靶向猪CLTC基因miRNA的预测与验证

Prediction and validation of miRNA targeting the porcine CLTC gene

网格蛋白介导的胞吞是病毒侵入细胞的重要途径,网格蛋白重链(clathrin heavy chain,CLTC)是形成网格蛋白小窝结构的重要组成部分。针对CLTC基因的转录后调控特别是调控猪Sus scrofa CLTC的miRNA目前还不太清楚。本研究旨在筛选出调控猪CLTC基因的miRNA。利用生物信息学方法预测出6个靶向猪CLTC基因的miRNA,将猪CLTC基因3′UTR克隆至双荧光素酶报告基因载体psi CHECK2中获得双荧光素酶报告基因重组载体psi CHECK2-CLTC-3′UTR。将预测得到的miRNA分别和重组载体psi CHECK2-CLTC-3′UTR共转染到细胞中,以乱序序列作为阴性对照(NC),检测miRNA对重组质粒荧光素酶活性的影响。结果发现miR-205,miR-1,miR-129-5p和miR-206均能够显著抑制荧光素酶活性(P<0.05)。在猪肾上皮细胞系PK15细胞中超表达miR-1和miR-129-5p后,定量PCR(q-PCR)结果显示:猪CLTC基因的表达量显著下调。突变了psi CHECK2-CLTC-3′UTR载体中这4个miRNA的种子序列的结合位点发现:miR-1对突变质粒中的荧光素酶无显著抑制作用。表明miR-1与猪CLTC基因有直接的靶向关系,并通过其种子序列抑制CLTC基因的表达。

Clathrin heavy chain（CLTC）, an important component of clathrin-coated pits, plays an important role with virus invasion of cells; however, post-transcriptional gene regulation of the CLTC gene, especially porcine CLTC gene regulation by miRNA has not yet been clearly elucidated. This study aimed to screen miRNAs that target the CLTC gene. First, bioinformatics predicted that miR-205, miR-1, miR-129-5p, miR-206, miR-19 a, and miR-19 b targeted the porcine CLTC gene. Then porcine CLTC 3′UTR was cloned into the psi CHECK2 vector, and the dual luciferase reporter recombinant vector psi CHECK2-3′ UTR was constructed.The prediction of miRNA and the recombinant vector psi CHECK2-3′ UTR were co-transfected into cells,respectively, with the scramble sequence of miRNA as a negative control（NC）; then the luciferase activity was detected. A quantitative PCR（q-PCR） was also used to determine the expression of CLTC m RNA levels. Then to verify whether miRNA regulated the porcine CLTC gene through seed sequences, binding sites of psi CHECK2-3′ UTR with seed sequence were mutated. Results showed that miR-205, miR-1, miR-129-5p, and miR-206 were able to significantly inhibit luciferase activity（P〈0.05）. At the same time there was an overexpression of miR-1 and miR-129-5p in PK15 cells, and the q-PCR showed that the expression of CLTC m RNA level was significantly reduced（P〈0.05）. Verification of whether the four miRNA（miR-205, miR-1,miR-129-5p, and miR-206） regulated the porcine CLTC gene through seed sequences showed that miR-1mutant plasmid did not inhibit the luciferase activity. Thus, the results demonstrated that miR-1 inhibited porcine CLTC gene expression through its seed sequence binding with CLTC 3′UTR.