assays further confirmed the pro-metastatic effects of HOTAIR. Moreover, the pro-metastatic effects of HOTAIR were partially mediated by the regulation of certain matrix metalloproteinases (MMPs) and epithelial-to-mesenchymal transition (EMT)-related genes. CONCLUSIONS Our data suggest that HOTAIR plays a vital role in EOC metastasis and could represent a novel prognostic marker and potential therapeutic target in patients with EOC.

Long non-coding RNA ANRIL predicts poor prognosis and promotes invasion/metastasis in serous ovarian cancer

Recent studies have highlighted the role of long non-coding RNAs (lncRNAs) in carcinogenesis and have suggested that genes of this class might be used as biomarkers in cancer. However, whether lncRNAs are involved in serous ovarian cancer (SOC) remains largely unknown. In the present study, we focused on lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) and investigated its expression pattern, clinical significance, and biological function in SOC. We found that ANRIL levels were elevated in SOC tissues compared with normal controls and were highly correlated with advanced FIGO stage, high histological grade, lymph node metastasis, and poor prognosis. Multivariate analysis further revealed that ANRIL is an independent prognostic factor for predicting overall survival of SOC patients. In vitro, we compared differential ANRIL levels between SOC parental cell lines (SK-OV-3, HO8910) and highly metastatic sublines (SK-OV-3.ip1, HO8910-PM). Notably, ANRIL was highly expressed in both SK-OV-3.ip1 cells and HO8910-PM cells. SiRNA-mediated ANRIL silencing in these cells impaired cell migration and invasion. Based on the metastasis-related mRNA microarray analysis and subsequent western blotting confirmation, we found that MET and MMP3 are key downstream genes of ANRIL involved in SOC cell migration/invasion. Together, our data suggest that lncRNA ANRIL plays an important role in SOC invasion/metastasis and could represent a novel biomarker for predicting poor survival as well a promising therapeutic target.

The long non-coding RNA HOTAIR promotes the proliferation of serous ovarian cancer cells through the regulation of cell cycle arrest and apoptosis.

HOX transcript antisense RNA (HOTAIR) is a well-known long non-coding RNA (lncRNA) whose dysregulation correlates with poor prognosis and malignant progression in many forms of cancer. Here, we investigate the expression pattern, clinical significance, and biological function of HOTAIR in serous ovarian cancer (SOC). Clinically, we found that HOTAIR levels were overexpressed in SOC tissues compared with normal controls and that HOTAIR overexpression was correlated with an advanced FIGO stage and a high histological grade. Multivariate analysis revealed that HOTAIR is an independent prognostic factor for predicting overall survival in SOC patients. We demonstrated that HOTAIR silencing inhibited A2780 and OVCA429 SOC cell proliferation in vitro and that the anti-proliferative effects of HOTAIR silencing also occurred in vivo. Further investigation into the mechanisms responsible for the growth inhibitory effects by HOTAIR silencing revealed that its knockdown resulted in the induction of cell cycle arrest and apoptosis through certain cell cycle-related and apoptosis-related proteins. Together, these results highlight a critical role of HOTAIR in SOC cell proliferation and contribute to a better understanding of the importance of dysregulated lncRNAs in SOC progression.

Prognostic value of centromere protein-A expression in patients with epithelial ovarian cancer

Altered expression of centromere protein-A (CENP-A) is observed in various types of human cancers. However, the clinical significance and pathological role of CENP-A in epithelial ovarian cancer (EOC) remains unclear. The main objective of this investigation was to clarify the relationships between CENP-A expression and the clinicopathological features of patients with EOC. Real-time quantitative PCR and Western blot were performed to examine CENP-A expression in 20 pairs of fresh-frozen EOC tissues and corresponding noncancerous tissues. Using immunohistochemistry, we performed a retrospective study of the CENP-A expression levels on 120 archival EOC paraffin-embedded samples. Prognostic outcomes correlated with CENP-A were examined using Kaplan–Meier analysis and Cox proportional hazards model. Our results showed that the expression levels of CENP-A mRNA and protein in EOC tissues were both significantly higher than those in noncancerous tissues. By immunohistochemistry, the data revealed that high CENP-A expression was significantly correlated with pathological grade (P = 0.02) and International Federation of Gynecology and Obstetrics stage (P = 0.006). Consistent with these results, we found that high expression of CENP-A was significantly correlated with poor survival in EOC patients (P < 0.001). Furthermore, Cox regression analyses showed that CENP-A expression was an independent predictor of overall survival. Our data suggest that CENP-A could play an important role in EOC and might serve as a valuable prognostic marker and potential target for gene therapy in the treatment of EOC.

Expression and clinical significance of estrogen-regulated long non-coding RNAs in estrogen receptor α-positive ovarian cancer progression.

Estrogen (E2) has long been implicated in epithelial ovarian cancer (EOC) progression. The effects of E2 on cancer progression can be mediated by numerous target genes, including coding RNAs and, more recently, non-coding RNAs (ncRNAs). Among the ncRNAs, long ncRNAs (lncRNAs) have emerged as new regulators in cancer progression; therefore, our aim was to determine whether the expression of any lncRNAs is regulated by E2 and, if so, whether a subset of these lncRNAs have some clinical significance in EOC progression. A microarray was performed to identify E2-regulated lncRNAs in E2 receptor (ER) α-positive EOC cells. Bioinformatics analyses of lncRNAs were conducted, focusing on gene ontology and pathway analyses. Quantitative real-time polymerase chain reactions were performed to confirm the expression of certain lncRNAs in ERα-positive EOC tissues. The correlation between certain lncRNA expression and clinicopathological factors as well as prognosis in ERα-positive EOC patients was then analyzed. We showed that 115 lncRNAs exhibited significant changes in E2-treated SKOV3 cells compared with untreated controls. Most of these lncRNAs were predicated to have potential to contribute to cancer progression. Notably, three candidates (TC0100223, TC0101686 and TC0101441) were aberrantly expressed in ERα-positive compared to ERα-negative EOC tissues, showing correlations with some malignant cancer phenotypes such as advanced FIGO stage and/or high histological grade. Furthermore, multivariate analysis indicated that TC0101441 was an independent prognostic factor for overall survival. Taken together, these results indicate for the first time that E2 can modulate lncRNA expression in ERα-positive EOC cells and that certain lncRNAs are correlated with advanced cancer progression and suggestive of a prognostic indicator in ERα-positive EOC patients. Knowledge of these E2-regulated lncRNAs could aid in the future understanding of the estrogenic effect on EOC progression and may assist in the clinical design of new target therapies based on a perspective of lncRNA.

Effects of oestrogen on long noncoding RNA expression in oestrogen receptor alpha-positive ovarian cancer cells

Although oestrogen (E2) signalling has long been implicated in epithelial ovarian cancer (EOC) progression, the underlying mechanisms remain unknown. Long noncoding RNAs (lncRNAs) play a major role in cancer progression; therefore, our aim was to explore whether any lncRNA is regulated by E2 and plays some potential roles in the hormonal regulation of EOC progression. Here, we reported that E2 significantly dysregulated 115 lncRNAs (fold change ≥1.5, P<0.05) in E2 receptor (ER) alpha (ERα)-positive EOC SKOV3 cells compared with E2-untreated controls based on the microarray analysis. E2 regulation of the expression of 58 lncRNAs was bioinformatics predicted to be ERα-mediated; this was confirmed for two candidates. Both TC0101441 and TC0101686 were dysregulated by E2 in another ERα-positive PEO1 cells but not in ERα-negative A2780 cells. Additionally, the modulation of TC0101441 and TC0101686 expression by E2 was abrogated by the ER inhibitor ICI 182, 780 and short hairpin RNAs targeting ERα (ERα-shRNA). Further study of the two lncRNA expression indicated that ERα-positive EOC tissues had lower expression of TC0101686 and higher expression of TC0101441 compared to ERα-negative tissues. Particularly, elevated TC0101441 expression was correlated with lymph node metastasis, showing a metastatic potential. Results of in vitro assays further confirmed the pro-metastatic effect of TC0101441 and revealed that knockdown of TC0101441 also impaired E2-induced EOC cell migration/invasion by at least partly, regulating MMP2 and MMP3. Together, our findings demonstrate, for the first time, that E2 modulates lncRNA expression in ERα-positive EOC cells and that this regulation is sometimes ERα-mediated. Furthermore, our findings reveal that TC0101441contributes to E2-induced EOC cell migration/invasion. These results may shed a new insight into estrogenic effect on EOC progression by providing a perspective of lncRNA.

E2F1-regulated long non-coding RNA RAD51-AS1 promotes cell cycle progression, inhibits apoptosis and predicts poor prognosis in epithelial ovarian cancer

Long non-coding RNA RAD51 antisense RNA 1 (RAD51-AS1, also known as TODRA) has been shown to be down-regulated by E2F1, a key cell cycle and apoptosis regulator, in breast cancer. Little is known regarding the role of RAD51-AS1 in disease. Here, we investigate the role of RAD51-AS1 in epithelial ovarian cancer (EOC). Using luciferase reporter and chromatin immunoprecipitation experiments, we verified RAD51-AS1 as a target of E2F1 under negative regulation in EOC. We then examined RAD51-AS1 expression in EOC samples using in situ hybridization (ISH). RAD51-AS1 was localized to the nucleus and found to be a critical marker for clinical features that significantly correlated with poor survival in EOC patients. RAD51-AS1 was also an independent prognostic factor for EOC. Overexpression of RAD51-AS1 promoted EOC cell proliferation, while silencing of RAD51-AS1 inhibited EOC cell proliferation, delayed cell cycle progression and promoted apoptosis in vitro and in vivo. RAD51-AS1 may participate in carcinogenesis via regulation of p53 and p53-related genes. Our study highlights the role of RAD51-AS1 as a prognostic marker of EOC. Based on its regulation of the tumor suppressor p53, RAD51-AS1-based therapy may represent a viable therapeutic option for EOC in the near future.

Downregulated long non-coding RNA CLMAT3 promotes the proliferation of colorectal cancer cells by targeting regulators of the cell cycle pathway

Over-expression of long non-coding RNA (lncRNA)-CLMAT3 is significantly associated with colorectal liver metastasis and is an independent predictor of poor survival for colorectal cancer patients. However, as little is known regarding the role of this gene in the proliferation of colorectal cancer in vitro, we investigated the involvement of lncRNA-CLMAT3 in colorectal cancer cell proliferation. In this study, we demonstrate that lncRNA-CLMAT3 expression was significantly increased in colorectal cancer cells compared with a normal intestinal mucous cell line and that inhibition of lncRNA-CLMAT3 suppressed colorectal cancer cell proliferation in vitro. We also found that this reduced colorectal cancer cell proliferation due to lncRNA-CLMAT3 knockdown is associated with G0/G1 cell-cycle arrest induction and apoptosis enhancement. Furthermore, lncRNA-CLMAT3 knockdown enhanced Cdh1 expression and resulted in p27Kip accumulation via increased Skp2 protein ubiquitination. Taken together, our findings suggest that reducing lncRNA-CLMAT3 inhibits colorectal cancer cell proliferation by affecting cell cycle components.

Downregulation of paraoxonase 3 contributes to aggressive human hepatocellular carcinoma progression and associates with poor prognosis

Paraoxonase (PON) enzymes possess antioxidant properties and protect against cardiovascular diseases. As a member of PON family, PON3 is primarily synthesized in the liver and poorly investigated. This study aimed to examine the expression of PON3 in human hepatocellular carcinoma (HCC) and investigate the clinical significance and biological function of PON3 in HCC patients. We first analyzed PON3 expression in 50 paired HCC samples (HCC tissues vs matched para-cancerous tissues) and 160 clinical HCC specimens by using immunohistochemistry (IHC). Our results showed that the expression of PON3 was downregulated in HCC and significantly associated with tumor-node-metastasis (TNM) stage, tumor size, and tumor number. Kaplan-Meier survival and Cox regression analyses showed that PON3 was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR). Finally, we aimed to reveal the biological function of PON3 in HCC growth and metastasis, and our results showed that overexpression of PON3 potently inhibited growth and metastasis of HCC. Collectively, our study demonstrated that PON3 exhibited tumor-suppressive effects toward HCC and it might serve as a novel prognostic marker in HCC.

Goserelin promotes the apoptosis of epithelial ovarian cancer cells by upregulating forkhead box O1 through the PI3K/AKT signaling pathway

Gonadotropins, including luteinizing hormone (LH) and follicle stimulating hormone (FSH), are conducive to the growth of ovarian cancer based on the ‘gonadotropin theory’ and are regulated by gonadotropin-releasing hormone (GnRH). The present study was carried out to investigate the effect of goserelin, a GnRH agonist, on the apoptosis of epithelial ovarian cancer (EOC) cells and the underlying in vitro and in vivo mechanisms. Through flow cytometry, Hoechst staining and TUNEL staining, we demonstrated that goserelin promoted the apoptosis of EOC cells both in vitro and in vivo. Through human apoptosis gene PCR array, we verified that the promotion of EOC cell apoptosis by goserelin was linked to the upregulation of members of the tumor necrosis factor (TNF) and TNF receptor superfamilies, which have been identified as downstream targets of forkhead box O1 (FOXO1). Goserelin enhanced FOXO1 expression, and siRNA-mediated knockdown of FOXO1 abrogated the induction of apoptosis by goserelin. Moreover, goserelin decreased AKT activity, and FOXO1 upregulation by goserelin was dependent on the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vivo, the expression of key factors in the PI3K/AKT/FOXO1 pathway was consistent with that observed in vitro. In conclusion, our data suggested that goserelin may promote EOC cell apoptosis by upregulating FOXO1 through the PI3K/AKT signaling pathway. We believe that GnRH agonists may be potential antitumor agents, and key factors in the PI3K/AKT-FOXO1 pathway may also be novel therapeutic targets for the treatment of EOC.