circ-LRP6调控miR-515-5p/IL-6通路影响卵巢癌细胞增殖、迁移和侵袭★

circ-LRP6 regulates miR-515-5p/IL-6 pathway to affect the proliferation, migration and invasion of ovarian cancer cells★

摘要:

目的 探讨环状RNA低密度脂蛋白受体相关蛋白6（circ-LRP6）对卵巢癌细胞增殖、迁移和侵袭的影响及其分子机制。方法 选取44例卵巢癌患者的癌组织及癌旁组织，培养正常人卵巢上皮细胞HUM-CELL-0088及卵巢癌细胞系SKOV3、OVCAR3、A2780，采用实时荧光定量PCR检测卵巢癌组织和细胞中circ-LRP6、微小RNA-515-5p（miR-515-5p）的表达水平；双荧光素酶报告实验检测miR-515-5p与circ-LRP6之间的靶向关系；将circ-LRP6干扰表达载体、miR-515-5p过表达载体、miR-515-5p抑制表达载体与circ-LRP6干扰表达载体转染至卵巢癌细胞SKOV3中，蛋白质印迹法（Western blotting）检测细胞周期蛋白D1（Cyclin D1）、基质金属蛋白酶（MMP）2、MMP9、白细胞介素6受体（IL-6R）蛋白的表达；CCK-8法检测SKOV3细胞活性；Transwell小室法检测细胞迁移和侵袭。结果 与癌旁组织及HUM-CELL-0088细胞相比，卵巢癌组织和SKOV3、OVCAR3、A2780细胞系中circ-LRP6表达水平显著升高，miR-515-5p表达水平显著降低（P<0.05）。双荧光素酶报告实验证实circ-LRP6可靶向负调控miR-515-5p的表达。低表达circ-LRP6或过表达miR-515-5p均可抑制Cyclin D1、MMP2、MMP9、IL-6R的表达，降低细胞活性，抑制细胞迁移和侵袭（P<0.05）。同时，抑制circ-LRP6和miR-515-5p表达可上调SKOV3细胞中Cyclin D1、MMP2、MMP9、IL-6R的表达，增强细胞活性，促进细胞迁移和侵袭（P<0.05）。结论 抑制circ-LRP6表达可能通过上调miR-515-5p抑制IL-6，从而抑制卵巢癌SKOV3细胞的增殖、迁移和侵袭。

Abstract:

Objective To explore the effects of circular RNA low-density-lipoprotein receptor-related protein 6 (circ-LRP6) on the proliferation, migration and invasion of ovarian cancer cells and the molecular mechanism.Methods The cancer tissues and adjacent tissues were collected from 44 ovarian cancer patients. The normal human ovarian epithelial cells HUM-CELL-0088 and ovarian cancer cell lines SKOV3, OVCAR3, A2780 were cultured. The real-time fluorescence quantitative PCR was used to detect the expression levels of circ-LRP6 and microRNA-515-5p (miR-515-5p) in ovarian cancer tissues and cells. Dual luciferase reporter assay was used to detect miR-515-5p and circ-LRP6 relationship. The circ-LRP6 interference expression vector, miR-515-5p overexpression vector, miR-515-5p suppression expression vector and circ-LRP6 interference expression vector were transfected into the SKOV3 cells. Western blotting was used to detect the expressions of Cyclin D1, matrix metalloproteinase (MMP) 2, MMP9, interleukin 6 receptor (IL-6R) protein expression. CCK-8 was used to detect the activity of SKOV3 cells, and the Transwell chamber was used to detect the migration and invasion of SKOV3 cells.Results Compared with adjacent tissues and HUM-CELL-0088 cells, the circ-LRP6 expression level was increased, while the miR-515-5p expression level was decreased in ovarian cancer tissues and SKOV3, OVCAR3, A2780 cell lines (P<0.05). The dual luciferase reporter assay verified that circ-LRP6 could target to and negatively regulate the expression of miR-515-5p. Inhibition of circ-LRP6 expression or overexpression of miR-515-5p could suppress the expressions of Cyclin D1, MMP2, MMP9 and IL-6R, attenuate the cell viability, and reduce the number of migrating and invasive cells (P<0.05). At the same time, low expression of miR-515-5p could up-regulate the expression levels of Cyclin D1, MMP2, MMP9 and IL-6R in SKOV3 cells, enhance the cell viability, and increase the number of migrating and invasive cells (P<0.05).Conclusion Inhibition of circ-LRP6 expression may inhibit the proliferation, migration and invasion of ovarian cancer SKOV3 cells by up-regulating miR-515-5p and then inhibiting IL-6.