| Ovarian cancer(OC)is one of the three major malignant tumours in the female reproductive system,with the greatest mortality rate among gynaecological malignant tumours,severely affecting women’s lives and health.The most frequent histological type of OC is epithelial ovarian cancer(EOC),which accounts for approximately 90% of all OC.More than 70%EOC patients were at advanced stage at the time of diagnosis due to a lack of particular tumour markers and early symptoms.Currently,surgery combined with platinum-containing chemotherapy is the primary treatment for EOC.Despite the fact that numerous adjuvant therapy options such as targeted therapy and immunotherapy have been used to treat EOC in recent years,the5-year survival rate of patients with advanced EOC has not improved appreciably.As a result,it is critical to investigate the molecular mechanisms of EOC occurrence and development in order to identify more effective therapeutic targets for improving the prognosis of EOC patients.Rab11a belongs to the Rab small molecule GTP enzyme family,which is widely expressed in mammals.Rab11 a is found in the endosome,the trans Golgi apparatus,and the plasma membrane,where it is involved in ligand and receptor transport,cytokinesis,cytokine release,and intracellular signal transduction.In recent years,more and more researches have focused on the role of Rab11 a in tumors.Rab11 a has been found in studies to stimulate tumour cell proliferation and invasion,thereby playing a role in the occurrence and development of diverse malignant tumours as a "cancer-promoting factor." Rab11 a has been found to be significantly expressed in a variety of tumours,including non-small cell lung cancer,breast cancer,and pancreatic cancer.However,there is currently no in-depth research on the precise role and related mechanism of Rab11 a in EOC.This topic focuses on the aforementioned issues,and the study findings may lead to new ideas and tactics for the pathophysiology and treatment of EOC.Part One The expression and clinicopathological characteristics of Rab-11a in epithelial ovarian cancer tissuesObjective: To analyze the difference between Rab11 a expression in OC and normal ovarian tissue and the effect of Rab11 a on the prognosis of OC,and to confirm it through collecting clinical samples.To further analyze the expression of Rab11 a in EOC and its relationship with clinicopathological features.Methods:1.Log in to the GEPIA database to analyze the difference in Rab11 a expression between ovarian cancer samples and normal ovarian tissue samples.Based on TCGA and GEO databases,the impact of Rab11 a on the prognosis of OC was analyzed by Kaplan-Meier survival analysis.2.A total of 59 cases of EOC tissue after surgical resection and 60 cases of normal ovarian tissue from oophorectomy due to non-ovarian benign diseases between January 2021 and October 2022 were collected from Hebei General Hospital and the Second Hospital of Hebei Medical University.3.Real time quantitative PCR(RT-q PCR)was used to determine the expression level of Rab11 a m RNA in EOC tissue and normal ovarian tissue.Further,according to the clinicopathological characteristics of EOC,the relative expression of Rab11 a in different patients’ age,histopathology type,tumor diameter,pathological staging and grading,and tissues with or without lymph node metastasis was analyzed to clarify the relationship between Rab11 a and the clinicopathological characteristics of EOC.4.The statistical analysis software of Statistical Product and Service Solutions 26.0(SPSS)is adopted to sort out,count and analyze the data.Graph Pad Prism 8.0 graphing software is used to plot the Rab11 a expression levels of the different variables.P<0.05 is considered to be with a statistically significant difference.Results:1.The GEPIA database showed that the expression level of Rab11 a in OC tissues was higher than that in normal tissues.Kaplan-Meier survival analysis showed that the overall survival of OC patients with high Rab11 a expression was significantly shorter than those with low Rab11 a expression(HR=1.17,P=0.029),and the progression free survival of OC patients with high Rab11 a expression was shorter than those with low Rab11 a expression,but there was no significant statistical difference between them(HR=1.15,P=0.061).2.The RT-q PCR results showed that the expression level of Rab11 a m RNA in EOC tissue was significantly higher than that in normal ovarian tissue(P<0.05),which is consistent with the expression trend of Rab11 a in OC in the GEPIA database.3.The expression level of Rab11 a in EOC samples was closely related to clinicopathological features.The results showed that the expression level of Rab11 a in EOC of FIGO stage III/IV was higher than that in EOC of FIGO stage I/II(P<0.001),and with the improvement of histological grading,the expression level of Rab11 a increases(P<0.001).In addition,the expression level of Rab11 a in patients with lymphatic metastasis was higher than that in patients without lymphatic metastasis.However,the expression level of Rab11 a in EOC had no significant correlation with the patient’s age,histopathology type and tumor diameter.Summary: Rab11 a is highly expressed in EOC tissues compared with normal ovarian tissues,and was related to FIGO stage,histological grade and lymphatic metastasis status.However,the expression level of Rab11 a in EOC had no significant correlation with the patient’s age,histopathology type and tumor diameter.Part Two The biological functions of Rab11 a in epithelial ovarian cancercellsObjective: To study the various biological functions of Rab11 a in EOC cells from the aspects of tumor cell proliferation,cell cycle distribution,invasion,and migration.Methods:1.Human normal epithelial ovarian cells HOSEpi C and OC cell lines A2780,SKOV-3,COC-1,OVCAR-3 were cultured.First,the m RNA expression level of Rab11 a in each cell was detected by RT-q PCR,and the cell line with higher expression of Rab11 a was selected for as H,and the cell with lower expression is designated as L.2.Using lipofectamine 2000,the sh-RNA of Rab11 a and its control sh-NC were transfected into H cells for knockdown;the Rab11 a overexpression plasmid and its control were transfected into L cells to build an overexpression model.The protein level of Rab11 a in each group of cells was verified by western blotting.3.The proliferation of cells in each group was detected by cell counting kit(CCK)-8;the cell cycle of each group was detected by flow cytometry(FCM);48h after transfection,the protein expression levels of PCNA and Cyclin D1 were detected by Western blotting.4.Wound healing assay was used to test the migration ability of cells in each group;transwell assay was used to test the invasion ability of cells in each group.ELISA kits were used to detect the concentrations of MMP-2 and MMP-9 in the supernatant of each group of cells.Results:1.Compared with HOSEpi C cells,the m RNA level of Rab11 a was significantly elevated in the four OC cell lines,with the highest abundance in OVCAR-3 cells and relatively lower level in A2780 cells.Therefore,we downgraded the expression of Rab11 a using sh-RNAs in OVCAR-3 cells,and employed the overexpressed plasmids to upregulate Rab11 a in A2780 cells for subsequent experiments.2.The results of western blotting showed that the expression of Rab11 a protein was significantly lowered in the OVCAR-3 cells with sh-RNA transfection than that in the control group,while,the expression of Rab11 a protein was significantly higher in A2780 cells with plasmid transfection than that in the control group,the difference is statistically significant(P<0.05).3.The results of CCK-8 assay showed that downwarded Rab11 a dramatically decreased cell viability of OVCAR-3 cells.By contrast,Rab11 a overexpression increased the cell viability of A2780 cells.Cell cycle analysis indicated that,the proportion of OVCAR-3 cells with sh-RNA transfection in G0/G1 phase was significantly increased,while the corresponding proportion of cells in S phase and G2 phase phase was decreased(P<0.05).On the contrary,overexpression of Rab11 a in A2780 cells displayed the opposite effects.In addition,the results of western blotting showed Rab11 a deficiency led to decreased expression of PCNA and Cyclin D1 in OVCAR-3 cells with sh-RNA transfection,while overexpression of Rab11 a upregulated expression levels of these proteins significantly(P<0.05).4.The results of the cell scratch test and the transwell chamber test show that,when compared to the negative control group,the healing rate of the scratch area in the OVCAR-3 cells with sh-RNA transfection group is significantly lower.Furthermore,the number of cells passing through the chamber was significantly lower than in the negative control group.On the contrary,in A2780 cells,the healing rate of the scratch area in the Rab11 a plasmid transfection group was significantly higher than that in the negative control group,as was the number of cells passing through the chamber in the Rab11 a plasmid transfection group.The above findings were confirmed by quantitative analysis of mobility and the number of invasive cells.Furthermore,ELISA was used to detect MMP-2 and MMP-9 levels in the supernatant of OC cells.MMP-2 and MMP-9 levels in the supernatant of OVCAR-3 cells with sh-RNA transfection were significantly reduced(P<0.05).Overexpression of Rab11 a,on the other hand,significantly increased the levels of MMP-2and MMP-9 in the supernatant of A2780 cells.Summary: Rab11 a can play a “cancer promoting” role in epithelial ovarian cancer cells,overexpression of Rab11 a can promote the proliferation,migration and invasion of EOC cells.Inhibition of Rab11 a can decrease the proliferation,migration and invasion of EOC cells.Part Three Rab11 a promotes proliferation,migration,and invasion ofepithelial ovarian cancer cells by regulating autophagyObjective: To preliminary exploration of the potential mechanism of Rab11 a in the occurrence and development of EOC,and clarify whether Rab11 a participates in the malignant biological behavior of EOC through autophagy.Methods:1.LC3 immunofluorescence staining was performed on OVCAR-3 cells transfected with Rab11 a sh-RNA and A2780 cells transfected with Rab11 a overexpression plasmid to observe the LC3 positive staining cells in each group.Western blotting was used to detect the relative expression levels of autophagy related proteins Beclin1,LC3I/II,and p62 in each groups.2.The overexpressed A2780 cells were treated with 3-methyladenine(3-MA,an autophagy inhibitor),and the protein levels of LC3I/II and p62 were detected by western blot;the cell viability of cells in each group was detected by CCK-8;the cell migration ability was detected by wound healing assay;the invasive ability of cells was detected by transwell test.Results:1.The results of LC3 immunofluorescence revealed that the number of LC3 positive stained cells in OVCAR-3 cells with Rab11 a down-regulation decreased.On the other hand,the number of LC3 positive stained cells in A2780 cells with Rab11 a overexpression increased.Beclin1,LC3I/II,and p62 protein expression levels were determined using western blotting.The results showed that compared with the control group,the expression of LC3 II and Beclin1 decreased and the expression of p62 significantly increased in OVCAR-3 cells with Rab11 a down-regulation(P<0.05).On the contrary,in A2780 cells with Rab11 a overexpressed,the expression of LC3 II and Beclin1 proteins increased,while the expression of p62 decreased(P<0.05).2.After using 3-MA to treat A2780 cells with Rab11 a overexpressed,Western blotting revealed that 3-MA treatment reversed the increase in LC3 II protein expression and decrease in p62 expression caused by Rab11 a overexpression(P<0.05).Furthermore,the CCK8 test,cell scratch test,and Transwell test revealed that 3-MA treatment significantly reduced the proliferation,migration,and invasion of EOC cells caused by Rab11 a overexpression(P<0.05).The above findings were confirmed by quantitative analysis of mobility and the number of invasive cells.Summary: Rab11 a can promote the proliferation,migration and invasion of EOC cells by inducing autophagy,thereby participating in the occurrence and development of EOC.Conclusions:1.When compared to normal ovarian tissue,Rab11 a is highly expressed in EOC tissue,which is related to FIGO staging,histological grading,and lymph node metastasis,but has no significant correlation with patients’ age,histopathological type,or tumor diameter.2.Rab11 a has been shown to be “cancer-promoting” in EOC cells.Overexpression of Rab11 a has been shown in vitro to promote the proliferation,migration,and invasion of EOC cells.Downwarded Rab11 a can inhibit the proliferation,migration,and invasion of epithelial ovarian cancer cells.3.Rab11 a may promote the proliferation,migration,and invasion of EOC cells by inducing autophagy,thereby contributing to the occurrence and progression of EOC.4.The mechanism of Rab11 a on the occurrence and progression of EOC can provide molecular biomarkers for early diagnosis and judge prognosis,and for new targets for subsequent treatment. |
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