miR-370-3p靶向HDAC4调节卵巢癌SKOV3细胞的生长和代谢研究

MicroRNA-370-3p targets histone deacetylase 4 to regulate growth and metabolism of ovarian cancer SKOV3 cells

目的:研究miR-370-3p对卵巢癌(ovarian cancer,OC)细胞的生长和代谢的作用及机制。方法:选取2017年2月至2019年2月23例于郑州人民医院行OC切除术患者的组织标本,RT-qPCR检测OC组织和细胞中miR-370-3p和组蛋白脱乙酰酶4(his⁃tone deacetylase 4,HDAC4)mRNA的相对表达量,双荧光素酶试验检测miR-370-3p与HDAC4的靶向关系,蛋白印迹法检测Ki-67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、裂解caspase-3、Bax、HDAC4蛋白表达水平,CCK-8检测细胞增殖,流式检测细胞凋亡率,Seahorse XF24分析仪分析细胞糖酵解能力和线粒体功能,建立移植瘤动物模型,测定移植瘤体积和重量,免疫组织化学法检测Ki-67和caspase-3表达水平。结果:在OC组织和细胞中miR-370-3p低表达,HDAC4高表达。miR-370-3p靶向下调HDAC4表达。miR-370-3p过表达显著减少SKOV3细胞增殖,增加细胞凋亡,下调PCNA和Ki-67蛋白表达,上调裂解caspase-3和Bax蛋白表达,降低糖酵解速率和糖酵解能力,升高基础呼吸速率和最大呼吸速率。在小鼠体内,miR-370-3p过表达减小移植瘤体积和重量,增加Ki-67阳性细胞比率,减少caspase-3阳性细胞比率,降低糖酵解速率和糖酵解能力,升高基础呼吸速率和最大呼吸速率。结论:miR-370-3p通过靶向下调HDAC4来抑制OC细胞的生长和代谢。

Objective:To investigated the effects of microRNA(miR)-370-3p on growth and metabolism of ovarian cancer(OC)SKOV3 cells and aimed to elucidate the underlying mechanisms.Methods:Expression of miR-370-3p and histone deacetylase 4(HDAC4)mRNA in OC tissues and cells was analyzed using reverse transcription-quantitative polymerase chain reaction(RT-qPCR).The relationship between miR-370-3p and HDAC4 was assessed using dual luciferase assay.Relative expression of Ki-67,proliferating cell nuclear antigen(PCNA),cleaved caspase-3,Bax,and HDAC4 was analyzed using Western blot.Further,cell proliferation was detected using Cell Counting Kit-8 assay,and apoptosis rates were assessed using flow cytometry.Cellular glycolysis capacity and mitochondrial functions were analyzed using a Seahorse XF24 analyzer.Furthermore,xenograft models were established by transplanting SKOV3 cells into,in which tumor volume and weight were recorded,and expression of Ki-67 and caspase-3 was analyzed using immunohistochemistry.Results:MiR-370-3p and HDAC4 showed low and high expression,respectively,in the OC tissues and cells.Further,miR-370-3p overexpression was found to downregulate HDAC4 expression.It also reduced cell proliferation,glycolysis rate,glycolysis capacity,and PCNA and Ki-67 protein expression,but increased apoptosis,basal and maximum respiration rates,and cleaved caspase-3 and Bax protein expression;these results were statistically significant at P<0.01.In vivo,miR-370-3p overexpression reduced the size and weight of SKOV3 cell-induced tumors,proportion of caspase-3-positive cells,glycolysis rate,and glycolysis capacity,but increased the proportion of Ki-67-positive cells and basal and maximum respiration rates;these results were also statistically significant at P<0.01.Conclusions:MiR-370-3p overexpression can inhibit OC cell growth by downregulating HDAC4 and alter OC cell metabolism.