沉默 MKRN1 基因表达对人胚胎肾细胞 HEK-293T 增殖及凋亡的影响.

BACKGROUND: It has been reported that makorin ring finger protein 1 (MKRN1) knockdown can inhibit the growth of tumor cells, but whether MKRN1 plays a role in the body as a tumor-related protein needs further study. The siRNA-mediated gene silencing is transient, and therefore, the duration is short and the conventional lentivirus technique also has an off-target effect. A cell line stably silencing MKRN1 gene constructed using Tet-on lentivirus system has reversible advantages and can avoid the above shortcomings, which is of great significance for the further study on the biological function of MKRN1. OBJECTIVE: To construct a MKRN1 gene silencing vector mediated by the Tet-on lentivirus system in order to study the effect of MKRN1 gene silencing on the proliferation and apoptosis of human embryonic kidney cells HEK-293T. METHODS: Using RNAi technique, three short hairpin RNAs (shRNAs) were designed for MKRN1 gene, namely sh1, sh2, and sh3, and three shRNA sequences were ligated into tetracycline-induced expression vector pTripz. The correct sequence was verified by sequencing. HEK-293T cells were transfected with the three-plasmid system and cultured in the complete medium containing 1 mg/L doxycycline for lentivirus packaging. The virus solution was collected at 48 and 72 hours and concentrated to infect HEK-293T cells. Forty-eight hours after infection, the virus infection efficiency was observed by fluorescence microscope. The knockout efficiency of MKRN1 was detected by real-time fluorescence quantitative PCR and western blot assay. The successful construction of tetracyclineinduced expression system was detected by western blot assay. Colony formation test and western blot assay were used to detect the effect of silencing MKRN1 expression on the proliferation and apoptosis of HEK-293T cells. RESULTS AND CONCLUSION: (1) Three MKRN1-shRNAs, named sh1, sh2, and sh3, were successfully inserted into the Tet-on vector, and the sequence of each vector was correct. HEK-293T cells were successfully transfected with lentivirus. (2) RT-qPCR and western blot results indicated that the MKRN1 knockdown level of the first sequence of the three shRNA sequences was significantly decreased (P < 0.05), and the expression of the target gene was rescued in the silent group without doxycycline induction. (3) The proliferation of HEK-293T cells decreased after MKRN1 silencing detected by colony formation experiments (P < 0.05). Western blot assay results revealed that after MKRN1 knockdown, the expression of proliferating cell nuclear antigen was down-regulated, the expression of BAX was increased, the expression of Bcl2 was down-regulated, and the Bcl2/BAX ratio was down-regulated (P < 0.001). (4) To conclude, the HEK-293T cell line stably silencing MKRN1 can be constructed by using Tet-on lentiviral vector system, and the silencing of MKRN1 can inhibit the proliferation of HEK293T cells and promote their apoptosis. The construction of stably silencing MKRN1 HEK-293T cells can lay a foundation for the further study on the biological function of MKRN1 and also provide a methodological reference for the study of the biological function of other genes. [ABSTRACT FROM AUTHOR]