Experimental Study On The Role Of Human IL-24Gene In CD133Positive Laryngeal Carcinoma Cells

Objective To clone human IL-24gene， construct its eukaryoticexpression vector; isolate CD133positive cells from Hep-2cell line(CD133+Hep-2)， study the expression of IL-24gene in CD133+Hep-2cells to discuss its role.Methods Normal human peripheral blood mononuclear cells wereseparated. Total RNA was extracted by Trizol method， and was reversetrancripted into cDNA by RT-PCR. Primer P1、P2were designed toamplify human IL-24gene， then TA cloned into pMD19-T simplevector after agarose gel electrophoresis and gene sequencing were correct.Human IL-24gene and its eukaryotic expression vector were digestedthrough NheI and XhoI double enzyme. Eukaryotic expression vectorpIRES2-ZsGreen1-hIL-24of human IL-24gene was constructed andverified through enzyme digestion and gene sequencing. CD133positivecells CD133+Hep-2were sorted out from Hep-2cells by flow cytometry(FCM)， and the correct pIRES2-ZsGreen1-hIL-24was transferred intoCD133+Hep-2cells mediated by the liposome2000before verifying itsexpression. The effects of human IL-24gene on the growth of CD133+Hep-2cells were detected by MTT and FCM. Tumor forming ability ofCD133+Hep-2cells transfected with human IL-24gene was detectedthrough the nude mice transplantation tumor experiment.Results Human IL-24gene with621bp was successfully cloned in humanperipheral blood mononuclear cells. DNA sequencing result was consistent with that reported in GenBank. Enzyme digestion and genesequencing verification of recombinant expression plasmidpMD19-T-hIL-24and pIRES2-ZsGreen1-hIL-24were correct.Recombinant plasmid pIRES2-ZsGreen1-hIL-24was successfullytransferred into CD133+Hep-2cells using liposome mediated method.Human IL-24gene was verified to integrate into transfected cells andexpress stablely. MTT test results showed that the cell growth wasinhibited significantly in the experimental group with IL-24genetransfected compared with the control group and the empty vectorgroup(P<0.05). FCM results showed that the cell apoptosis rate wasincreased significantly in the experimental group compared with thecontrol group and the empty vector group (P<0.05). Nude micetransplantation tumor experiment verified that the volume and weight ofthe experimental group were significantly decreased compared with thecontrol group and the empty vector group in nude mice (P<0.05).Conclusions The expression of human IL-24gene could inhibit theproliferation of CD133+Hep-2cells， promote their apoptosis andsuppress the growth of their xenografts in nude mice.