黄芩苷联合奥沙利铂调控miR-433-3p/SRC对胃癌细胞增殖和侵袭的影响

Mechanism of regulation of miR-433-3p/SRC by baicalin combined with oxaliplatin on proliferation and invasion of gastric cancer cells

目的探究黄芩苷联合奥沙利铂调控miR-433-3p/SRC对胃癌细胞增殖和侵袭的影响。方法将SGC-7901细胞分为对照组、黄芩苷(100、200、300μmol·L^(-1))组、奥沙利铂(33μmol·L^(-1))组,联合用药(300μmol·L^(-1)黄芩苷+33μmol·L^(-1)奥沙利铂)组,miR-433-3p组、miR-NC组、anti-miR-433-3p组、anti-miR-NC组、pcDNA-SRC组、si-SRC组,miR-433-3p+联合用药组、anti-miR-433-3p+联合用药组、pcDNA-SRC+联合用药组、si-SRC+联合用药组、miR-433-3p+pcDNASRC+联合用药组。CCK-8检测细胞增殖能力;Transwell小室法检测细胞侵袭能力;流式细胞仪检测细胞凋亡率;实时荧光定量PCR(qRT-PCR)检测细胞中miR-433-3p表达;双荧光素酶报告检测miR-433-3p和SRC的靶向关系;Western blotting检测细胞中SRC蛋白表达。结果与对照组相比,黄芩苷(100、200、300μmol·L^(-1))组、奥沙利铂组、联合用药组细胞增殖抑制率、凋亡率和miR-433-3p表达显著增加(P<0.05),细胞侵袭数目、SRC蛋白表达显著减少(P<0.05);与黄芩苷300μmol·L^(-1)组、奥沙利铂组相比,联合用药组细胞增殖抑制率、凋亡率显著增加,细胞侵袭数目显著减少;与黄芩苷300μmol·L^(-1)组比较,miR-433-3p表达显著增加(P<0.05);与奥沙利铂组比较,SRC蛋白表达显著减少(P<0.05)。与对照组比较,miR-433-3p组细胞中miR-433-3p表达显著升高,SRC蛋白表达显著降低,anti-miR-433-3p组细胞中miR-433-3p表达显著降低,SRC蛋白表达显著升高(P<0.05);miR-433-3p+联合用药组细胞增殖抑制率和凋亡率显著高于联合用药组,细胞侵袭数目显著少于联合用药组(P<0.05),anti-miR-433-3p+联合用药组细胞增殖抑制率和凋亡率显著低于联合用药组,细胞侵袭数目显著多于联合用药组(P<0.05)。miR-433-3p组SRC-WT荧光素酶活性显著低于miR-NC组(P<0.05)。si-SRC组细胞中SRC蛋白表达显著低于对照组(P<0.05),pcDNA-SRC组细胞中SRC蛋白表达显著高于对照组(P<0.05);与pcDNA-SRC组相比,miR-433-3p+pcDNA-SRC组细胞中SRC蛋白表达显著降低(P<0.05)。si-SRC+联合用药组细胞增殖抑制率和凋亡率显著高于联合用药组,细胞侵袭数目显著少于联合用药组(P<0.05),pcDNA-SRC+联合用药组细胞增殖抑制率和凋亡率显著低于联合用药组,细胞侵袭数显著高于联合用药组(P<0.05)。与pcDNA-SRC+联合用药组相比,miR-433-3p+pcDNA-SRC+联合用药组细胞增殖抑制率和凋亡率显著降低(P<0.05),细胞侵袭数目显著升高(P<0.05)。结论黄芩苷联合奥沙利铂可通过miR-433-3p靶向调控SRC抑制胃癌细胞的增殖和侵袭,诱导胃癌细胞凋亡。

Objective To investigate the effect of baicalin combined with oxaliplatin to regulate miR-433-3p/SRC on the proliferation and invasion of gastric cancer cells.Methods SGC-7901 cells were divided into control group,baicalein(100,200,and 300μmol·L^(-1))group,oxaliplatin group(33μmol·L^(-1)),and combination therapy group(300μmol·L^(-1) baicalein and 33μmol·L^(-1) oxaliplatin).They were divided into miR-433-3p group,miR-NC group,anti-miR-433-3p group,anti-miR-NC group,pcDNA-SRC group,si-SRC group,miR-433-3p+combination therapy group,anti-miR-433-3p+combination therapy group,pcDNA-SRC+combination therapy group,si-SRC+combination therapy group,and miR-433-3p+pcDNA-SRC+combination therapy group.CCK-8 method was used to detect cell proliferation ability,transwell assay was used to detect cell invasion ability,detection of cell apoptosis rate using flow cytometry,real time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of miR-433-3p in cells,double luciferase assay was used to detect the targeting relationship between miR-433-3p and SRC,and Western blotting was used to detect the expression of SRC protein in cells.Results Compared with control group,the cell proliferation inhibition rate,apoptosis rate and miR-433-3p expression increased and the cell invasion number and SRC protein expression decreased in baicalin(100,200,and 300μmol·L^(-1)),oxaliplatin and combination therapy groups(P<0.05).Compared with the oxaliplatin group and baicalin 300μmol·L^(-1) group,the combination therapy group showed a significant increase in cell proliferation inhibition rate and apoptosis rate,while the number of cell invasions decreased significantly.Compared with baicalin 300μmol·L^(-1) group,the expression of miR-433-3p significantly increased in combination therapy group(P<0.05)Compared with the oxaliplatin group,the expression of SRC protein was significantly reduced in combination therapy group(P<0.05).Compared with control group,miR-433-3p expression was elevated and SRC protein expression was decreased in cells of miR-433-3p group,and miR-433-3p expression was decreased and SRC protein expression was increased in cells of anti-miR-433-3p group(P<0.05).The cell proliferation inhibition rate and apoptosis rate of the miR-433-3p+combination therapy group were significantly higher than those of the combination therapy group,and the number of cell invasions was significantly lower than that of the combination therapy group(P<0.05).The cell proliferation inhibition rate and apoptosis rate of the anti miR-433-3p+combination therapy group were significantly lower than those of the combination therapy group,and the number of cell invasions was significantly higher than that of the combination therapy group(P<0.05).The luciferase activity of SRC-WT in the miR-433-3p group was significantly lower than that in the miR-NC group(P<0.05).The expression of SRC protein in si-SRC group cells was significantly lower than that in the control group(P<0.05),while the expression of SRC protein in pcDNA-SRC group cells was significantly higher than that in the control group(P<0.05).Compared with the pcDNA-SRC group,the expression of SRC protein in the miR-433-3p+pcDNASRC group cells was significantly reduced(P<0.05).The cell proliferation inhibition rate and apoptosis rate of the si-SRC+combination therapy group were significantly higher than those of the combination therapy group,and the number of cell invasions was significantly lower than that of the combination therapy group(P<0.05).The cell proliferation inhibition rate and apoptosis rate of the pcDNA-SRC+combination therapy group were significantly lower than those of the combination therapy group,and the number of cell invasions was significantly higher than that of the combination therapy group(P<0.05).Compared with the pcDNASRC+combination group,the miR-433-3p+pcDNA-SRC+combination group showed a significant decrease in cell proliferation inhibition rate and apoptosis rate(P<0.05),and a significant increase in cell invasion number(P<0.05).Conclusion Baicalin combined with oxaliplatin can inhibit the proliferation and invasion of gastric cancer cells through miR-433-3p-targeted regulation of SRC and induce apoptosis of gastric cancer cells.