Construction Of Recombinant Retroviral Vector Expressing SHRNA Targeting MCPH1 Gene And Establishment Of Its Stable CASKI Cell Line

Cervical cancer is one of common malignant tumors in women, worldwide, it is the second leading cause of cancer deaths among women. Experts say more than 200,000 women die from cervical cancer every year. These deaths are most common in developing countries. Therefore, research on cervical cancer has drawn increasing attention.Current mainstream treatments of cancer are surgery, radiotherapy and chemotherapy. Even though scientists and medical staff have continuously find ways to improve these three treatments, many patients are still not healed by these treatments. Gene therapy, which is the introduction of genetic material into a patient's tissues with the intent to achieve therapeutic benefit, is the only way to tackle cancer in the 21st century.MCPH1 is one of six genes causing primary microcephaly when non-functional mutations exist in the homozygous state. The gene encodes the BRCT-domain containing protein microcephalin /BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage.RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens.In this study, we have investigated the efficacy of preformed shRNA to modulate the expression of MCPH1 gene in CaSki cells, and established the cell lines with stable silencing MCPH1 gene expression in cervix cancer cell line.1. To screen effective siRNA sequences targeting MCPH1 gene. METHODS: We generated two siRNA sequences targeting against MCPH1. then the annealed duplexes were were transfected into CaSki cells. The effect of siRNA on the transcription and translation of MCPH1 gene was analyzed by RT-PCR and Western blot.RESULTS: Transcription level of MCPH1 gene decreased significantly, the amount of Microcephalin protein also decreased significantly. The siRNA sequence 1 group decreased more than siRNA sequence 2 group. CONCLUSION: siRNA can inhibit the expression of MCPH1 gene, and the silence effect of siRNA sequence 1 was better than siRNA sequence 2.2.To construct the recombinant retroviral vector with MCPH1 gene targeted shRNA, and establish its stable CaSki cell line.METHODS: For preparation of duplexes, sense- and antisense-stranded oli- gonucleotides were mixed and annealed, the annealed duplexes were cloned into the sites of BglII and Hindâ…¢of the pSUPER retroviral vector for constructing the recombinant retrovirus plasmid. Then the plasmid was packaged through PT67 cells in order to prepare the recombinant retrovirus expressing shRNA of MCPH1 gene. Retrovirus serum was collected and used to directly transfect CaSki cells. After puromycin screening, cells with stable expression of shRNA were cultured. The silencing effect of MCPH1 gene were determined by RT-PCR, real time PCR and Western blot assay. RESULTS: Sequencing revealed that shRNA was successfully cloned into the pSUPER retrovirus vector. We got two vectors called pSUPER- shRNA-MCPH1 and Negative control. After being transfected into CaSki cells, the pSUPER-shRNA-MCPH1 was found to inhibit the MCPH1 gene effectively.CONCLUSION: The pSUPER-shRNA-MCPH1 retrovirus vector was successfully constructed. It shows effective inhibition on the expression of MCPH1 at mRNA and protein levels, which is the basis for further study of molecular functions of MCPH1, may be potentially useful in cervix cancer gene therapy.