鸡miR-17-92基因簇靶基因ZFPM2的鉴定及功能分析

发布时间：2018-02-28 19:21

本文关键词： miR--基因簇 ZFPM 鸡前脂肪细胞细胞增殖　出处：《遗传》2017年04期 　论文类型：期刊论文

【摘要】：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(P0.01);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(P0.01或P0.05)。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(P0.01);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(P0.01或P0.05),但这些抑制剂对突变型ZFPM2报告基因的活性无明显影响。q RT-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(P0.01或P0.05)。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。
[Abstract]:MiR-17-92 gene cluster plays an important role in many physiological and pathological processes in mammals. In order to reveal the mechanism of miR-17-92 gene cluster promoting the proliferation of preadipocytes, CCK-8 cell proliferation assay was used to analyze the effect of interfering ZFPM2 on preadipocytes. Interfering with ZFPM2 could significantly promote the proliferation of preadipocytes, and in accordance with the results of CCK-8 analysis, interfering with ZFPM2 could significantly increase the expression of mRNA in the cell proliferation marker gene PCNA1Ki67, Cyclin D1. Further, the bioinformatics analysis of chicken ZFPM2 gene was carried out. It was found that there were two potential binding sites in miR-17-92 gene cluster 4 members of miR-17-92 gene cluster, miR-17-5, miR-20aanmiR-19a and miR-19b, in order to verify whether the miR-17-92 gene cluster was targeted to chicken ZFPM2 gene. The luciferase reporter gene vector psi-CHECK2-ZFPM2-3 UTR-WT) and its mutant luciferase reporter gene vector, psi-CHECK2-ZFPM2-3UUTR-MUTN, were constructed. Overexpression of mi-17-92 gene cluster could significantly inhibit the activity of wild type ZFPM2 reporter gene (P0.01), and transfection of miR-17-5 pnmiR-20a and miR-19a could significantly increase the activity of wild type ZFPM2 reporter gene (P0.01 or P0.05N), but these inhibitors could significantly increase the activity of mutant ZFPM2 reporter gene. There was no significant effect on activity. Q RT-PCR analysis showed that miR-17-5 pmmiR-19a and inhibitor of miR-20a could significantly increase the expression level of mRNA of endogenous ZFPM2 gene (P0.01 or P0.05A). Although the difference is not significant, However, both the inhibitors of miR-17-5p and miR-19a tended to reduce the proliferation of ZFPM2 interference fragments. The results showed that the miR-17-5pmiR-20aanmiR-19a and miR-19b targeted the ZFPM2 miR-17-92 gene cluster to promote the expression of ZFPM2 gene, at least partly by inhibiting the expression of ZFPM2 gene. Proliferation of preadipocytes.
【作者单位】：农业部鸡遗传育种重点实验室;黑龙江省普通高等学校动物遗传育种与繁殖重点实验室;东北农业大学动物科学技术学院;
【基金】：国家自然科学基金项目(编号:31372299) 农业部产业体系(编号:CARS-42)资助~~
【分类号】：S831

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