ssc-miR-204 Targeted Regulation the Expression of DLG5 Gene in Intestinal Porcine (Sus scrofa) Epithelial Cells
WANG Wei1, XIE Kai-Hui1, LUO Rui-Rui1, GAO Xiao-Li1, WANG Peng-Fei1, ZHANG Juan-Li1, YANG Jiao-Jiao1, ZHANG Bo1, MA Yan-Ping3, GUN Shuang-Bao1,2,*
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 Gansu Research Center for Swine Production Engineering and Technology, Lanzhou 730070, China; 3 Gansu Agricultural University Library, Lanzhou 730070, China
Abstract：Based on early study, the high-throughput sequencing found that ssc-miR-204 was significantly differentially expressed in piglets infected with Clostridium perfringens type C in the resistance and susceptibility group. This study aims to verify the targeted regulatory relationship between miR-204 and discs large homolog 5 gene (DLG5). In the present study, the TargetScan software was used to predict the targeted binding sites and conservation between miR-204 and DLG5 gene. The wild-type and mutant-type pmirGLO dual-luciferase reporter vector in the 3'UTR region of DLG5 gene was constructed and the targeting relationship between miR-204 and DLG5 confirmed by dual luciferase activity experiment. Further, the regulation of miR-204 on DLG5 expression was detected by qRT-PCR, Western blot and cell immunofluorescence experiments after transfected with miR-204 mimic and inhibitor in intestine porcine epithelial cells, respectively. The results showed that the miR-204 mature seed region sequence was complementary to the nucleotide sequence of DLG5 at 3'UTR 1 333~1 339, and was highly conserved among multiple species (such as human (Homo sapiens), pig, chimpanzee (Pan troglodytes), rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), cattle (Bos taurus), etc). Through double enzyme digestion and sequencing identification, this experiment successfully constructed pmirGLO-DLG5-3'UTR dual luciferase vector; the results of dual luciferase reporter gene experiment showed that miR-204 mimic could significantly reduce the luciferase activity of DLG5 gene (P<0.01), the two had a target relationship. Then, the results of qRT-PCR and Western blot indicated that after transfected with miR-204 mimic, the expression level of DLG5 was significantly inhibited (P<0.01). On the contrary, after transfected with miR-204 inhibitor, the expression level of DLG5 was significantly upregulated (P<0.01). The immunofluorescence results showed that compared with the mimic NC group (89.90±3.818), DLG5 fluorescence intensity was significantly reduced (62.65±3.790, P<0.01) after transfected with miR-204 mimic. While, comparing with the inhibitor NC group (85.52±6.220), the fluorescence intensity of DLG5 was significantly enhanced (102.80±3.588, P<0.05) after transfected with miR-204 inhibitor. From the above results, it could be seen that miR-204 could target the DLG5 gene, and miR-204 had a negative regulatory effect on the expression of DLG5. The results of this study can provide a basis for the further verification of the functions of miR-204 and DLG5 genes at the cellular level, and provide a basis for the role of miRNAs in the regulation of C. perfringens type C diarrhea of piglets.
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