miR-486-3p对乳腺癌细胞MCF-7凋亡的调控作用研究

Regulation of miR-486-3p on Cell Apoptosis of Breast Cancer Cell Line MCF-7

为探讨miR-486-3p对乳腺癌细胞MCF-7凋亡的调控作用,采用qRT-PCR法和Western blot法测定24例乳腺癌组织和癌旁正常组织miR-486-3p和凋亡相关蛋白的表达水平;采用qRT-PCR法测定乳腺癌细胞MCF-7、HBL101和正常乳腺细胞MCF10A中miR-486-3p的表达水平。将MCF-7、HBL101和MCF10A细胞分为正常对照组、模拟物对照组、miR-486-3p模拟物组、抑制物对照组和miR-486-3p抑制物组,各组细胞转染后进行培养,采用CCK-8法测定各组细胞增殖情况,采用细胞划痕法测定MCF-7和MCF10A细胞的迁移情况,采用Transwell法和流式细胞术测定各组细胞侵袭和凋亡情况,采用qRT-PCR和Western blot法测定凋亡相关蛋白mRNA和蛋白的表达水平。该研究得出乳腺癌组织中miR-486-3p表达水平较癌旁正常组织显著降低(P<0.05),乳腺癌组织中Bcl-2蛋白表达水平较癌旁正常组织显著增加(P<0.05),而Bax和Caspase-3蛋白表达水平较癌旁正常组织显著降低(P<0.05)。乳腺癌细胞MCF-7和HBL101中miR-486-3p表达水平较正常乳腺细胞MCF10A显著降低(P<0.05),其中以乳腺癌细胞MCF-7中miR-486-3p表达水平最低。miR-486-3p模拟物组乳腺癌细胞MCF-7和HBL101中miR-486-3p的表达水平较模拟物对照组显著升高(P<0.05),miR-486-3p抑制物组miR-486-3p的表达水平较抑制物对照组显著降低(P<0.05)。miR-486-3p模拟剂组乳腺癌MCF-7和HBL101细胞在24 h、48 h和72 h时的吸光度值较模拟物对照组显著降低(P<0.05),而miR-486-3p抑制物组在24 h、48 h和72 h时吸光度值较抑制物对照组显著升高(P<0.05)。miR-486-3p模拟剂组乳腺癌MCF-7细胞划痕宽度显著宽于模拟物对照组(P<0.05),而miR-486-3p抑制物组乳腺癌MCF-7细胞划痕宽度显著窄于抑制物对照组(P<0.05)。miR-486-3p模拟剂组乳腺癌MCF-7细胞穿膜细胞数量较模拟剂对照组显著降低(P<0.05),而miR-486-3p抑制物组穿膜细胞数量较抑制物对照组显著升高(P<0.05)。miR-486-3p模拟剂组乳腺癌MCF-7细胞凋亡数量较模拟物对照组显著升高(P<0.05),而miR-486-3p抑制物组细胞凋亡数量较抑制物对照组显著降低(P<0.05)。miR-486-3p模拟剂组乳腺癌MCF-7细胞中Bcl-2 mRNA和蛋白表达水平较模拟物对照组显著降低(P<0.05),Bax和Caspase-3表达水平显著升高(P<0.05),miR-486-3p抑制物组Bcl-2表达水平较抑制物对照组显著升高(P<0.05),Bax和Caspase-3表达水平显著降低(P<0.05)。总之,miR-486-3p是乳腺癌的抑癌基因,可能通过调节凋亡相关蛋白Bcl-2、Bax和Caspase-3 mRNA及蛋白的表达,而对乳腺癌细胞的凋亡起促进作用。

To investigate the regulatory effect of miR-486-3p on apoptosis in breast cancer cells MCF-7 and its related mechanism,the expressions of miR-486-3p and apoptosis-related proteins in breast cancer tissues and adjacent normal tissues from 24 breast cancer patients were examined employing qRT-PCR and Western blot,respectively.miR-486-3p expression in breast cancer cells MCF-7,HBL101 and normal breast cells MCF10A was assayed by qRT-PCR.The groups of normal control,mimics control and miR-486-3p mimics,inhibitor control and miR-486-3p inhibitor were set in the experiments.After cell culture and transfection,cell proliferation was tested by CCK-8.Cell migration and invasion were assessed by wound healing and transwell assays.Cell apoptosis was evaluated by flow cytometry.The mRNA and protein expressions of Bcl-2,Bax and Caspase 3 were measured by qRT-PCR and Western blot.The results showed that in normal controls,compared with adjacent normal tissues,miRNA-486-3p expression in breast cancer tissues was significantly decreased (P<0.05).Bcl-2 protein level was remarkably increased (P<0.05),but Bax and caspase-3 protein levels were markedly reduced in breast cancer tissues (P<0.05).Compared with normal breast cells MCF10A,miR-486-3p expression was obviously reduced in breast cancer cells MCF-7 and HBL101 (P<0.05).miR-486-3p expression was lowest in MCF-1 cells.In the miRNA-486-3p mimic group,miR-486-3p expression was higher compared to the control group in MCF-7 and HBL101 cells (P<0.05).The optical density values of MCF-7 and HBL101 cells were significantly declined at 24 h,48 h and 72 h (P<0.05).Moreover,MCF-7 cells had wider scratches (P<0.05).There were fewer transmembrane cells (P<0.05) and more apoptotic cells of MCF-7 cells (P<0.05).Bcl-2 level in MCF-7 cells was lower (P<0.05),but the levels of Bax and Caspase-3 were higher (P<0.05).In the miR-486-3p inhibitor group,miR-486-3p expression was decreased compared to the normal group in MCF-7 and HBL101 cells (P<0.05).The optical density values of MCF-7 and HBL101 cells were significantly higher at 24 h,48 h and 72 h (P<0.05).Additionally,MCF-7 cells had narrower scratches (P<0.05).There were more transmembrane cells (P<0.05) and fewer apoptotic cells of MCF-7 cells (P<0.05).Furthermore,Bcl-2 expression was obviously increased (P<0.05),but the levels of Bax and Caspase-3 were reduced in MCF-7 cells (P<0.05).Overall,these results indicated that miR-486-3p served as an antioncogene in breast cancer that might promote breast cancer apoptosis by regulating the mRNA and protein levels of Bcl-2,Bax and Caspase-3 related to apoptosis.