The Influence Of Radiosensitivity Of Human Hepatoma Carcinoma Cell By Selective Inactivation Of N-Ras Gene

ObjectiveThe primary hepatocelluar carcinoma is most common maligancies in our country,The death of primary hepatocelluar carcinoma was 18.8% of total malignanttumor.The mainstay of HCC treatments today consists ofsurgery,radiotherapy, transcatherater arterial chemotherapy. The surgery is most maintreatments of the HCC,But the majority of HCC patients have no chance of surgicaltreatment because of late stage of disease, the poor hepatocelluar function,and specialsite and so on.The patients who get surgery was 5-10% of total HCC patients.So it isimportant to seek a new effective treatment appoach. As auxiliary treatment methodof surgery-radiotherapy and chemotherapy, Although therapeutic response toradiotherapy and transcatherater arterial chemotherapy had been great improved, butthe effect was still limited,the key factor was thought about that radiotherapy andtranscatherater arterial chemotherapy were improve suppression gene expression tocause the carcinoma cell to resistent of chemotherapy and radiotherapy. So seek amethod of the gene interfering method to increase treatment effect appears extremelyimportant.At present,The ocurrance of carcinoma was thought about the gene mutationwhich control cell proliferation,cell differentiation and apotosis.Ras gene was paid close attention in these gene because of it was most common activedgene,proto-oncogene Ras is multiple function cytokine,it's constraction and functionhave extraordinary conservation in nature,it has extreme function in multiple vitalmovement.Also it was one of first confirmed gene which connect with the currence ofcarcinoma.Ras gene family incloud three member,one of which has one pointmutation were activate Ras gene which lead to protein 21 excessive expression,thesignal of transduction was introducued in cell.These lead to the cell canceration orcell transformation.Gu first obtained N-Ras gene from hepatocelluar carcinoma genebank in 1988,thia also improved that N-Ras gene has close connection withhepatocelluar carcinoma.In many creature cell,exogenous and endogenous double RNA may causehomologization mRNA degradation,it lead to this gene silence,this phenomenon werecalled RNA interference, this gene slience occurrenced after transcription,so it alsowas called post transcription gene silence.The technology of RNAi has characteristicon rigorous sequence specificity, high performance, definitive effect of interference,rare side effect and so on. opposite to antisense oligonucleotides,MCAB,RNAi hasdistinguished specificity.moreover it has gene silence in short time.This technologywhich lead gene silence was widespread applicated on creature,futhermore the targetgene expression was cut down highly significant. Andrew Z.Fire from US StanfordUniversity medical college and Craig C.Mello form Massachusetts University cancercenter which obtained Nobel physiological science/medical science prize Becausethis technology was found them.MethodAfter cell cultivate, the carcinoma cell creep on glass plate,In order to know ifthe hepatocelluar carcinoma cell(HepG2 and MHCC97-H) have N-Ras gene mutationthrough the method of immunohistochemistry. Design small RNA interference series was supplied by ofgene bank. After RNA interfering we definite if the N-Rasinterferce series transfected in carcinoma cell through fluorescent microscope.wethrough many methods which are western blot,RT-PCR,immunohistochemistry todetect the effect of RNAi by N-Ras protein expression,mRNA N-Ras absorbancecompared with GADPH.calculate the inhibition rate of RNAi N-Ras.Detect each holeabsorbance at wavelength 570nm (A optimum) thorugh immunodetection equipment,calculate the inhibition rate of RNAi N-Ras and the growth of carcinoma cell.we usedflow cytometry to detecet the variation of cell cycle and the rate of cell apotosis.Atlast we research the number of cell colony after different dose radiation through thecontrast group which have no RNAi interference.We got radioactive biologicalparameter through setting up the L-Q linear model and one-hit multitarget model byUsing Graphpad prism 4.0 sofeware, and identify if the RNA interferce N-Ras geneincrease the radiosensitivity ofhepatoma carcinoma cell HepG2 and MHCC97-H.Result1. The expression of N-ras protein of Hepatoma carcinoma cell HepG2 andMHCC97-H were powerful positive.It illustrated that the HCC has close connectwith N-Ras gene mutation.2. The average efects of transfection of Hepatoma carcinoma cell HepG2 andMHCC97-H respectively was 93% and 91%.through the rate of green fluorescentcells to total cells3. The inhibition of RNAi N-Ras gene of Hepatoma carcinoma cell HepG2 andMHCC97-H respectively was (61.3±3.351)% and (96.9%±0.159)%,It havesignificant difference contrast with the group of blank plasmid by RT-PCR whichcan show the variation of density of mRNA.4. The inhibition of RNAi N-Ras gene of Hepatoma carcinoma cell HepG2 andMHCC97-H respectively was (63.7%±0.041)% and (89.8±0.012)%,It have significant difference contrast with the group of blank plasmid by western blotwhich can show the variztion of density of protein.5. The inhibition of RNAi N-Ras gene of Hepatoma carcinoma cell HepG2 andMHCC97-H respectively was 60% and almost 100% by immunohistochemistry.6. The speed of growth of the Hepatoma carcinoma cell HepG2 and MHCC97-HWere Slow down after RNA interference.MHCC97-H cell show significant differenceon the day after RNAi three days contrast with the group which have not RNAinterference. HepG2 cell show significant difference on the day after RNAi the oneday contrast with the group which have not RNA interference.7. The inhibition of growth of the Hepatoma carcinoma cell HepG2 and MHCC97-Hrespectively was 21.9% and 23.8%8. The G1 phase proportion of has significant difference after RNAi contrast withuntransfected group. RNAi also enhenced cell apotosis.9. Hepatoma carcinoma cell HepG2 and MHCC97-H after RNAi received differentdose irradiation, The value of D₀ Dq SF2α/βof Hepatoma carcinoma cell HepG2and MHCC97-H after RNAi contrast with untransfected respectively was2.38±0.05vs2.33±0.10;3.00±0.077vs3.34±0.015,2.88±0.087vs2.28±0.018;2.89±0.042vs1.83±0.22,0.824±0.001vs0.742±0.020;0.817±0.008vs0.716±0.013,0.938±0.132vs2.98±1.232; 4.210±1.536vs9.837±2.234.The variation of the valueof SF2 and Dq have significant difference.The SER of Hepatoma carcinoma cellHepG2 and MHCC97-H after RNAi respectively was 1.10VS1.15.Conlusions1. The occurrence and development of Hepatoma carcinoma cell has close connectwith N-Ras gene.2. The confirmed siRNA targeting mRNA of human N-Ras gene was synthesizedand insert in Hepatoma carcinoma cell HepG2 and MHCC97-H can silence N-Ras gene expression,vary distribute of the cell cycle-G1 phase increased,and enhancecell apotosis, the growth speed of the different status P53 Hepatoma carcinomacell slow down evidently.3. The investment first apply RNA interferce technology to study the different statusP53 Hepatoma carcinoma cell radiosensitize after RNAi,The reaserch indicatedthat RNA interference N-Ras gene can change the radioactive biologicalparameter of the two cell,enhanced radiosensitivity of the Hepatoma carcinomacell HepG2 and MHCC97-H.