| Ovarian cancer is the highest mortality gynecological cancer, which has a serious threat to the health and lives of women. Epigenetics in ovarian cancer has drawn more interest. EZH2is a methylase, a catalytic sub unit of the PRC complex and plays epigenetic roles through methylation of histone3lysine27. Histone3lysine27(H3K27) methylation could induce transcriptional repression, and participate in the regulation of gene expression. Several literatures showed that EZH2were overexpressed in various tumor tissues, such as breast cancer, ovarian cancer, prostate cancer, and colon cancer. DZNep is a widely-methyl transferase inhibitor. As an inhibitor of EZH2, the potential therapeutic role of DZNep in a variety of tumors has increasingly gained the attention of the researchers. Transgenic mice are a good model to study tumor development. The gene knockout technology provides a method to study a particular gene function in mice.PART I:Research of EZH2in the proliferation and clone formation of A2780cancer cellsBackground:Currently, researches on the biological characteristics of ovarian cancer, clinical treatment and medical technology have gained great progress.The morbidity and mortality of ovarian cancer is still high. EZH2is a methylase, which belongs to PRC complexes, and leads to histone H3lysine27methylation. Several researches showed that EZH2were overexpressed in several tumors, such as breast cancers, ovarian cancers, prostate cancers, colon cancers and so on. Based on previous studies, we may speculate that EZH2could play an important role in the proliferation and colony formation of A2780cancer cells.Objective:This study is aimed to find the role of EZH2in the proliferation and colony-forming ability of A2780after knocking down EZH2, and to explore the role of EZH2in ovarian cancer progression.Methods:The lentiviral infection was used to build stable knocked down EZH2A2780cell line. MTT assay was used to test the proliferation of A2780-shEZH2. Colony formation assay was applied to detect the clone formation ability of A2780-shEZH2cell lines. Real-time quantitative PCR was used to detect the mRNA expression of EZH2, CDKN2A and DAB2IP. Results:(1) we built A2780-shEZH2-1and A2780-shEZH2-2, which could stabilize down-regulated EZH2successfully.(2) MTT assay:Compared with A2780shctrl, the inhibition rate of A2780shEZH2-1and A2780shEZH2-2were72.9±4.13%and64.9±3.26%respectively in the24hours;49.9±3.37%and49±4.05%in the48hours,56.1±2.18%and57.9±2.17%in the72hours. Compared with A2780shctrl, the two cell lines have significant differences (P<0.05).(3) Colony forming experiment: Compared with A2780shctrl, A2780shEZH2-1and A2780shEZH2-2, two groups of ovarian cancer cell colony formation were significantly reduced (P<0.05). The control group, A2780shEZH2-1and A2780shEZH2-2colony forming ability were9.8%,2.0%and1.9%respectively.(4) Real-time quantitative PCR results:Compared with A2780shctrl, the mRNA expression of EZH2, CDKN2A, DAB2IP in A2780shEZH2-1and A2780shEZH2-2are respectively62.8±8.12%and65.3±5.31%,230±4.31%and237±4.42%,269±7.62%and297±6.52%. CDKN2A and DAB2IP mRNA in A2780shEZH2-1and A2780shEZH2-2are significantly increased (P<0.05).Conclusion:(1)Successfully set up two stable EZH2knock-down A2780-shEZH2cell lines. As a good experimental model, it could be used to study the relation between EZH2and ovarian cancer biology.(2)Long-term lenti-virus particles pLKO.1-shEZH2could specific silence EZH2expression of A2780ovarian cancer cell line efficiently.(3)Long-term lenti-virus particles pLKO.1-shEZH2could significantly inhibit the proliferation and colony formation of A2780.(4) Long-term lenti-virus particles pLKO.1-shEZH2could significantly decrease the expression of tumor suppressor gene CDKN2A and DAB2IP, and promote the progression of ovarian cancer. PART II:the role of DZNep in the proliferation, apoptosis, and migration of A2780ovarian cancer cellsBackground:DZNep, a cyclopentanyl analog of3-deazaadenosine, is a widely methylation transferase inhibitor. It could reduce the expression of EZH2, SUZ12and EED and inhibit27lysine methylation of histone3. Recently, researchers found that DZNep could suppress EZH2expression, and inhibit cell migration and invasion of glioma and melanoma cancer cells. Avan found that inhibition of EZH2by DZNep and combined with gemcitabine can significantly reduce migration, proliferation, apoptosis, migration of ductal adenocarcinoma cells. Chiba et al used shRNA interference and DZNep to inhibit the EHZ2expression of liver stem cells, and found both could inhibit the growth of liver cancer stem cells and sphere formation. The cause of these changes may be related with impaired liver stem cell self-renewal capacity by interfering EZH2. Crea et al found DZNep could increase the anti-metastatic gene expression, and inhibit the metastasis. Currently, DZNep have a promising prospects in therapy of breast cancer, lung cancer, and brain tumors.Objective:We want to decrease EZH2by using chemical inhibitors DZNep and observe its effect on proliferation, migration, apoptosis of A2780ovarian cancer cells. We want to explore the possibility of DZNep in the treatment of ovarian cancer, and provide a new therapeutic strategy for the treatment of ovarian cancer.Methods:(1) MTT assay was used to detect effect of different concentrations (2μM,4μM,6μM,8μM, and10μM) of DZNep on proliferation of A2780;(2) Flow cytometry was detected whether5μM DZNep could induce apoptosis of A2780;(3) Would healing assay was used to test the migration of DZNep on A2780cancer cells;(4) Real-time quantitative PCR was applied to detect the expression of the migration-related factors E-cadherin and mir-101in the DZNep-treated A2780cancer cells.Results:(1)DZNep could inhibit the proliferation of A2780:DZNep could inhibit proliferation of A2780in a concentration-dependent manner.(2) DZNep could induce the apoptosis of A2780:compared with the control group, after5μM DZNep treatment for24h,12.27±1.2%of A2780cancer cells initiate apoptosis (P<0.05);(3) DZNep could inhibit the migration of A2780:in DZNep group after the treatment with48h, scratch distance of A2780was significantly wider than that in the control. There was significantly different between the two groups (P<0.05);(4)Compared with the control group, EZH2, the expression of E-cadherin and miR-101in the DZNep treatment group respectively is32.7±1.1%(P<0.05),14.6times(P<0.05), and1.16times(P>0.05). Conclusion:(1) DZNep could inhibit proliferation of A2780in a concentration-dependent manner.(2) DZNep could induce the apoptosis of A2780.(3) DZNep could increase the expression of E-cadherin by reducing the of EZH2, thereby inhibited migration of A2780.(4) DZNep didn’t change the mir-101and inhibited migration of A2780. Part III:The effect of EZH2on the formation of tumors in LSL K-rasG12D/p53fl/fltransgenic miceBackground:Transgenic animal can be inserted into the target gene for gene over-expression studies, or inserted a gene fragment of shRNA to reduce the expression of target gene, which are knowed as knockin and knockout mice, respectively. Gene knockout offers an possibility to study the function of a particular gene in mice. Transgenic mice were used in the research of mechanism of ovarian cancer. Several papers showed that there were similar gene changes in endometriosis and ovarian cancer, such as p53, PTEN, K-ras, HNF-land so on. Daniela et al showed that K-ras and PTEN played an important role in the endometrioid ovarian cancer. Kinross used the LSL K-rasG12Dand PTEN transgenic mice and validate that the PI3K/Akt inhibitor PF-04691502could cure the ovarian cancer. Johnson et al used LSL K-ras G12D transgenic mice as a lung cancer model. In their experiments, Cre could induce the K-ras mutation and leads to the lung cancer.Therefore, LSL K-ras G12D transgenic mice could act as a model for studying tumor mechanism in vivo.Objective:Knock down EZH2in the LSL K-rasG12D/p53fl/fl transgenic mice, and explore the role of EZH2in transgenic mice tumor formation.Methods:Feed LSL K-rasG12D and p53fl/fl transgenic mice and set up K-rasGI2D/p53fl/fl transgenic mice. Set up Cre-shEZH2plasmid. Inject virus through ovarian bursa and explore the role of EZH2in the endometriosis and ovarian cancer. Inject virus into the lung and explore the role of EZH2in the lung cancer. Results:(1)Set up LSL K-rasG12D/p53fl/fl transgenic mice successfully.(2)Three Cre-shEZH2-pgk plasmids were set up:Cre-shnontarget, Cre-shEZH2-1,Cre-shEZH2-3. Realtime PCR and Western blot showed that shEZH2-land shEZH2-3could significantly decrease the expression of EZH2. Western blot showed that those plasmids could express Cre enzyme in DF-1.(3)Transgenic ovarian cancer model: After injection of Cre-shnontarget and Cre-shEZH2-1virus into the ovarian bursa, there was no difference in the ovary. There was no proliferation of epithelial ovarian cells in HE slide. There was no change of endometriosis and endometrioid ovarian cancer.(4) Transgenic lung cancer model:After injection of Cre-shnontarget and Cre-shEZH2-1virus into the lung, there are several tumor nodules in both groups. Compared with the Cre-shnontarget group, there are less tumor nodules in the transgenic mice injected with Cre-shEZH2-1. Also, the tumor nodules are smaller than those of control group.Conclusion:(1)Set up the LSL K-rasG12D/p53fl/fl transgenic mice and provide the animal models for studying some gene.(2)Setup the Cre-shEZH2plasmid which could silence the EZH2and provide a new toolsfor EZH2.(3)Setup LSL K-ras G12D/p53fl/fl transgenic mice lung cancer models.(4)Silencing the EZH2could inhibit the progression of lung cancer. It implies that EZH2could act as a new target for lung cancer gene therapy.(5)Explore the methods on virus injection into the ovarian bursa, and provide experience for the follow-up construction of transgenic mice animal models of ovarian cancer. |
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