Study On The Biological Function And Potential Mechanism Of PRR11 Gene In Human Ovarian Cancer

Background:Ovarian cancer,as a common malignant tumour of the female reproductive system,has a slightly lower incidence than endometrial and cervical cancer.According to statistics,there are about 530,000 new cases of ovarian cancer in the world every year,and about 130,000new cases of ovarian cancer every year in China.Besides,ovarian cancer is also one of the most lethal malignant tumors in the world,and the most lethal tumors in gynecological tumors.Ovarian cancer has a high degree of malignancy,without typical clinical symptoms in the early stage.It is difficult to identify the malignancy degree and histological types of the tumor before operation,which leads to the loss of the best treatment opportunity.In addition,with the characteristics of small size,deep location,easy expansion,high drug resistance,recurrence,and abdominal metastasis,ovarian cancer exhibits poor overall prognosis,especially in stage III-IV patients.The 5-year survival rate is only about 25%-30%.In the past20 years,although the clinical application of radical surgery,tumor reduction therapy,multiple adjuvant drugs combined with chemotherapy and radiotherapy has brought new hope to patients with ovarian cancer,the drug resistance caused by chemotherapy still brings serious side effects to patients,and the overall survival rate remains low.Therefore,in-depth study of molecular mechanisms such as malignant drug resistance,recurrence,and metastasis of ovarian cancer,exploring early screening methods and more effective treatment for advanced-stage is still the focus of basic oncology research.In recent years,with the development of molecular biology,a series of mutations or inactivations of tumor suppressor genes（P53,BRCA,ARID1A）,abnormal activation of oncogenes（Ras,Her2,c-fms）and epigenetic changes（methylation level,histone modification）have been found to be closely related to the clinical characteristics,distant metastasis and recurrence of ovarian cancer.PRR11 gene,as a newly discovered candidate oncogene,is located in the 17q22 region of the human chromosome.Its biological structure consists of a binary nuclear localization signal,two proline-enriched regions and zinc finger domains.PRR11 has a wide range of targets,including membrane protein receptors,adhesion molecules,or growth factors and transcription factors,plays an important role in tumorigenesis and malignant progression.The effect of PRR11 on malignant tumors was first studied in lung cancer and involved in the regulation of cell cycle progression.Targeted knockdown PRR11 expression leads to down-regulation of RRM1 and RRM2 expression and decrease of DNA synthesis rate,resulting in cell cycle arrest in S phase,and thus significantly reduced cell proliferation.In addition,PRR11 is widely expressed in gastrointestinal malignant tumors,and the expression rate from high to low is esophageal squamous cell carcinoma,gastric cancer,duodenal cancer,colorectal cancer,liver cancer,which is an important prognostic indicator.Furthermore,PRR11 also promotes local infiltration and distant metastasis of cancer cells by regulating the mesenchymal transition of epithelial cells of various malignant tumors,such as gastric cancer and hepatocellular carcinoma.However,the expression of PRR11 in ovarian cancer and its function and mechanism in the pathogenesis of ovarian cancer remains unclear in the domestic and foreign literature.In this study,we intend to explore the expression and distribution of PRR11 in ovarian cancer tissues through clinical sampling,and analyze the relationship between PRR11 and clinicopathological characteristics of ovarian cancer patients and further investigate the role and mechanism of PRR11 in the biological behavior of ovarian cancer in vitro experiments,thus to provide new ideas and targets for clinical treatment of ovarian cancer.Part Ⅰ.The expression and relationship with clinicopathology of PRR11 in Ovarian CancerObjectives:The purpose of this study was to investigate the expression and distribution of PRR11 in clinical samples of ovarian cancer and to analyze the correlation between PRR11expression and clinical characteristics of patients with ovarian cancer,preliminarily exploring the significance of PRR11 in ovarian cancer progression.Methods:1.Real-time PCR and Western Blot assays were performed to clarify the expression of PRR11 in 51 cases of ovarian cancer and normal ovarian epithelium.2.Immunohistochemistry was preformed to validate the expression and localization of PRR11 protein in ovarian cancer tissues.The correlation of clinical features（age,histological grade,lymph node metastasis,FIGO stage and histological type）was further analyzed according to the expression intensity of PRR11 protein.3.The Kmplotter website analyze the correlation between the expression of PRR11 and the prognosis of patients with ovarian cancer.Results:1.Compared with normal ovarian epithelial tissues,PRR11 expression in ovarian cancer tissues increased at either the protein or m RNA level.2.PRR11 protein is mainly located in the cytoplasm of ovarian cancer cells.Clinical data analysis showed that PRR11 expression was positively correlated with FIGO stage（P=0.026）,lymph node metastasis（P=0.029）,but not associated with age（P=0.649）,histological grade histological types（P=0.869）and of patient（P=0.803）.3.The overall survival of patients with higher PRR11 expression was significantly lower than that of patients with lower expression of PRR11.Conclusions:The high expression of PRR11 in ovarian cancer tissues is positively correlated with the clinical malignant phenotype of patients.High expression of PRR11indicates a shorter overall survival and a worse prognosis of patients with ovarian cancer.PRR11 may be involved in the progression of ovarian cancer.Part Ⅱ.The expression of PRR11 in ovarian cancer cells and its effect on the proliferation of ovarian cancer cellsObjectives:The purpose of this study was to analyze the effects of silencing or overexpressing PRR11 gene on the proliferation of ovarian cancer cells.Methods:1.The expression of PRR11 protein in human normal ovarian epithelial cells I0SE80,human ovarian cancer cell lines Caov3,SKOV3,OVCAR3,and HO-8910 was detected by Western blotting.2.siRNA targeting PRR11 silencing was designed,synthesized,and transfected into human ovarian cancer HO-8910 cells with Lipofectamine^TM RNAIMAX according to the manufacturer’s instructions.Real-time PCR and Western blotting were used to demonstrate the interference efficiency of PRR11 at the level of m RNA and protein,respectively.3.PRR11 overexpression recombinant vector was constructed in vitro and was transfected into human ovarian cancer Caov3 cells with Lipofectamine^TM2000 according to the manufacturer’s instructions.Real-time PCR and Western blotting were used to demonstrate the overexpression efficiency of PRR11 at the level of m RNA and protein,respectively.4.CCK-8 and clonogenic experiments were used to analyze the effect of PRR11interference on proliferation and clone formation of ovarian cancer HO-8910 cells.5.CCK-8 and clonogenic experiments were to demonstrate the effect of overexpression of PRR11 on the proliferation and cloning ability of ovarian cancer Caov3 cells.6.The effects of silencing or overexpression of PRR11 gene on the expression of c-myc and cyclin D1 were detected by Western blotting at the protein level.Results:1.Compared with human normal ovarian epithelial cell line I0SE80,PRR11 expression showed high expression in ovarian cancer cells.In four ovarian cancer cells,PRR11expression level was the lowest in Caov3 cell line and the highest in HO-8910 cell line.2.RNAi silencing PRR11 expression significantly inhibited the proliferation and cloning ability of HO-8910 cells.3.Overexpression of PRR11 significantly increased the proliferation and cloning ability of Caov3 cells.4.After PRR11 overexpression,the expression of proliferation-related genes c-myc and cyclin D1 in Caov3 cells was significantly increased.5.After RNAi silencing PRR11 expression,the expression of proliferation-related genes c-myc and cyclin D1 in HO-8910 cells was markedly decreased.Conclusions:PRR11 is highly expressed in ovarian cancer cells.PRR11 promotes the malignant proliferation of ovarian cancer cells by positively regulating the proliferation of related proteins c-myc and cyclin D1.Part Ⅲ.Effect of PRR11 on invasion and migration of ovarian cancer cells.Objectives:The purpose of this study was to investigate the effect of PRR11 silencing or overexpression on the invasion and migration of ovarian cancer cells.Methods:1.Transwell invasion and migration analysis were used to analyze the effect of PRR11interference on the invasion and migration of ovarian cancer HO-8910 cells.2.Transwell invasion and migration analysis were performed to investigate the effect of overexpression of PRR11 on invasion and migration of ovarian cancer Caov3 cells.3.The effect of silencing or overexpression of PRR11 gene on the expression of MMP-2 and TIMP-2 was detected by Western blotting at the protein level.Results:1.RNAi silencing PRR11 expression markedly suppressed the invasion and migration of HO-8910 cells.2.Overexpression of PRR11 markedly promoted the invasion and migration ability of Caov3 cells.3.When PRR11 expression was silenced by siRNA transfection,MMP-2 expression was down-regulated,and TIMP-2 expression was up-regulated in HO-8910 cells.4.After PRR11 overexpression,the expression of MMP-2 in Caov-3 cells was significantly increased and the protein level of TIMP-2,an MMP-2 inhibitor,was decreased markedly.Conclusions:Highly expressed PRR11 enhances the invasion and migration abilities of ovarian cancer cells by mediating the increase of MMP2/TIMP-2 ratio.Part Ⅳ.The mechanism of PRR11 exerted biological function of ovarian cancer cellsObjectives:The purpose of this study was to analyze the molecular mechanism of PRR11 regulating the proliferation,invasion,and migration of ovarian cancer cells.Methods:1.The effect of PRR11 silencing on the expression of Akt,phosphorylated Akt,totalβ-catenin and nuclearβ-catenin in HO-8910 cells was detected by Western blot.2.The expression of Akt,phosphorylated Akt,total and nuclearβ-catenin in Caov3cells were detected by Western blot after transfection with PRR11 overexpression recombinant vector and PI3K specific inhibitor LY294002 treatment.Besides,CCK8 assay,clone formation assay and Transwell assay were further used to detect the changes of proliferation,clone formation and invasion and migration abilities of Caov3 cells.Results:Western blot analysis revealed that knockdown of PPR11 reduced p-Akt,total and nuclearβ-catenin protein levels in HO-8910 cells,whereas increased PRR11 exerted the opposite functions in Caov3 cells.Besides,LY294002,a specific inhibitor of PI3K was added to the Caov3 cells with PRR11 up-regulation,and the results suggested that the expression of p-Akt and protein levels of total and nuclearβ-catenin were reversed.Consistently,the enhancement of cell proliferation,clonogenicity,and metastasis by PRR11 up-regulation was suppressed by LY294002 treatment in Caov3 cells.Conclusions:Highly expressed PRR11 promotes the nuclear translocation ofβ-catenin by activating PI3K/Akt signal and then regulate the proliferation,invasion,and migration of ovarian cancer cells.