| Background and purpose:Ovarian cancer(OC)is one of the three most common gynecological malignant tumors in China.The difficulty of early diagnosis and chemotherapy resistance are the main reasons for poor prognosis.OC has an insidious onset and it is difficult to detect the symptoms at early stage,with 80%patients diagnosed at an advanced stage.The standard treatment is surgery combined with platinum-based chemotherapy.Although OC is sensitive to platinum-based chemotherapy drugs,chemotherapy resistance easily occurs.The application of targeted therapy has improved the prognosis of OC patients,but the benefit is limited and some patients may suffer the drug resistance.Therefore,exploring new therapeutic targets has always been a hot topic in OC research.Previous studies have shown that Retinoic Acid Receptor Gamma(RARG)plays an important role in the occurrence and development of various malignant tumors.However,its biological function in OC is still unclear.This study aims to explore the expression level of RARG in OC and its correlation with prognosis,and further investigate its role in the progression of OC.Methods:This study respectively obtained sample data of 88 normal ovarian tissues and 379 OC tissues from the The Genotype Tissue Expression(GTEx)and The Cancer Genome Atlas(TCGA)databases.The transcriptome data and relevant clinicalpathological information were integrated and analyzed,and RARG with elevated transcriptional level in OC was identified.According to the median value of RARG,OC patients were divided into RARG mRNA high-and low-level groups,and Kaplan-Meier survival analysis was performed.The accuracy of RARG mRNA level in predicting 1-,5-,and 9-year survival outcomes of OC patients were evaluated by ROC curves.The univariate and multivariate Cox regression analysis(RARG,age,FIGO pathological stage,tumor histological grade)were performed to identify the prognostic risk factors of OC patients.Then,a survival prediction model was established,and calibration plots was used to evaluate the consistency between the Nomogram-predicted rates and actual survival rates.The differentially expressed genes between high and low RARG mRNA level groups were performed,GSEA enrichment analysis was used and the relationship with tumor immune infiltration was investigated.qRT-PCR was performed to verify the difference in RARG mRNA levels between 10 pairs of normal ovarian tissues and OC tissues.A total of 49 OC tissues and 30 normal ovarian tissues were selected for immunohistochemistry to detect RARG protein expression levels.The survival prognosis of 47 OC patients were followed up,and Kaplan-Meier survival analysis was performed,based on RARG IRS scores.The univariate and multivariate Cox regression analysis were performed to identify the effects of CA125,age,IRS score,initial tumor reduction and platinum-resistant recurrence on the overall survival of OC patients.The chi-square test method was used to compare the expression of RARG in various related factors.Specific siRNA transient knockdown and lentiviral interference shRNA plasmid were used to suppress RARG expression in A2780 and SKOV3 cells.The cell proliferation ability,colony formation ability,cell cycle,and tumor growth in nude mice subcutaneous transplantation tumor model were observed.Immunohistochemistry was used to detect the differences in RARG,Ki-67 and PCNA protein expression levels between the stable knockdown group and the control group.Results:Database analysis results showed that RARG mRNA level was significantly higher in OC tissue than that in normal ovarian tissue(P<0.001).Subgroup analysis revealed that RARG mRNA levels were significantly higher in FIGO Ⅲ-Ⅳ OC patients.compared with FIGO Ⅰ-Ⅱ OC patients(P=0.027).The OC patients with higher RARG mRNA levels had poorer prognosis(P<0.001).The AUC values of RARG for predicting 1-,5-and 9-year survival were 0.659,0.616 and 0.627,respectively.The univariate and multivariate Cox regression analysis indicated that elevated RARG mRNA level was an independent risk factor for poor prognosis in OC patients(P=0.004).The Nomogram incorporating RARG and age was constructed,and the calibration curves for 1-,3-,and 5year survival rates were close to the standard curves.The GSEA enrichment analysis suggested that RARG was involved in multiple cancer related signaling pathways,including PI3K-Akt signaling pathway and tight junctions.Immune cell infiltration analysis demonstrated that the infiltration levels of CD4 memory activated T cells(P=0.018)and Neutrophils(P=0.028)in low RARG mRNA level group were higher than that in high mRNA level group.The results of qRT-PCR and immunohistochemical experiments revealed that RARG mRNA level(P<0.05)and protein level(P<0.01)in OC tissues were higher than that in normal tissues,and the patients with higher RARG protein levels had a worse prognosis(P<0.001).The univariate and multivariate Cox regression analysis indicated that age higher than 65 years old(HR=8.883,95%CI=2.264-34.855,P=0.002).IRS score higher than or equal to 4(HR=5.450,95%CI=1.091-27.211,P=0.039).the patients who received unsatisfactory tumor reduction(HR=3.236,95%CI=1.057-9.909,P=0.040)and platinum resistance(HR=7.956,95%CI=1.843-34.345,P=0.005)were independent risk factors for predicting poor prognosis in OC patients.The expression of RARG protein was associated with chemoshensitivity(P=0.046).Downregulation of RARG expression inhibited the proliferation and colony formation ability of A2780 and SKOV3 cells but didn’t affect the cell cycle.The volume and weight of transplanted tumors in sh-RARG group respectively decreased 69.66%(P<0.01)and 72.22%(P<0.01),compared with the control group.Compared with the control group,protein expression levels of RARG,Ki-67 and PCNA were significantly decreased in the sh-RARG group.Conclusion:The mRNA level and protein expression level of RARG are higher in ovarian cancer than that in normal ovarian tissue,and high RARG expression is an independent risk factor for poor prognosis in OC patients,highlighting RARG as a biomolecular marker for predicting the prognosis of OC patients.The proliferation ability decreased by inhibiting RARG expression,suggesting that RARG may act as a potential molecular target for the treatment in OC patients. |
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