| BACKGROUNDInvasion and migration are the important characteristics of the metastasis ofmalignant tumor, account for about90%of all ovarian carcinomas patients died fromthe tumor recurrence and metastasis. Ovarian cancer is one of the most severegynecological malignancies known and has now become the most lethal of allgynecological malignancies. Because of hidden clinical symptoms,75%of patientshas been in later period when diagnosis. Although more than80%of the patients hasreceived benefit after first-line treatment, whereas the5-year survival rate of patientswith stage III/IV ovarian cancer is between25%and30%.Therefore, to understanddeeply the molecular mechanism of the the occurrence and development of ovariancancer and seek new treatment methods are urgent.Current gene therapy has provideda new hope for the treatment of ovarian cancer. In recent years the rise of RNAinterference technology which has strong gene silencing effect after transcriptional.Due to its high efficiency and specificity show a strong advantage in basic research.However,how to exert the most advantages of clinical targeted therapy will still be aproblem.RhoA gene is one of the members of the Rho protein family, which acted as a"switch" gene of invasion and metastasis of malignant tumor is involved in metastasisof many malignances. The activation of RhoA gene may cause cells transform tobecome cancer. Recent study indicate that RhoA is associated with the movement andshape of the cell,cell growth and proliferation,invasion and migration, cell apoptosisand Tumor angiogenesis formation. Therefore, gene therapy that target RhoA gene isexpected to become a new direction in the therapy targeting ovarian cancer. In our previous study, we has successfully cloned a novel RhoA gene stabledown-regulation cell line by using lentivirus mediated gene silencing technologycalled RNA interference. Reduce expression of RhoA gene by lentivirus vectormediated RNAi may inhibit the proliferation, invasion and migration ability ofovarian cancer cell. Because of evaluating the feasibility and effectiveness ofinterventions for ovarian cancer in vitro experiment is not entirely true，to establish anude mice transplantation tumor model for simulation of the ovarian cancer pathologyand evaluating treatment is necessary and vital. Therefore, on the basis of preciousstudy, ovarian cancer nude mice transplantation tumor model was built to expore theinhibitory effects of RhoA gene silence on malignant biological behaviors of humanovarian cancer in nude mice intraperitoneal xenograft, and expore the effect andmechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer innude mice subcutaneous xenograft in vivo.Chapter I Inhibitory effects of RhoA gene Silence on malignantbiological behaviors of human ovarian cancer in nude miceintraperitoneal xenograftOBJECTIVE: To establish intraperitoneal xenograft model of human ovariancancer and investigate effects of RhoA gene silence on the malignant biologicalbehaviors of ovarian xenograft in nude mice in vivo.METHODS:1.21female nude mice were randomly assigned to three groups:HO8910-RhoA-shRNA group, HO8910-RhoA-NC group and HO8910group. Thestable knockdown of RhoA cell line, negative control cell line and ovarian cancercell line HO8910were inoculated respectively into abdominal cavity of eachgroup to establish intraperitoneal xenograft model of human ovarian cancer.2. Abdominal circumference were recorded every two days, the nude mice weresacrificed four weeks post-inoculation. The ascitic volume, dissemination position number, disseminated tumor number, tumor weight and tumor growth inhibitionrate were recorded.3. Histopathological analysis for xenograft tissues were observed by HE staining.4. The expression of RhoA mRNA and protein of xenograft tissues were detectedrespectively by real-time qPCR and Western blot.5. Cell apoptosis in tumor tissues were observed by TUNEL method and apoptoticindex (AI) were counted.RESULTS:1. Abdominal circumference in HO8910-RhoA-shRNA group growth delay werestatistically significant (P<0.05).2. The HO8910-RhoA-shRNA group had decreased in ascitic volume (P=0.01),dissemination position number (P<0.001), disseminated tumor number (P<0.001)and tumor weight (P<0.001), with tumor growth inhibition rate of70.62%.3. The relative expression of RhoA mRNA and protein were both significantlydecreased in HO8910-RhoA-shRNA group (P<0.001).4. AI were significantly increased in HO8910-RhoA-shRNA group (P<0.001).CONCLUSION: RhoA gene silenced by Lentivirus-mediated RNAi cansignificantly suppress the malignant biological behaviors of human ovarian cancer innude mice xenograft.Chapter II Effect and mechanism of gene therapy of lentivirusmediated RhoA shRNA on ovarian cancer xenograft in vivoOBJECTIVE: To investigate treatment effect of lentivirus mediated RhoAshRNA on xenograft tumor of ovarian cancer in nude mice in vivo and underlyingmechanism.METHODS:1. Human ovarian cancer cell line HO8910were inoculated to establishsubcutaneous xenograft model of human ovarian cancer. Tumor-bearing nudemice were abandonly assigned to three groups: Lenti-RhoA-sh group, Lenti-NC(Negative control）group and PBS（Phosphate Buffered Saline）group;lentivirus mediated RhoA shRNA, negative control lentivirus and PBS wererespectively injected in the three groups. Effects of treatment were observed bytumor growth curve, tumor volume，tumor weight, and tumor inhibition rate.2. Xenograft tissues and liver, spleen, lung, and renal tissues were histopathologicalanalysis by HE staining.3. The changes of RhoA gene expression in xenograft tissues after lentivirusmediated RhoA shRNA treated were detected by real-time qPCR,immunohischemistry and Western blot assay.4. Cell apoptosis in xenograft tissues were examined by TUNEL method andapoptotic index (AI) were counted.RESULTS:1. Compared with Lenti-NC group and PBS group, the growth speed of xenograft inLenti-RhoA-sh group delayed significantly from post-injection9days（P=0.000）.Tumor volume (338±114) mm3statistically significant decreased in theLenti-RhoA-sh group when compared with Lenti-NC group（1190±332）mm3andPBS group（1101±396）mm3(P=0.000,P=0.001).Tumor weight (0.23±0.11）gstatistically significant decreased in the Lenti-RhoA-sh group when comparedwith Lenti-NC group（0.79±0.19）g and PBS group（0.74±0.17）g （P=0.000）.2. Real-time qPCRresult displayed that the expression of RhoA mRNA（0.30±0.05）statistically significant decreased in the Lenti-RhoA-sh group compared withLenti-NC group (0.95±0.06) and PBS group(1.00±0.11)(P=0.000).Western blotresult displayed that the expression of RhoA protein (0.14±0.06) statisticallysignificant decreased in the Lenti-RhoA-sh group compared with Lenti-NCgroup(0.78±0.14) and PBS group (0.75±0.13)3. TUNEL staining displayed that AI significantly increased in theLenti-RhoA-sh(（20.9±3.4）%compared with Lenti-NC group（5.2±2.0）%andPBS group（6.0±2.1）%（P=0.000）.CONCLUSIONS:1. Lentivirus mediated RhoA shRNA may effectively down-regulate of theexpression of RhoA in xenograft, inhibit the growth of subcutaneous xenografttumor of ovarian cancer in nude mice in vivo 2. The underlying mechanism of treatment of Lentivirus mediated RhoA shRNA inhuman ovarian cancer xenograge in vivo is by the means of increasing the cellapoptosis.3. No obvious toxicity in lentivirus mediated gene therapy was found in animalexpresiments in vivo, it indicates lentivirus mediated gene therapy may be arelatively safe treatment. |
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