miR-124-3p与miR-506-3p在蛋白C降低中的作用及机制研究

目的探讨miR-124-3p和miR-506-3p在蛋白C(protein C,PROC)降低中的作用及机制。方法将PROC 3'-UTR野生型(PROCwt)和PROC 3'-UTR突变型(PROCmut)克隆构建到含萤火虫荧光素酶报告质粒中(Luc-PROCwt和Luc-PROCmut),利用双荧光素酶报告体系检测相对荧光素酶酶活性,明确miR-124-3p和miR-506-3p与PROC的靶基因关系及其紧密性。将miR-124-3p mimics和miR-506-3p mimics及其抑制基因(inhibitor)分别转染至HL-7702细胞,检测对照组(NC)、miR-506-3p组、miR-124-3p组、miR-506-3p抑制组和miR-124-3p抑制组的细胞增殖率,PROC mRNA表达水平,PROC、COX-2、Bcl-2和Bax蛋白表达水平和细胞凋亡水平,并采用单因素和双因素方差分析比较各组间的差异。结果双荧光素酶报告体系检测显示,NC+Luc-PROCwt组、miR-506-3p+Luc-PROCwt组、miR-124-3p+Luc-PROCwt组、NC+Luc-PROCmut组、miR-506-3p+Luc-PROCmut组和miR-124-3p+Luc-PROCmut组荧光素酶活性分别为6.98±0.07、2.01±0.05、2.67±0.06、8.13±0.26、6.91±0.14和8.41±0.13。与NC+Luc-PROCwt组相比,miR-506-3p+Luc-PROCwt组与miR-124-3p+Luc-PROCwt组的荧光素酶活性均明显降低,差异均有统计学意义(P均<0.01);与NC+Luc-PROCmut组相比,miR-506-3p+Luc-PROCmut组荧光素酶活性明显降低,miR-124-3p+Luc-PROCmut组荧光素酶活性明显升高,组间比较,差异亦均有统计学意义(P<0.01和P=0.0181)。CCK-8检测显示,NC组、miR-506-3p组、miR-124-3p组、miR-506-3p抑制组和miR-124-3p抑制组96 h时细胞增殖率分别为0.506±0.016、0.323±0.021、0.329±0.011、0.570±0.007和0.562±0.007。与NC组相比,miR-506-3p组和miR-124-3p组细胞增殖率均有所下降,组间差异均有统计学意义(P均<0.01);miR-506-3p抑制组和miR-124-3p抑制组细胞增殖率均有所上升,组间差异亦均有统计学意义(P均<0.01)。RT-PCR检测显示,NC组、miR-506-3p组、miR-124-3p组、miR-506-3p抑制组和miR-124-3p抑制组PROC mRNA表达量分别为1.00±0.08、0.31±0.09、0.34±0.04、1.73±0.28和1.75±0.36。与NC组相比,miR-506-3p组和miR-124-3p组PROC mRNA表达水平均明显降低,组间差异均有统计学意义(P=0.0026和0.0032);miR-506-3p抑制组和miR-124-3p抑制组PROC mRNA表达水平均明显上升,组间差异亦均有统计学意义(P=0.0018和0.0016)。Western-blot检测显示,与NC组相比,miR-506-3p组与miR-124-3p组PROC与Bcl-2蛋白表达水平下调,COX-2和Bax蛋白表达水平上调;miR-506-3p抑制组和miR-124-3p抑制组PROC与Bcl-2蛋白表达水平上调,COX-2和Bax蛋白表达水平下调。Annexin V-FITC/PI检测显示,NC组、miR-506-3p组、miR-124-3p组、miR-506-3p抑制组和miR-124-3p抑制组细胞凋亡率分别为(10.27±0.57)%、(21.50±1.44)%、(13.52±0.40)%、(1.12±0.08)%和(7.63±0.40)%。与NC组相比,miR-506-3p组和miR-124-3p组细胞凋亡率均明显升高,组间差异均有统计学意义(P均<0.01);miR-506-3p抑制组和miR-124-3p抑制组细胞凋亡率均明显降低,组间差异亦均有统计学意义(P均<0.01)。结论PROC是miR-506-3p和miR-124-3p的靶基因,miR-506-3p和miR-124-3p可能通过抑制肝细胞增殖、促进肝细胞凋亡和抑制PROC mRNA表达引发PROC蛋白表达水平降低。

miR-124-3p and miR-506-3p regulated the expression of protein C by targeting PROC

Objective To explore the role and mechanism of miR-124-3p and miR-506-3p in the deficiency of protein C(PROC).Methods The wild-type(PROCwt)and mutant-type(PROCmut)of PROC 3'-UTR were cloned and constructed into luciferase reporter plasmids(Luc-PROCwt&Luc-PROCmut).The relative luciferase activity was detected by double luciferase reporter system and the targeting relationship between miR-124-3p/miR-506-3p and PROC clarified.And miR-124-3p mimics,miR-506-3p mimics and inhibitor(suppressor gene)were transfected into HL-7702 cells.Rates of cell proliferation and apoptosis,level of PROC mRNA and protein levels of PROC,COX-2,Bcl-2 and Bax in control group(NC),miR-506-3p,miR-124-3p,miR-506-3p inhibitor and miR-124-3p inhibitor groups were compared.Results The luciferase activity was significantly lower in miR-506-3p+Luc PROCwt/miR-124-3p+Luc PROCwt group than that in NC+Luc PROCwt group(2.01±0.05 vs.6.98±0.07,2.67±0.06 vs.6.98±0.07,P<0.01).As compared with NC+Luc PROCmut group,the luciferase activity of miR-506-3p+Luc PROCmut group decreased markedly(6.91±0.14 vs.8.13±0.26,P<0.01)while that of miR-124-3p+Luc PROCmut group increased(8.41±0.13 vs.8.13±0.26,P<0.05).Cell proliferative rate of miR-506-3p/miR-124-3p group was significantly lower than that of NC group at 96h in CCK-8 test(0.323±0.021 vs.0.506±0.016,0.329±0.011 vs.0.506±0.016,P<0.01);Cell proliferative rate of miR-506-3p/miR-124-3p inhibitor group was significantly higher than that of NC group at 96h in CCK-8 test(0.570±0.007 vs.0.506±0.016,0.562±0.007 vs.0.506±0.016,P<0.01).As compared with NC group,the levels of PROC and Bcl-2 was down-regulated and the levels of COX-2 and Bax became up-regulated in miR-506-3p and miR-124-3p groups.However,the level of PROC/Bcl-2 was up-regulated while that of COX-2/Bax down-regulated in miR-506-3p/miR-124-3p inhibitor group.In RT-PCR test,the level of PROC mRNA of miR-506-3p/miR-124-3p group was significantly lower than that of NC group(0.31±0.09 vs.1.00±0.08,P=0.0026;0.34±0.04 vs.1.00±0.08,P=0.0032);the level of PROC mRNA of miR-506-3p/miR-124-3p inhibitor group was significantly higher than that of NC group(1.73±0.28 vs.1.00±0.08,P=0.0018;1.75±0.36 vs.1.00±0.08,P=0.0016).In Annexin V-FITC/PI test,cell apoptotic rate of miR-506-3p/miR-124-3p groups was significantly higher than that of NC group(21.50±1.44)%vs.(10.27±0.57)%,(13.52±0.40)%vs.(10.27±0.57)%,P<0.01],cell apoptotic rate of miR-506-3p/miR-124-3p inhibitor group was significantly lower than that of NC group(1.12%±0.08%vs.10.27%±0.57%,7.63%±0.40%vs.10.27%±0.57%,P<0.01).Conclusions miR-506-3p and miR-124-3p may lead to a deficiency of protein C by targeting the gene of protein C through inhibiting the proliferation of hepatocytes,promoting the apoptosis of hepatocytes and repressing the expression of PROC mRNA.