Micro RNA-370 Suppresses Proliferation And Promotes Endometrioid Ovarian Cancer Chemosensitivity To CDDP By Negatively Regulating ENG

Objective: Micro RNAs(mi RNAs) are a class of non-coding RNAs that post-transcriptionally inhibit gene expression. In this study, we discovered that micro RNA-370(mi R-370) was down-regulated in endometrioid ovarian cancer cells. In IGROV1 and TOV112 D endometrioid ovarian cancer cells, mi R-370 suppressed cellular viability and colony formation. mi R-370 also enhanced endometrioid ovarian cancer cell chemosensitivity to c DDP. Endoglin(ENG) was directly and negatively regulated by mi R-370. In addition, hypermethylation was a potential mechanism of mi R-370 epigenetic silencing. We conclude that mi R-370 acts as a tumor suppressor in endometrioid ovarian cancer via ENG regulation.Methods: First, q RT-PCR and Northern blotting analysis confirmed mi R-370 dysregulation in both endometrioid ovarian cancer cell lines and tissues. These data led us to presume a refined regulation of endometrioid ovarian cancer cells by mi R-370. A p EGP-mi R vector-based mi R-370 ectopic expression plasmid and asynthesized mi R-370 inhibitor(mi R-370 ASO) were obtained to alter mi R-370 levels in the IGROV1 and TOV112 D endometrioid ovarian cancer cell lines. Our experiment indicated that the elevated mi R-370 reduced, whereas mi R-370 suppression strengthened IGROV1 and TOV112 D cell colony formation. mi R-370 also alleviated endometrioid ovarian cancer cell viability over a wide time span(from 48 h to 96 h after transfection; Fig. 2C). These data revealed a role of mi R-370 as a tumor suppressor in endometrioid ovarian cancer. Annexin V- and 7-AAD-based apoptosis analyses revealed that blocking mi R-370 reduced IGROV1 cell apoptosis. Next, we detected mi R-370 activity in these two cell lines with two common chemotherapeutic agents, c DDP and Taxol. Flow cytometry analyses indicated that the apoptotic and dead cell ratio increased when mi R-370 was elevated and was suppressed by blocking mi R-370. When Taxol was used instead of c DDP, mi R-370 did not influence apoptosis of these cells. With the help of the Target Scan Human andmi Randa databases, we predicted that the 3′UTR of ENG bears a potential binding site for mi R-370. The following, we introduced a bioluminescent reporter assay in IGROV1 cells to confirm the direct regulation of mi R-370 on ENG m RNA. Furthermore, endogenous ENG levels were also inversely correlated with mi R-370 levels, as detected by Western blotting in IGROV1 and TOV112 D cells. These data indicated thatmi R-370 directly regulates ENG expression via the response element within the ENG 3′UTR. To evaluate whether the negative role of mi R-370 on endometrioid ovarian cancer was because of its regulation of ENG, we overexpressed mi R-370, which subsequently strengthened ENG expression in IGROV1 and TOV112 D cells. These data demonstrate that mi R-370 attenuates the malignant phenotypes of endometrioid ovarian cancer cells by negatively regulating ENG. To continuously overexpress mi R-370 in endometrioid ovarian cancer cells, we constructed an IGROV1 cell line that stably expresses mi R-370. The IGROV1/mi R-370 cells were injected into the axillary fossae of nude mice. We determined that the tumors derived from mi R-370-expressing IGROV1 cells were smaller than those derived from the control cell line regardless of whether c DDP was applied, though c DDP could generally reduce the tumor volume. These data further illustrate the negative role of mi R-370 in endometrioid ovarian cancer growth in vivo.Results: In this study, we focused on the regulatory role of the mi RNA mi R-370 in endometrioid ovarian cancer. First, mi R-370 was down-regulated in endometrioid ovarian cancer tissues or cell lines compared with normal ovarian epithelial cells, indicating its potential tumor suppressor role in thismalignancy. Second, in the IGROV1 and TOV112 D endometrioid ovarian cancer cell lines, mi R-370 suppressed cell proliferation and colony formation. The in vivo studies also revealed that the xenograft tumors derived by IGROV1 cells with mi R-370 overexpression exhibited a lower growth rate than the control groups. Third, mi R-370 overexpression in endometrioid ovarian cancer cells could sensitize these cells to the chemotherapeutic agent c DDP, further highlighting the potential antitumor role of mi R-370. However, when cells were treated with Taxol, another chemotherapeutic agent, mi R-370, did not affect its chemosensitivity. This interesting result drew our attention to identifying the functional target gene of mi R-370 and to illuminating the mechanism of mi R-370 and its target gene in the regulation of endometrioid ovarian cancer.Conclusions: Collectively, our research revealed a new regulatory pathway in endometrioid ovarian cancer cells. Specific DNA region hypermethylation reduced mi R-370 levels and elevated the expression of its direct target gene ENG. ENG overexpression contributes to the enhanced malignancy in endometrioid ovarian cancer cells including high proliferation, low apoptosis and/or cell death, and enhanced c DDP chemoresistance. This knowledge may shed light on the mechanisms by which mi RNAs regulate in ovarian cancer and may also aid the development of novel therapeutic strategies to overcome this malignancy.