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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(n 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(n P<0.01和n P＝0.018 1)。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(n 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(n 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(n P＝0.002 6和0.003 2)；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(n P＝0.001 8和0.001 6)。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(n 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(n P均<0.01)。n 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。“,”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, n 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,n P<0.01) while that of miR-124-3p+ Luc PROCmut group increased (8.41±0.13 vs. 8.13±0.26,n 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,n 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,n 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,n P=0.0026; 0.34±0.04 vs. 1.00±0.08, n 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, n P=0.0018; 1.75±0.36 vs. 1.00±0.08, n 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)%, n 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%,n P<0.01).n 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.