The Effects Of LAPTM4B On Stemness Maintenance Of CD133-positive Liver Cancer Stem Cells

ObjectiveThe characteristics of high metastasis of liver cancer is an important factor which limits the therapeutic effect of liver cancer.Liver cancer stem cells(LCSCs)play a crucial role in the development,recurrence,metastasis and drug resistance of liver cancer.Our research group found stronger invasion and metastasis ability occurred in those liver cancer cells who highly express lysosome-associated protein transmembrane 4 beta(LAPTM4B).We confer that LAPTM4B may influence the stemness maintenance of LCSCs,and drive them to malignant transformation.In this study,we sorted CD133~+LCSCs by magnetic activated cell sorting(MACS),aimed at study the effects of LAPTM4B on stemness maintenance of LCSCs and probed into the molecular mechanism of LAPTM4B on stemness regulation of LCSCs.To provide theoretical foundation for prognostic judgement and combined chemo-radiotherapy in liver cancer.Methods1 MACS was used to sort CD133~+LCSCs from the HepG2 and Huh7 cells.Flow cytometry and Western Blot were performed to evaluate the proportion of CD133~+cells in HepG2 and Huh7 liver cancer cell lines before and after sorting.CCK-8 assay was used to monitor the proliferation of CD133~+HepG2 and CD133~+Huh7 cells.We tested the ability of colony forming of CD133~+HepG2 and CD133~+Huh7 cells by colony formation assay.We cultured the CD133~+HepG2 and CD133~+Huh7 cells in conditioned medium,and observed the monoclonal formation characteristics of them.The expressions of related genes in CD133~+HepG2 and CD133~+Huh7 cells were screened by qPCR and Western Blot.2 The LAPTM4B shRNA plasmid expression vector targeting LAPTM4B was designed and constructed,and the recombinant plasmid and lentiviral vector packaging auxiliary plasmids VSVG,RRE and REV transfected 293FT cells,then produced lentiviral vectors LV-shLAPTM4B.3 The lentiviral vector constructs a stable CD133~+HepG2 and CD133~+Huh7 cells which interferes with LAPTM4B expression,detected its jamming efficiency by Western Blot and qPCR,colony formation assay and sphere formation efficiency were used to evaluate the effects of LAPTM4B on the stemness maintenance of CD133~+LCSCs.Results1 Flow cytometry results showed the percentage of CD133~+cells was 92.35±5.15%after sorting by MACS,and CD133~+Huh7 cells was 95.80±2.19%.Western Blot results showed that the expression of CD133 protein between the CD133~+cells and CD133~-cells was significantly different.The isolated CD133~+HepG2 and CD133~+Huh7 cells were cultured in a conditioned medium,we found there were tumor spheres formed,whereas the CD133~-cells could not.CCK-8 assay and colony formation assay results showed that the proliferation and clone forming ability of CD133~+HepG2 cells and CD133~+Huh7 cells were higher than CD133~-cells.Western Blot and qPCR results showed that the related genes EpCAM,SOX9,OCT4 and Nanog were significantly up-regulated in CD133~+HepG2 cells and CD133~+Huh7 cells compared with CD133~-cells.These results illustrated that the CD133~+HepG2 and CD133~+Huh7 cells were high purity and possessed some CSCs-like properties and abilities2 The results of DNA sequencing and identification showed that plasmid with target silencing LAPTM4B gene shRNA fragment was constructed correctly.Lentiviral vectors were successfully packaged and infected with CD133~+HepG2 and CD133~+Huh7 cells.The knockdown efficiencies of the selected stable cell lines were89.74%±2.20%and 69.70%±1.64%.The expression of LAPTM4B protein in the interference group was significantly lower than LV-ctrl group.3 In the colony formation assay results showed that CD133~+HepG2 and CD133~+Huh7cells infected with LV-shLAPTM4B possessed much lower colony formation efficiency than CD133~+HepG2 and CD133~+Huh7 cells infected with LV-ctrl.The sphere formation efficiency showed that the spheroid formation ability of CD133~+HepG2 and CD133~+Huh7 cells infected with LV-shLAPTM4B was significantly reduced compared with LV-ctrl group.Western Blot and qPCR results showed that the expressions of related genes were down-regulated in CD133~+HepG2 and CD133~+Huh7 cells infected with LV-shLAPTM4B.Conclusion1 We successfully sorted the CD133~+HepG2 and CD133~+Huh7 cells by MACS,the purity of CD133~+HepG2 and CD133~+Huh7 cells was high and they possessed some CSCs-like properties and abilities.2 The successful construction of recombinant lentiviral vector LV-shLAPTM4B had high infection efficiency and could stabilize the inhibition of LAPTM4B expression in CD133~+HepG2 and CD133~+Huh7 cells.3 The results demonstrated that knocking down the expression of LAPTM4B could decrease the stemness of CD133~+liver cancer stem cells and down regulation the expression of related genes in vitro.Those indicated that LAPTM4B plays an important role in regulating the stemness of CD133~+liver cancer stem cells.