Construction of miR-331-3p overexpression vector and its effect on cell proliferation
(2.Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai’an, Shandong, China 271018)
(3.Weifang University of Science and Technology, Weifang, Shandong, China 262700)
【Abstract】To investigate the effect of miR-331-3p on the proliferation of porcine renal epithelial cells (PK15) and its mechanism, the pcDNA 3.1 (+) overexpression vector of miRNA-331-3 p (pcDNA 3.1 (+)-miR-331–3p) was constructed. PK15 cells were divided into four groups, including experimental group, experimental control group, inhibitor group, and inhibitor control group. Experimental group and experimental control group were transfected with pcDNA 3.1 (+)-miR-331-3p and pcDNA 3.1 (+), respectively. Inhibitor group and inhibitor control group were transfected with miR-331-3p inhibitor and miR-331-3p negative control (miR-331-3p NC), respectively. Above all, CCK-8 reagent was used to plot the cell proliferation curve and Propidium (PI) staining was used to detect the proportion of cell stages. Then, its expression changes were detected by quantitative real-time PCR that included the growth inhibitory protein family member 5 (ING5), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 3 (CDK3), cyclin-dependent kinase 4 (CDK4), Cyclin B and cyclin-dependent kinase inhibitor 1 A (CDKN1A). The results showed that the expression of miRNA-331-3p was significantly increased in the experimental group. The cell proliferation curve showed that the number of cells in experimental group was significantly higher than that in experimental control group or inhibitor control group at the 48th and 72nd hour (P < 0.05). Simultaneously, inhibitor group was significantly lower than experimental control group or inhibitor control group in the number of cells at the 48th and 72nd hour (P < 0.05), but there was no significant difference between the experimental group and the control group. Compared with the experimental control group, the proportion of cells of experimental group at G0/G1 phase was decreased, the proportion of S phase and G2/M phase increased, and the inhibitor control group showed the opposite trend. Simultaneously, the expression levels of CDK2, CDK3, CDK4 , and Cyclin B genes in the experimental group were significantly increased, while ING5 and CDKN1A genes inhibiting proliferation showed a significant downward trend. These results demonstrate that the miR-331-3p overexpression vector was successfully constructed, and miR-331-3p has the ability to promote the proliferation of PK15 cells. The study lays a solid foundation for further research for its role in pig growth and development.
【Keywords】 miR-331-3p; cell proliferation; overexpression vector; PK15; pig;
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