pWPXL

miR152 is involved in various biological functions and the development of diseases. The study here explores the role of mir-152 in cell proliferation and colony formation of liver cancer stem cells. It was found that exogenous overexpression of mir-152 inhibited cell proliferation and colony formation of CD133+ hep3B cells. KIT is a direct target of miR-152, and miR-152 downregulates the protein expression of KIT by directly binding to the 3' untranslated region of KIT. Downregulation of KIT by specific siRNA inhibited the proliferation and colony formation of CD133+ hep3B cells, which was similar to the inhibitory effect of miR-152. Furthermore, exogenous expression of KIT impaired the inhibitory effects of miR-152 on cell proliferation and colony formation.

In this study, the pri-miR-152 sequence was amplified from normal human genomic DNA and constructed into the lentiviral expression vector pWPXL to generate pWPXL-miR-152. The 3'UTR sequence of KIT was amplified from normal human genomic DNA and subcloned into the psiCHECK-2 vector. The KIT coding sequence was amplified, the lentiviral expression vector pWPXL was constructed, and pWPXLmiR-KIT was generated. Then, pWPXL-miR-152 or a mixture of pWPXL, psPAX2, and pMDG2 was transfected into HEK293T cells to generate miR-152 overexpressing lentivirus (lenti-miR152) and mock lentivirus (lentiNC). CD133+ cells were infected with recombinant lentivirus. The expression of miR-152 in infected cells was determined by qRT-PCR.

Figure 1. mRNA expression of miR-152 in CD133+ hep3B cells was determined by qRT-PCR. (Huang H, et al. 2015)