OBJECTIVETo construct a lentiviral expression vector of has-miR-338-3p and verify its target gene.

METHODSThe pre-miR-338-3p was synthesized and inserted into pLV-THM, and the recombinant plasmid pLV-THM-miR-338-3p was confirmed by restriction endonuclease analysis and DNA sequencing. 293T cells were co-transfected with the lentiviral vector pLV-THM-miR-338-3p, psPAX2 and pMD2.G, and the supernatant containing the lentivirus particles was harvested to determine the virus titer and used to infect SW-620 cells. Flow cytometry was employed for sorting the GFP-positive cells. The expression of miR-338-3p was determined using real-time RT-PCR and the expression of SMO protein was detected with Western blotting in the infected SW-620 cells. The invasiveness of the infected SW-620 cells was assessed using Transwell assay.

RESULTSRestriction enzyme digestion and DNA sequencing demonstrated successful construction of the lentiviral vector pLV-THM-miR-338-3p. SW-620 cells infected with pLV-THM-miR-338-3p showed a significantly increased expression of miR-338-3p, and the overexpression of miR-338-3p suppressed the expression of SMO protein and the invasiveness of the cells.

CONCLUSIONThe successful construction of the lentiviral vector pLV-THM-miR-338-3p and the establishment of a SW-620 cell line with miR-338-3p overexpression provide the basis for further study of the molecular function of miR-338-3p in colorectal carcinoma. MiR-338-3p can suppress SMO gene expression to inhibit the invasiveness of colorectal carcinoma cells.