关键词: 微RNAs, RNA干扰, microRNA-196b, 慢病毒感染, 骨髓基质细胞, 海绵吸附慢病毒载体

Abstract: Objective To construct miR- 196b sponge lentiviral vector, and laid the foundation for studying the function of miR- 196b in bone marrow stromal cells. Methods Based on the miR- 196b mature sequence, a sequence consisting of 6 tandem repeats of the complementary sequence of miR- 196b was designed, and which was cloned into pUC19 plasmid by using reverse PCR. Then the six-repeat sequence was cut and subcloned into pLVX- shRNA2 lentiviral vector. The lentivirus was packaged using 293T cells, and titer determination was done. The pLVX-shRNA2 lentivirus was used as the control group, and the 196b-sponge-pLVX lentivirus was the experimental group. Then ST2 cells were infected with the viruses, and the infection efficiency was calculated. The protein level of forkhead box O1 (FoxO1) was detected by Western blot assay. Results The identity of the sponge sequence was verified by sequencing. The titer of the sponge virus was 1 × 108 PFU/mL, and the infection efficiency reached 80% . Compared with the control group, the expression level of FoxO1 protein was significantly increased (P < 0.05). Conclusion The miR-196b sponge lentiviral vector is successfully constructed, and which has the capability to inhibit endogenous miR-196b.

Key words: microRNAs, RNA interference, microRNA-196b, lentivirus infections, bone marrow stromal cells, sponge lentiviral vector