Expression And Prognosis Of C-erbB2 In Ovarian Carcinoma Cells & Specific Silencing (RNAi) Of C-erbB2 Expression By ShRNAs In SKOV3 Cells

C-erbB2 can be called "neu" gene, it is confirmed in 1985 and is relativewith v-C-erbB2.It can encode a glycoprotein of 185kD and is extremelysimilar to EGFR. It is relative with the development of human, tumors bygene amplification and the overexpression of protein production. There areabout 20%～40% of ovarian cancer which show higher amplification andoverexpression . Researchers have confirmed that the family of C-erbB2 palysan important part in the generation and development of malignanttumour.Their overexpression in ovarian cancer is not only closly correlatedto its generation, development and aversion,but also the drug fast ofchemotherapy,insensitivity to drugs and unfavourable prognosis.We have the ascendency that our department is researching ovariancancer.We refer to a lot of case files of patients who are suffered with ovariancancer and make research of the histochemistry of C-erbB2.We havecultured SK-OV3 cells while identificated the cells.We have tried someexperiments by applying the technique of RNAi。RNAi can specificly inhibitgene expresstion。We make sure 3 target sites according to VEGF1-5 exons。We have synthesized 5 pairs of siRNA by the transcription of T7 RNApolymerase in vitro.After transfecting the SK-OV3 cells,they inhibited theexpresstion of C-erbB2。We also make research of the expresstion ofC-erbB2 and sensitivity of chemotherapeutics。The studies are useful to thepathogenesygene and gene therapy of ovarian cancer。The family of EGFR is composed of C-erbB1,C-erbB2, C-erbB3 andC-erbB4。All of them have the activity of TPK。Many growth factor andgrowth factor receptors are oncoprotein 。It is considered that they paly angreat role in the development of cell cycle,cell survival and apoptosis .Ifgrowth factor receptors are independent on growth factors,anti-oncogenep53 and the activation of pRb,they can cause the loss of control ,nodifferentiation and apoptosis is inhibited.Because C-erbB2 is located in plasma membrane.So its diffusion islimited in two-way. Even if moderate overexpression of C-erbB2 ,receptorstructure can be actived.We can find that the C-erbB2 of some humancarcinomas is 10-100 times than that of normal glandular epithelium byhistochemistry dyeing.There is amplification and overexpression of C-erbB2 in human'stumours.It shows that C-erbB2 plays an important role in the generation oftumour. C-erbB2 trangenic mice provide a direct evidence. C-erbB2 canoverexpress in some tumors.It play a great part in malignant transformationand can promote malignant tumor meta stasia.It can also influence thegeneration and development of tumors.The overexpression of C-erbB2 is relatived with the generation,development,prognosis and drug sensitivity.C-erbB2 can not only reflectthe biology activity of malignant tumor ,but also produce a new method tocure tumor.We can take some measures to cure tumor:1)introduce antisenseoligonucleotides into cells to make parts of C-erbB2 inactivation;2)Viasome Rho factor,we can inhibit the expression of C-erbB2 on transcriptionallevel:3)Use antibody or other ligand to bind with C-erbB2 and inactiveC-erbB2 or lower its activity.My result of immunochemical show : Positive expression rate ofC-erbB2protein in ovarian cancer was 76.7%, significantly higher than thatin benign tumors and in normal controls(P<0 01);no significant correlationwas observed between benign tumors and normal control;No significantcorrelation was observed between positive rate of C-erbB2 protein andpathological type or grade in ovarian cancer;Positive expression rate of C-erbB2 protein in stageⅢ～IV of patient was much higher than that instageⅠ～Ⅱ of patient(P<0 01);and positive rate of C-erbB2 in patient withamount of ascites volume≥500ml was much higher than that with ascitesvolume<500ml(P<0 01);Positive expression rate of C-erbB2 protein in thatpatients with lymphnode metastasis or omentum involvement wassignificantly higher than without metastasis, and positive expression inpatients with tumor residue≥2cm was higher than that in those with none(P<005) The cumulate survival rate in patients with positive expression rate of C-erbB2 was lower than that in patients with negative expression(P<0 05).RNA interference (RNAi) –the post transcriptional gene silencing (PTGS)is one of the most exciting discoveries of the past decade in functionalgenomics. Duplexes of 21-nt RNAs, known as short-interfering RNAs(siRNAs), efficiently inhibit gene expression by RNA interference (RNAi)when introduced into mammalian cells. This phenomenon is now beingexploited as a powerful tool for reverse genetics, and shows great promisefor therapeutic applications. Angiogensis is thought to depend on a balancebetween endogeneous positive and negative regulatory molecules.Our experiment want to use siRNA to effectly interfere the expressionof C-erbB2 in SK-OV3 cells so that settle the base that we can cure theovarian cancer.We design the oligo-DNA template which contains T7-promotoraccording to C-erbB2 exons .By groping the annealing temperature ,wedesign the hairpin sequence.To evaluate the the inhibitory effection of siRNA,we can transfect it into SK-OV3 cells。Apply CCK-8 to count the cell growthdynamic so that observe the inhibitory effection。We can use semi-quantitationPCR and Western-Blot to evaluate the the inhibitory effection。RNAi result :inhibiting rate siRNAb> siRNAa> siRNAc;after RNAi Carboplatin (CBP)was used that had show the most remarkable inhibiting rate. Conclusion:Theexpression of C-erbB2 protein is correlated with the carcinogenesis andprogress of ovarian cancer, and can be regarded as a biomarker for predictingprognosis.