鸡miR-17-92基因簇靶基因ZFPM2的鉴定及功能分析

摘要

The miR-17-92 cluster plays important roles in a variety of physiological and pathological processes in mammals. Previously, we showed that miR-17-92 cluster promotes chicken preadipocyte proliferation; however, the mechanism for its action is unknown. In order to explore the mechanism by which miR-17-92 cluster promotes chicken preadipocyte proliferation, CCK8 proliferation assay was performed to determine the effect of ZFPM2 knockdown on chicken preadipocyte proliferation. The results showed that ZFPM2 knockdown significantly promoted chicken preadipocyte proliferation (P<0.01). Consistent with the CCK8 results, the mRNA levels of cell proliferation marker genes, i.e., Cyclin D1, PCNA and Ki67, were markedly increased in the si-ZFPM2-transfected preadipocytes (P<0.01 or P<0.05). Bioinformatics analysis showed that there were two potential miRNA binding sites for the four individual members of miR-17-92 cluster in the ZFPM23′UTR, one for miR-17-5p and miR-20a and the other for miR-19a and miR-19b. To test whether ZFPM2 is a target for the miR-17-92 cluster, the ZFPM23′UTR reporter (psi-CHECK2-ZFPM2-3′UTR-WT) and its mutant reporter (psi-CHECK2-ZFPM2-3′UTR-MUT) were constructed. Reporter assays showed that overexpression of miR-17-92 cluster significantly inhibited the luciferase reporter activity of psi-CHECK2-ZFPM2-3′UTR-WT (P<0.01), as compared with control vector (empty pcDNA3.1). Transfection of miR-17-5p, miR-19a and miR-20a inhibitors increased the reporter activities of psi-CHECK2-ZFPM2-3′UTR-WT (P<0.01 or P<0.05). In contrast, transfection of miR-17-5p, miR-19a, and miR-20a inhibitors had no obvious effect on reporter activity of psi-CHECK2-ZFPM2-3′UTR-MUT. Further qRT-PCR analysis showed that miR-17-5p, miR-20a and miR-19a inhibitors significantly elevated the endogenous ZFPM2 mRNA expression (P<0.01 or P<0.05). Cotransfection of either miR-17-5p or miR-19a inhibitor and siZFPM2 showed that both inhibitors tended to reduce only slightly the promoting effect of siZFPM2 on chicken preadipocyte proliferation. Taken together, these data demonstrated that ZFPM2 is a target of miR-17-5p, miR-20a, miR-19a, and miR-19b, and that miR-17-92 cluster promotes chicken preadipocyte proliferation at least in part by targeting ZFPM2 and inhibiting its expression.%miR-17-92基因簇在哺乳动物的许多生理和病理过程中发挥重要作用.本实验室前期研究发现,miR-17-92基因簇促进鸡前脂肪细胞的增殖,但其作用机制尚不清楚.为了揭示miR-17-92基因簇促进鸡前脂肪细胞增殖的作用机制,本研究采用CCK-8细胞增殖检测方法分析干扰ZFPM2对前脂肪细胞的影响,结果发现,干扰ZFPM2能显著促进鸡前脂肪细胞的增殖(P<0.01);与CCK-8分析结果相一致,干扰ZFPM2可致使细胞增殖标志基因PCNA、Ki67、Cyclin D1的mRNA表达量明显升高(P<0.01或P<0.05).进一步对鸡ZFPM2基因进行生物信息学分析,发现该基因mRNA的3′UTR有两个区域存在miR-17-92基因簇4个成员(miR-17-5p、miR-20a、miR-19a及miR-19b)的潜在结合位点.为验证miR-17-92基因簇是否靶作用于鸡ZFPM2基因,构建了鸡ZFPM2基因3′UTR区的荧光素酶报告基因载体(野生型)(psi-CHECK2-ZFPM2-3′UTR-WT)及其突变型的荧光素酶报告基因载体(psi-CHECK2-ZFPM2-3′UTR-MUT).报告基因活性分析显示,过表达mi-17-92基因簇能极显著地抑制野生型ZFPM2的报告基因活性(P<0.01);转染miR-17-5p、miR-20a及miR-19a的抑制剂均能显著地提高野生型ZFPM2报告基因的活性(P<0.01或P<0.05),但这些抑制剂对突变型ZFPM2报告基因的活性无明显影响.qRT-PCR分析发现,miR-17-5p、miR-19a及miR-20a的抑制剂能显著提高内源性ZFPM2基因mRNA的表达水平(P<0.01或P<0.05).共转染分析发现,尽管差异不显著,但miR-17-5p和miR-19a的抑制剂均倾向于降低ZFPM2干扰片段的促细胞增殖作用.本研究结果表明:miR-17-92基因簇成员miR-17-5p、miR-20a、miR-19a及miR-19b靶作用于ZFPM2;miR-17-92基因簇至少部分通过抑制ZFPM2基因表达从而促进鸡前脂肪细胞的增殖.