Preliminary Study On The Effects Of Brit1Gene Invovled In Cell Cycel And Apoptosis In Cervical Cancer Cell Line Hela

Cervical cancer is the most common gynecologic reproductive tractmalignant tumor. According to a report in the global scope, more than200,000women died from cervical cancer every year. In recent years,cervical cancer incidence was significantly increased and showed youngertrend. Cervical cancer has become a serious disease among women. Studieson cervical cancer become hotspots in the area of cancer research.The functional study of BRIT1is a hot spot worldwide. BRIT1(BRCT-repeat inhibitor of hTERT expression) is also known as MCPH1(microcephalin).This gene is also called cerebellum disease gene, which isone of the main virulence genes causing primary microcephary with thecharacteristics of reduced brain volume and mental retardation. BRIT1playsan important role in the response of DNA damage and its basic function hasdocumented. Thus, some researchers speculate that BRIT1may be of greatimpotence in tumorigenesis，however its mechanism is not clear. In thepresent study, we preliminarily studied the effects on cell cycle andapoptosis by knocking down BRIT1or over-expressing BRIT1in cervical cancer cell line HeLa.1. Designing of siRNA targeting BRIT1and to study the efficiency ofsiRNA in silencing of BRIT1on cell cycle and apoptosis in HeLa cells.Objective: To design siRNA targeting BRIT1and transfect in HeLacells to study the effect on cell cycle and apoptosis in HeLa cells. Method:Three siRNAs and one negative control siRNA were designed and thentransfected into HeLa cells. The efficiency of siRNA on the transcriptionand translation of BRIT1gene were analyzed by RT-PCR and Westernblot. The cell cycle and apoptosis were assayed by Flow cytometry(FACS).Results: Transcription level of BRIT1gene was decreased significantly, theexpression of BRIT1protein was also decreased significantly. Theefficiency of siRNA sequence1and2were more significant than siRNAsequence3. Effects of the knockdown of BRIT1gene on the cell cycle andapoptosis have no statistical significance. Conclusion: Transfection ofsiRNA can inhibit the expression of BRIT1gene, and the silencingefficiency of siRNA sequence1and2were better than siRNA sequence3,which would lay a foundation for constructing the recombinant retroviralvector with BRIT1gene targeted shRNA for further study on the functionof BRIT1in cervical cancer. Considering the cell cycle and apoptosischange have no statistical significance, so we are not studying theconstruction of retroviruses carrier in this paper.2. To study the effects of over-expressed BRIT1on cell cycle and apoptosis in HeLa cells.Objective: To study t the effects of overexpressed BRIT1on cell cycleand apoptosis in HeLa cells. Methods: The over-expression recombinantvector of BRIT1and the negative control vector were constructed. Therecombinant vectors were confirmed by enzyme analysis and DNAsequencing. The recombinant vectors were then transfected into HeLa cellsby Lipofectamine2000.The mRNA level of BRIT1was detected by RT-PCRand Real time PCR. The protein level of BRIT1was examined by Westernblotting. The cell cycle and apoptosis were assayed by Flowcytometry(FACS). Results: The over-expressed recombinant vector ofBRIT1was successfully constructed, which can effectively increase themRNA level and protein level of BRIT1gene after transfection. Flowcytometry results showed the apoptosis rate of cells transfected withover-expressed recombinant vector of BRIT1was（12.37±0.19）%,whichwas significantly higher than negative control group （1.81±0.22）%andwithout disposal group （2.06±0.10）%. Effects of over-expressed BRIT1gene on the HeLa cell cycle have no statistical significance. Conclusion:Over-expression of BRIT1gene can induce apoptosis in cervical cancer cellline HeLa, which would establish a favorable foundation for further study onthe function of BRIT1in cervical cancer.