The Research On CDC25A Inhibition Affecting The Proliferation Of CNE-2 Cell In Radiotherapy By RNAi

【Background】The nasopharyngeal carcinoma(NPC)is a high incidence of malignant tumors in southern China.the radiation therapy has been regraded as the standard treatment of NPC,which is the consensus for the scholars.Whether in the southern China with a high incidence of NPC and Southeast Asian countries, or in the western countries with a lower incidence of NPC,the NPC has a similar clinical and biologic behavior.As the special anatomical location of the nasopharyngeal,NPC infiltrating a strong growth characteristics,it is difficult to be early detected in the clinic,so most patients were diagnosed in the later period.As the radiotherapy equipment,techniques,physics and radiation biology research constantly developing recently,the simply radiation therapy for NPC have greatly increased,howere,there is high failure rate of radiotherapy treatment.Different scholars have different reports,patients with different clinical stages is main reason,but they all agreed that the local recurrence and distant metastasis leaded to the failure of radiotherapy treatment.A study pointed out that there might be the accelerate proliferation of tumor cells phenomenon in the NPC radiotherapy,The results in the clinical study also suggested that the accelerated proliferation migth occur four weeks after radiotherapy treatment.To explore the mechanism of the accelerated proliferation of tumor cells in NPC radiotherapy,the older generation of the author made a lot of researchs. The experimental studying on the NPC cell line CNE-2 cells confirmed that there was the accelerate proliferation phenomenon of tumor cells in the irradiation(mid-late stage)of the NPC cell line,and found that the remarkable difference gene was CDC25A,which difference was more than two times between the expression of the tumor cells at the highest activity proliferation phase and the minimum phase in the irradiation,so I choose CDC25A as the target gene to research.I used the RNA interference technology,which got the 2006 Nobel Prize in Physiology,to study CDC25A.RNAi is a newly developed effective method for gene closed expression,a gene silencing induced by double-stranded RNA(dsRNA)and leading the double-stranded RNA into cells.The activiting small interfering RNA(siRNA)was produced by the enzyme Dicer,and the RNA of a specific homologous mRNA degradated,as a result,the specific post-transcriptional gene expressed gene silencing.It characteristics with specificity,high efficiency inhibition,which couid be of proliferation and inherited.RNAi technology is an alternative to the anti-RNA and gene knocking out technology in gene function study,because of its unique mechanisms and the simple technical process.As new method of gene therapy research,the RNAi technology has been widly applied and rapid developed,however,there aren't any reports on NPC radiotherapy and accelerated proliferation by RNAi technology.【Purpose】Using RNA interference technology silent gene of CDC25A of NPC cell line(CNE-2),to find out the growth and proliferation changes of the NPC cell line(CNE-2)in irradiation,after the gene CDC25A silencing.To exposure the impact of NPC cell line(CNE-2)by gene CDC25A,to further understand the molecular mechanism of the accelerated proliferation of NPC cell line(CNE-2) in irradiation,to provide theory basis for the unconventional segmentation radiation therapy of NPC.【Method】1.Constructing the cell lines of the suppression CDC25A expression by RNAi technology:Constructing siRNA expression vector by siRNA expression vector,the constructed Psilence4.1-CDC25A carrier will be transfected into the CNE-2 by cationic liposome-transfected,by the G418 toxicity test screening, positive clones limited dilution screening methods,and cultivate a CNE-2 cell line which could inhibit the CDC25A expression.2.Using Reverse transcriptase-polymerase chain reaction(RT-PCR)and Western Blot to certify the interference effects of selected cell lines in the mRNA levels and protein levels,detecting the expression changes of CDC25A of cells in radiation.3.Using Tetrazolium salt color method(MTT)and flow cytometry(FAM) to detect the growth and proliferation of cells and changes in the phase of the cell cycle distribution in the Co-γ-60,2 Gy/d for 5 days irradiation.[Results]1.The vector of Psilence4.1-CDC25A had been Constructed,and the carrier comply with the equirements by the sequencing certified.Finally,four CNE-2 cell lines with inhibition CDC25A expression were produced by transfection, screening,training,and 3 control cell lines were produced.2.The inhibition rate of Psilence4.1-CDC25A cell was 49.6%by RT-PCR, the inhibition rate was 52.5%by the Western Blot method;in the irradiation conditions,the results of RT-PCR,Western Blot Detection show that the CDC25A expression of Psilence4.1-CDC25A cells were lower than the control cells psilence4.1 in the first,third,fifth day.3.The result of MTT and the growth and survival curves of psilence4.1-CDC25A cells showed that show that survival scores of psilence4.1-CDC25A cells in the first day,second day,third day,fourth day,fifth day were 128%,110%,127%,138%,149%,the highest survival scores is which in the fifth day.The result of flow cytometry analysis(FCM)showed that, psilence4.1-CDC25A cells in the irradiation fifth day,the PI and SPF were the highest values.The PI values of the psilence4.1-Control cells is the highest in the forth day by irradiated,SPF value is the highest of the third day by irradiated; compared with the two,the trend of growth and proliferation of Psilence4.1-CDC25A cell have slowed.【Conclusion】1.To successfully produce the CNE-2 cells which inhibit the expression of CDC25A,named Psilence4.1-CDC25A cells,the control cells named psilence4.1-Control cells.2.whether in the non-irradiated conditions or in the irradiation conditions, the expression of CDC25A is downward after the gene CDC25Aof Psilence4.1-CDC25A cells interfered,which indicates that the interference of CDC25A is effective and stable.3.The trend of growth and proliferation of Psilence4.1-CDC25A slowed,and the peak of growth and proliferation delayed,which suggest that accelerated proliferation of tumor cells in NPC radiotherapy after interference of CDC25A,however,it doesn't say that accelerated proliferation of tumor cells was dispear.It is a good evidence to use unconventional fractionation radiotherapy in clinc.