七氟烷调控miR-96-5p/SIK1轴对胃癌细胞恶性生物学行为的影响

Impacts of sevoflurane on malignant biological behavior of gastric cancer cells by regulating miR-96-5p/SIK1 axis

目的 探讨七氟烷调控微小RNA(miR)-96-5p/盐诱导激酶1(SIK1)轴表达对胃癌(GC)细胞增殖、侵袭和凋亡的影响。方法 以0、1%、2%、3%、4%、5%v/v浓度的七氟烷处理GC细胞SGC7901细胞,CCK-8法检测SGC7901细胞增殖情况;将SGC7901细胞随机分为对照组(正常培养)、七氟烷组(3%v/v七氟烷干预)、七氟烷+miR-NC组(转染miR-NC后再以3%v/v浓度的七氟烷干预48小时)、七氟烷+miR-96-5p mimics组(转染miR-96-5p mimics后,以3%v/v浓度的七氟烷干预48小时),RT-qPCR检测各组细胞中miR-96-5p表达;流式细胞术检测细胞凋亡;划痕实验检测细胞迁移;Transwell小室实验检测细胞侵袭;Western blot检测细胞磷酸化盐诱导激酶1(SIK1)、基质金属蛋白酶(MMP)-2、MMP-9的表达;双荧光素酶报告基因实验验证miR-96-5p和SIK1的靶向关系。结果 与0浓度组相比,SGC7901细胞增殖抑制率在1%、2%、3%、4%、5%v/v浓度七氟烷处理下以剂量依赖性的方式显著增加(P<0.05),选取3%v/v浓度七氟烷进行后续实验;与对照组比较,七氟烷组细胞miR-96-5p表达水平、迁移、侵袭以及MMP-2、MMP-9蛋白表达水平显著降低,凋亡率和SIK1蛋白表达水平显著增加(P<0.05);激活miR-96-5p表达减弱了七氟烷对SGC7901细胞迁移和侵袭的抑制能力,降低细胞凋亡率(P<0.05);双荧光素酶报告基因结果表明miR-96-5p可靶向调节SIK1表达。结论 七氟烷可能通过下调miR-96-5p、上调SIK1抑制GC SGC7901细胞的增殖、迁移和侵袭,促进细胞凋亡。

Objective The objective of this study was to investigate the impacts of sevoflurane on the proliferation,invasion and apoptosis of gastric cancer(GC)cells by regulating the expression of microRNA(miR)-96-5p/salt induced kinase 1(SIK1)axis.Methods GC cell SGC7901 cells were treated with sevoflurane of 0%,1%,2%,3%,4%,5%v/v concentration,the proliferation of SGC7901 cells was detected by CCK-8 method;SGC7901 cells were randomly grouped into three groups:control group(Normal culture),sevoflurane group(3%v/v sevoflurane intervention),sevoflurane+miR-NC group(Transfected with miR-NC and then treated with sevoflurane at 3%v/v concentration for 48 h),sevoflurane+miR-96-5p mimics group(After transfection with miR-96-5p mimics,intervention was performed at 3%v/v concentration of sevoflurane for 48 h),RT-qPCR was applied to detect the expression of miR-96-5p in cells of each group;flow cytometry was applied to detect apoptosis;scratch test was applied to detect cell migration;Transwell cell test was applied to detect cell invasion;Western blot was applied to detect the expression of phosphorylate-induced kinase 1(SIK1),matrix metalloproteinase-2(MMP-2)and MMP-9;double luciferase reporter gene experiment was applied to verify the targeting relationship between miR-96-5p and SIK1.Results Compared with the 0%concentration group,the inhibition rate of SGC7901 cell proliferation was obviously increased in a dose-dependent manner under the treatment of 1%,2%,3%,4%,and 5%v/v concentration of sevoflurane(P<0.05),the 3%v/v concentration of sevoflurane was selected for subsequent experiments;compared with the control group,the expression level of miR-96-5p,migration,invasion,and the expression levels of MMP-2 and MMP-9 proteins in sevoflurane group were obviously decreased,the apoptosis rate and the expression level of SIK1 protein were obviously increased(P<0.05);activation of miR-96-5p expression weakened the inhibition of sevoflurane on the migration and invasion of SGC7901 cells,and decreased the apoptosis rate(P<0.05);the results of double luciferase reporter gene showed that miR-96-5p could target the expression of SIK1.Conclusion Sevoflurane may inhibit the proliferation,migration and invasion of GC SGC7901 cells and promote cell apoptosis by down-regulating miR-96-5p and up-regulating SIK1.