Construction Of Human MKRN1 Gene SIRNA Recombinant Adenovirus Vector, Eukaryotic Expression Vector And Its Expression In Cervical Caner Cells

Cervical cancer is one of the common gynecological malignancies in recent years. The rate of this cancer tends to rise every year. Although usage of surgery, radiotherapy, chemotherapy and other modern strategies have been able to increase the survival rate of the cervical cancer patient, the prognosis for this cancer still remains poor. Use of gene therapy to find a solution to this malignancy has become a research hotspot these days. Stable expression of normal human cells transfected with telomerase can divide infinitely. This suggests that telomere shortening can be used to control replicative senescence. Kim has found MKRN1 ubiquitin-mediated degradation of hTERT. His results indicate that MKRN1is involved in death of tumor cells. Hence, MKRN1 has a very close relationship with the tumor. At present, study on the MKRN1 is not very profound both inside and outside the country. The biological function and signal transduction pathway of MKRN1 is not clear. In this study, a recombinant adenovirus vector for siRNA targeting MKRN1 gene was constructed and transfected to hela cells. It provided a powerful tool to study the function of MKRN1 gene in cervical cells and a good experimental basis to be performed with animals models. Secondly, a eukaryotic expression pEGFP-N1-MKRN1 vector was also constructed. This laid a solid foundation and provided with a cell model for further research on the function of MKRN1 and its relationship to cell proliferation and apoptosis. This study includes three parts:1. Construction of human MKRN1 gene siRNA recombinant adenovirus vector. METHODS: First siRNA sequence targeting MKRN1 gene was synthesized and cloned into the shuttle plasmid pSES-HUS. It was then homogenously recombinated with adenovirus backbone plasmid pAdeasy-1 in E coli BJ5183. After which the adenovirus was packaged and amplified in HEK293 cells. The recombinant adenovirus vector was injected into hela cells. Presence of MKRN1 gene was detected by RT-PCR and Western blot. The telomerase activity in hela cells was detected by PCR-TRAP. RESULTS: Recombinant adenovirus vector containing siRNA targeting MKRN1 gene was successfully constructed. The recombinant adenovirus Adeasy-MKRN1-siRNA significantly inhibited the expression of MKRN1 and increased the telomerase activity in the hela cells. CONCLUSION: The recombinant adenovirus was successfully constructed, which had provided a valid tool to study the function of MKRN1 and its relationship with tumor. 2. Construction of Eukaryotic Expression Vector pEGFP-N1-MKRN1 and Its Expression in Siha Cells. METHODS:The cDNA of human MKRN1 was amplified by RT-PCR from the total RNA that has been isolated from the 293 cells, which was then inserted into pEGFP-N1 vector to construct the recombinant eukaryotic expression vector pEGFP-N1-MKRN1. The recombinant plasmid was then transferred into Siha cells through liposomes. Stably-transfected siha cells were selected by G418. Over expression of the MKRN1 in the transfected Siha cells was confirmed by Western blot. RESULTS: A 1477bp DNA fragment was successfully amplified from the 293 cells by performing RT-PCR. This DNA fragment matched with the length of cDNA encoding the MKRN1. Enzyme digestion analysis and DNA sequencing showed that the target gene was successfully cloned into recombinant vector. Green fluorescence was detected from transfected cells under fluorescent microscope study. Western blot analysis revealed that the fusion protein pEGFP-N1-MKRN1 could be expressed stably in the transfected Siha cells. CONCLUSION:The eukaryotic expression vector pEGFP-N1- MKRN1 was successfully constructed. Positive Siha cell clones expressing MKRN1 stably were obtained, which provided solid foundation and a cell model for further research on the function of MKRN1 and its relationship with cell proliferation and apoptosis.3. Effect of stably expressing MKRN 1 Gene on Human Siha Cells. METHODS:To use as experimental group, MKRN1 gene Siha stable expression cell lines were constructed after which MKRN1 gene transcription of hTERT gene, mRNA expression level of telomerase activity along with its effects on cell cycle was studied. RESULTS: In transfected Siha cells, stable expression MKRN1 significantly inhibited both the expression of hTERT gene mRNA levels as well as telomerase activity. The G1 phase cells increased significantly whereas the S-phase cells decreased significantly. CONCLUSION:These results indicate that MKRN1 stable expression can specifically reduce the hTERT mRNA level of cervical cancer cells, inhibit telomerase activity in cancer cells, inhibit cancer cell proliferation, and prevent cancer cells in G1 phase to enter S phase. This method can be useful in exploring new strategies to find a remedy for cervical cancer.