A Study Of The Impact Of Inhibiting The Expression Of CUL5 Gene With RNAi On The Status Of CNE2 Cell Proliferation During Continuous Irradiation

Background and objectives:Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China, especially in the southern part of the country. Owing to its special anatomic site and characteristic of radio-sensitivity, presently radiotherapy was considered a preferred means of treatment. Conventional radiotherapy has been prevailingly adopted in clinical practice of NPC and other malignant tumors treatment for long, and has achieved satisfactory effect. However, some NPC conventional radiotherapy failure occurs as a result of being out of control or local recurrence after the threatment. Many studies on the failure of the NPC conventional radiotherapy believe that the proliferation of tumor stem cells accelerated during radiotherapy; therefore it becomes crucial to make clear the mechanism and find out the solution to the accelerated proliferation of tumor stem cells during radiotherapy and improve the local control rate.Researches have shown evidence that the alteration of proliferative activities of irradiated cells has correlations with the cell cycle genes. In order to investigate into the mechanism of the accelerated proliferation of tumor cells in the process of NPC conventional radiotherapy, our team has in the first phase done research with various methods to study the proliferating status of CNE2 cell lines in the process of continuous fractioned irradiation, and confirmed that CNE2 cell lines had demonstrated accelerated proliferation in the midanaphase of the continuous fractioned irradiation. At the meantime, gene chip technique and RT-FQ-PCR have been adopted in bolting the differential genes (including CDC25A,CUL5,p21,DDA3,CCNG1,ABL1,CDKN1A,CKS2,CCNE1,CDC2,CDKN2C,CCND3) correlated with the CNE2 cell line proliferating status after irradiation. Among the differential genes CUL5 is one of the up-regulated gene during the activation of cell proliferation, and it is more than twice the expression of that during the lower activity of proliferation,so it is chosen to be the targeting gene for the experiment. This study probes the expression of CUL5 gene's influences on cell proliferative activity in NPC irradiations by means of RNA inhibition technology in the experiment in vitro.RNAi is a recently developed technology of gene interference which achieve target mRNA degradation through dsRNA specific mediation, thus inhibit target gene expression. This technology has been widely used in the field of gene function and the gene therapy of communicable diseases and cancer. Since currently the implementation of RNAi technology in radiotherapy generally focuses on its sensitization, this study makes use of RNAi technology's speciality, highly-effectiveness and having no cytotoxicity in researching directly into CUL5 gene function of CNE2 cell lines, and confirms the role CUL5 plays on NPC accelerated proliferation in continuous fractioned irradiation. Therefore, this study ventured to reveal the molecular biology mechanism of accelerated proliferation of NPC conventional radiotherapy. Methods:1.Construct, culture the cell lines that stably inhibit CUL5 gene expression: construct recombinant plasmid pGPU6/GFP/Neo-CUL5, transfect lipofectamine into CNE2, bolt and culture the NPC cell line CNE2-pGPU6/GFP/Neo-CUL5 that inhibit CUL5 expression by G418 test and limiting dilution assay.2.Verify the interfering effectiveness of CUL5 gene in the constructed cell line before and after irradiation with RT-PCR3.Detect the proliferation and alteration of the constructed cell line in a 5-day-continuous fractionated irradiation of 60Co-y2Gy by MTT and FCM test.Results:1.DNA sequencing showed that the recombinant plasmid pGPU6/GFP/Neo-CUL5 identified by restricted incision enzymes were successfully constructed. CNE2-pGPU6/GFP/Neo-CUL5 cell line with CUL5 silencing and negative control cell line CNE2-pGPU6/GFP/Neo-NC has been reared after transfecting, bolting and culturing.2.Detect NPC CUL5 gene expression by RT-PCR, analyze the result of electrophoresis strip through gel imaging system, corrected by internal parameterβ-actin indicated that, the gray scale results of CUL5 in the blank control group (CNE2 group), the negative control group (CNE2-pGPU6/GFP/Neo-NC group) and experimental group (CNE2-pGPU6/GFP/Neo-CUL5 group) are respectively 0.122,0.175,0.004 before irradiation, and the inhibition of the experimental group CUL5 expression was marked; the inhibition of the experimental group CUL5 expression, compared with the control groups, was still marked during irradiation of the 1st,3rd and 5th day. 3.Dynamic changes of CNE2-pGPU6/GFP/Neo-CUL5 cell lines and control cell lines proliferation were detected by MTT and flow cytometry(FC)assay during 60Co-y2Gy 5-day-continuous fractionated irradiation.The peak of CNE2-pGPU6/GFP/Neo-CUL5 cell lines proliferation occurred in the 1st day while the control cell lines occurred in the 3rd day, and the valleys of proliferation both occurred in the 5th day.Conclusions:1.CNE2-pGPU6/GFP/Neo-CUL5 cell line with CUL5 silencing and negative control cell line CNE2-pGPU6/GFP/Neo-NC have been successfully reared.2.Both pre-irradiation (or non-irradiation) and post-irradiation, expression of CUL5 in CNE2-pGPU6/GFP/Neo-CUL5 showed down regulation, which indicate the interference of CUL5 is stable and effective.3.CNE2-pGPU6/GFP/Neo-CUL5's proliferation declined during the five day continuous fractioned irradiation and exhibited a down-ward tendency day by day, which indicate NPC accelerated proliferation of irradiation changed after the inhibition of CUL5 expression, this will provide empirical basis and theoretical foundation for the search for the target of NPC gene therapy and its targeting medication.