环状RNA ciRS-7靶向miR-219a-5p影响微小隐孢子虫体外增殖的机制

The Mechanisms of Circular RNA ciRS-7 Affecting the Propagation of Cryptosporidium parvum in HCT-8 Cells via Targeting miR-219a-5p

旨在探究环状RNA ciRS-7靶向miR-219a-5p影响微小隐孢子虫(Cryptosporidium parvum)在HCT-8细胞中增殖的作用机制。以C.parvum感染HCT-8细胞为研究模型,利用Western blot检测HCT-8细胞中的自噬蛋白LC3B-II和p62的表达情况;利用qRT-PCR和双荧光素酶报告基因试验检测和验证ciRS-7与miR-219a-5p的靶向关系,利用qRT-PCR检测HCT-8细胞中C.parvum的荷虫量(C.parvum hsp70基因的mRNA水平)。结果显示:C.parvum感染诱导HCT-8细胞中LC3B-II和p62的蛋白质表达水平在感染后8、12、24、36和48 h显著上调;过表达ciRS-7显著抑制了C.parvum感染细胞中LC3B-II的蛋白质表达水平,但升高了p62的蛋白质表达水平,而干扰ciRS-7表达的作用相反;C.parvum感染诱导HCT-8细胞中miR-219a-5p的显著下调表达,且ciRS-7可靶向调节miR-219a-5p的表达;miR-219a-5p mimics显著增加了C.parvum感染细胞中LC3B-II的蛋白质表达水平,但降低了p62的蛋白质表达水平,而miR-219a-5p inhibitor的作用相反;miR-219a-5p mimics可逆转过表达ciRS-7对C.parvum感染细胞中LC3B-II蛋白质表达的抑制和p62蛋白质表达的促进作用;miR-219a-5p mimics显著抑制了感染细胞中C.parvum hsp70基因的mRNA表达水平,而miR-219a-5p inhibitor的作用相反;miR-219a-5p mimics逆转了过表达ciRS-7对感染细胞中C.parvum hsp70基因的mRNA表达水平的上调作用。ciRS-7通过靶向miR-219a-5p抑制C.parvum感染诱发的HCT-8细胞自噬,进而促进C.parvum在HCT-8细胞中的增殖。

The present study was carried out to investigate the impact of circRNA ciRS-7 on the propagation of Cryptosporidium parvum in HCT-8 cells via targeting miR-219 a-5 p.Using C.parvum-infecting HCT-8 cells as the model,we detected the expression of the autophagy associated-proteins of LC3 B-II and p62 in HCT-8 cells by using Western bolt assays.The targeting relationship between ciRS-7 and miR-219 a-5 p were detected and verified by using qRT-PCR and dual luciferase reporter assays,and the infection burden of C.parvum(the mRNA level of C.parvum hsp70 gene) in HCT-8 cells was determined by using qRT-PCR.C.parvum infection induced significantly up-regulation of the protein levels of LC3 B-II and p62 in HCT-8 cells at 8 h,12 h,24 h,36 h and 48 h post-infection.Overexpression of ciRS-7 markedly inhibited the protein level of LC3 B-II but promoted the protein level of p62 in C.parvum-infecting cells,whereas interfering the expression of ciRS-7 obtained the opposite results.C.parvum infection induced significant down-regulation of miR-219 a-5 p in HCT-8 cells,and ciRS-7 could target to regulate the expression of miR-219 a-5 p.miR-219 a-5 p mimics significantly increased the protein level of LC3 B-II but decreased the protein level of p62 in C.parvum-infecting cells,while miR-219 a-5 p inhibitor had the opposite effects,and the inhibition effect on the protein level of LC3 B-II and the promoted effect on the protein level of p62 by overexpression of ciRS-7 in C.parvum-infecting cells were reversed by miR-219 a-5 p mimics.miR-219 a-5 p mimics significantly inhibited the mRNA level of C.parvum hsp70 in infected cells,while miR-219 a-5 p inhibitor had the opposite effect.The upregulated effect of overexpression of ciRS-7 on the mRNA level of C.parvum hsp70 gene in infected cells was attenuated by miR-219 a-5 p mimics.The results revealed that ciRS-7 promoted the propagation of C.parvum in HCT-8 cells by targeting miR-219 a-5 p to inhibit C.parvum-induced cell autophagy.