Construction of shRNA lentivirus vector targeting rat Eap1 gene and Eap1 effects on Kiss1 gene at cellular level / 上海交通大学学报（医学版）

ABSTRACT

Objective·To construct effective shRNA-lentiviral vector targeting rat Eap1 gene and explore the effect of Eap1 on Kiss1 at cellular level. Methods·Four shRNA sequences targeting rat Eap1 mRNA and one negative control sequence were designed and synthesized, and then recombined with lentiviral vectors. After DNA sequencing identification, the shRNA recombinant vectors were co-transfected into 293T cells with pGag/Pol, pRev, pVSV-G to packaging lentiviral particles and the virus titers were determined. NRK-52E cells were transfected with all the four lentiviral-Eap1-shRNAs and the negative control lentivirus, and cells were harvested after 72 h. Real-time PCR and Western blotting was performed to detect the change of the mRNA and protein level of Eap1 gene. The most effective LV-Eap1-shRNA was used to infect NRK-52E cells [non-lentivirus and Eap1-shRNA(-) lentivirus as control groups], and then the level of Kiss1 mRNA expression was detected after infection for 72 h. Results·Seventy-two hours after transfection, the Eap1 mRNA and protein expression between blank group and LV-shRNA(-) group showed no significant difference; while Eap1 expression in all LV-Eapl-shRNA groups reduced significantly in comparing to the control groups. In addition, the lentiviral-Eap1-shRNA4 was the most effective in RNA interference. After blocking Eap1 expression in NRK-52E cells, the Kiss1 mRNA level was significantly increased comparing to the control groups. Conclusion·The effective Eap1-shRNA lentiviral vector is established successfully. The Kiss1 gene expression decreases after blocking Eap1 in NRK-52E cell line.