| Cervical cancer remains as one of the biggest killers of women worldwide. Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease that readily metastasize in the earlier period, hence newer therapeutical modalities are needed.Advanced in our understanding of the molecular biology of tumor cells have led to the development of new trestment strategies, including gene therapy. The molecular basis of cancer is now known to involve activation of dominant oncogenes and inactivation of tumor suppressor genes, these genetic events may represent novel targets for cancer therapy.IL-24 was identified through subtraction hybridization from a human melanoma cell line by P.B.Fisher in 1995. Up to now, a large body of data has accumulated to demonstrate that IL-24 results in growth suppression and induction of apoptosis in a broad range of cancer cells. RNA interference (RNAi) is a post-transcriptional regulatory manner of gene expression, it is a conservative mode in organism evolution, and it has the effect of counteract invading of virus and maintenance stablity of genome. RNAi could knockdown the expression of gene in mammalians, which provide a new approach for gene therapy of tumor. It was indicated that specifically inhibiting over-expression of oncogene, cancer related genes or mutant gene by RNAi and retained them in silence status could achieve the result of anticancer effect.In this study, we investigated the effect of hIL-24 gene inducing the apoptosis and reducing metastasis.of human cervical cancer CaSki cells and its possible mechanism, evaluated the effect of hIL-24 gene associated with HPV-16 E6 siRNA on inducing apoptosis of CaSki cells. And we observed the effect of gene therapy on the CaSki xenograft tumor in nude mice with hIL-24 expression recombinant plasmids, which providing experimental evidence for the treatment of cervical cancer by gene therapy. We develop this study according to following procedure:1. To clone hIL-24 gene and construct its eukaryotic expression vector, then transfect it into CaSki cells to express hIL-24 protein. METHODS: hIL-24 gene was acquired by RT-PCR from the total RNA of CaSki cell and cloned into plasmid pcDNA3.1(+) to generate the eukaryotic expression vector named pcDNA3.1(+)-hIL-24. pcDNA3.1(+)-hIL-24 was transfected into CaSki cells to express hIL-24 protein.RESULTS: PCR and sequencing revealed that hIL-24 gene was successfully cloned into plasmid pcDNA3.1(+) and its eukaryotic expression vector was constructed. The vector was able to express hIL-24 protein after transfected into CaSki cells.CONCLUSION: The hIL-24 eukaryotic expression vector was successfully constructed.2. To investigate hIL-24 gene inducing the apoptosis of human cervical cancer CaSki cells and its possible mechanism. METHODS: CaSki cells were transfected with hIL-24 eukaryotic expression vector by lipofectamine2000. The effect of hIL-24 on the transcription of HPV E6 gene was analyzed by RT-PCR. The effect of hIL-24 on P53 protein was analyzed by Western Blot. Apoptosis of CaSki cells induced by hIL-24 gene was analyzed by FACS. RESULTS: Transcription level of HPV E6 gene decreased significantly, the amount of P53 protein increased significantly and the apoptosis rate of CaSki cells increased significantly after hIL-24 transfection. CONCLUSION: HIL-24 gene can inhibit the transcription of HPV E6 gene, recover the activity of P53 protein, then induce the apoptosis of CaSki cells.3. To evaluate the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human cervical cancer cells.METHODS: We investgated the ability of metastasis of CaSki cells treated with hIL-24 by Cell migration assay and Cell invasion assay .We analysed the level of proteins associated with these processes by Western blot. RESULTS: Cervical cancer cells (CaSki) treated in vitro with hIL-24 migrated and invaded less than cells treated with PBS or vector control. MDA-7/IL-24 inhibited migration and invasion by down-regulating the production of matrix metalloproteinase-2 (MMP-2) and by up- regulating the production of E-cadherin relative to PBS and vector control. CONCLUSION: These results show that MDA-7/IL-24 inhibits invasion and migration of cervical cancer cells by down- or up-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.4. To evaluate the effect of HPV-16 E6 siRNA associated with hIL-24 gene on inducing apoptosis of CaSki cells.Methods: Transfecting CaSki cells with hIL-24 gene eukaryotic expression vector and HPV-16 E6 siRNA vector separately and associately. The transcription of HPV E6 gene was analyzed by RT-PCR, P53 protein was analyzed by Western blot, apoptosis of CaSki cells was analyzed by FACS. Results: After transfecting, transcription level of HPV E6 gene decreased in all the three group of cells, the co-transfection group was significant; the amount of P53 protein increased in all the three group of cells, the co-transfection group was significant; and the rate of apoptosis of CaSki cells increased in all the three group of cells, the co-transfection group was significant.Conclusion: Both HPV-16 E6 siRNA and hIL-24 gene can inhibit the transcription of HPV E6 gene, recover the activity of P53 protein, then induce the apoptosis of CaSki cells. HPV-16 E6 siRNA associated with hIL-24 gene can significantly induce apoptosis of CaSki cells with positive cooperativity.5. The inhibition effect of hIL-24 on CaSki cell xenograft tumor in nude mice.Methods: Obtain the cell line expression recombinant plasmids by G418 screening. The cell lines were innoculated on the back of Balb/c nude mice subcutaneouly, and the growth of tumors were observed; the mice were sacrificed and tumor were decoherenced and weighed, the inhibition ratio of tumor growth was calculated. CaSki cell was innoculated into nude mice subcutaneouusly and the CaSki xenograft tumor model was constructed, the recombinant plasmids were injectioned into the tumor admixed with liposome, the volume and weight of tumor were measured. The pathological change was observed by HE stain.Results: the cell lines expressing recombinant plasmids were obtained. The oncogenicity of CaSki cell transfected by pcDNA3.1(+)-hIL-24 was lower obviously; After treated by recombinant plasmid, the growth of tumor was inhibited, the weight and volume of the pcDNA3.1(+)-hIL-24 treated group were lower than that of control group(P<0.05). There were many necrosis zone in tumor tissue treated by pcDNA3.1(+)-hIL-24, and there were necrosis cell and apoptosis body could be found in the CaSki xenograft tumour.Conclusion: the ectopic production of MDA-7/IL-24 gene could degrade the oncogenicity of CaSki cell; the treatment of plasmid expressing hIL-24 could inhibite growth of CaSki xenograft tumor. |
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