Study On Biomarkers And Potential Drug Targets Of Gynecological Oncology

Cervical cancer(CC)has the highest incidence among malignant tumors of the female reproductive system.Approximately one-fifth of CC patients in the world come from China,which seriously threatens the life and health of Chinese women.More than 50%of patients in China are diagnosed with locally advanced CC at the time of discovery.This type of patient is usually accompanied by risk factors such as lymph node metastasis and distant metastasis.The treatment effect is poor,and the recurrence rate is high.The 5-year survival rate is less than 60%.At present,the understanding of the molecular mechanism of the occurrence and development of CC is insufficient.The treatment of locally advanced patients is still dominated by concurrent chemoradiotherapy,and there is a lack of clinical biomarkers that can predict the prognosis of patients.Screening and identifying reliable biomarkers and drug treatment targets can futher promote the individualized treatment of CC,which has important practical significance for improving the survival rate of patients and improving the quality of life.In order to eliminate the interference of tumor heterogeneity among different pathological subtypes,cervical squamous cell carcinoma(CSCC),the main pathological type of CC,was selected for this study.First,we screened and identified a molecule,TMOD3,with abnormally elevated transcription levels in CSCC from the TCGA database.Survival analysis showed that higher TMOD3 mRNA levels were positively correlated with poor prognosis of patients.Clinical tissue samples were used for qRTPCR,immunohistochemistry,and western blot experiments to further confirm that the mRNA level and protein expression of TMOD3 were significantly higher in tumors compared to normal tissues.To reveal the role of TMOD3 in CSCC,specific siRNA and lentiviral interfering RNA plasmids were used to inhibit the expression of TMOD3 in SiHa and CaSki(CSCC cells).It was found that downregulation of TMOD3 could inhibit cell proliferation and colony formation.When TMOD3 expression was restored in TMOD3 knockout cells,the proliferation activity of the cells was subsequently significantly restored.Knockout of TMOD3 significantly inhibited the growth of CSCC xenografts in vivo.In addition,inhibition of TMDO3 can significantly reduce the ability of cell migration and invasion.TMOD3 can positively regulate VEGFA and N-Cadherin,and negatively regulate E-Cadherin.To reveal the molecular mechanism by which TMOD3 promotes the progression of CSCC,this study combined GST pull-down assay and LC-MS/MS to identify the TMOD3 binding protein MELK.Downregulation of MELK expression can also inhibit cell proliferation and colony formation.Although the inhibition of TMOD3 did not affect the expression of MELK protein,the inhibition of MELK could downregulate the protein level of TMOD3.The above results indicated that TMOD3 is a downstream molecule that can bind to MELK.Further treatment of cells after MELK knockdown with the proteasome inhibitor MG 132 showed that inhibition of proteasome activity could restore the reduced TMOD3 protein level due to MELK inhibition.Cycloheximide tracking assay showed that inhibition of MELK expression accelerated the degradation of TMOD3 protein,and MELK overexpression enhanced the stability of TMOD3 protein.Finally,we introduced the MELK inhibitor MELK-8a dihydrochloride to investigate its antitumor effect in CSCC.The IC50 values of SiHa and CaSki cells treated with the inhibitor for 48 hours were 7.1194±0.6906 μM and 7.8355±0.3136 μM,respectively.The IC50 values of these two cells treated with the inhibitor for 72 hours were 5.1445±0.1158 μM and 5.1625±0.1054 μM,respectively.Treatment of cells with low concentrations(1μM and 2 μM)of MELK inhibitors significantly inhibited the colony formation ability of CSCC cells.In vivo,MELK inhibitors(3 mg/Kg)significantly inhibited the growth of CSCC xenografts and showed good drug safety.In summary,this study is the first to show that TMOD3 is abnormally overexpressed in CSCC,and its elevated transcription level is positively correlated with poor prognosis of patients.TMOD3 may be a biomarker for predicting prognosis of patients.MELK can promote the malignant proliferation of CSCC cells by enhancing the stability of TMOD3 protein,while the targeted inhibition of MELK or TMOD3 can reduce the cell proliferation activity.MELK and TMOD3 are potential therapeutic targets in CSCC,which have broad clinical application prospects.Endometrial cancer(EC)is one of the three major malignant tumors of the female reproductive system.Surgery,chemotherapy,radiotherapy and endocrine therapy are the main treatments for patients.With the development of the social economy and changes in the human lifestyle,the incidence of EC is increasing year by year.Screening and identification of biomarkers and drug therapeutic targets for individualized diagnosis and treatment can predict disease risk and improve patient efficacy,which has important practical significance.Previous studies have shown that estrogen/progesterone dysregulation is a key pathogenic factor for EC,and abnormal changes in this signaling pathway accelerate the development and progression of tumors.Based on this,this study aimed to screen estrogen/progesterone-related biomarkers in EC and construct a prognostic risk model.Firstly,we downloaded the clinical and transcriptome data of 397 patients with type I EC from the TCGA database.The cohort also matched the data of 23 normal samples.The 51 estrogen/progesterone-related gene sets were downloaded from the MSigDB database to construct the estrogen/progesterone-related gene expression matrix.Compared with normal tissues,GSEA showed that 9 out of the 51 estrogen/progesteronerelated gene sets were significantly enriched in EC.The R package "limma" was applied to analyze the differential expression of estrogen/progesterone-related genes between the normal and the tumor group,and a total of 2114 differentially expressed genes were screened.Based on the above differentially expressed genes and the survival information of patients,univariate and multivariate Cox regression analyses were performed to construct a prognostic risk model consisting of CDC25B,PRXL2A,GNG3,ITIH3,and SDHB.Compared with normal tissues,the transcription levels of CDC25B,PRXL2A,GNG3,and SDHB were higher in tumors,and the increased mRNA levels were positively correlated with poor prognosis of patients;while the mRNA level of ITIH3 was lower in tumors,and the depressed mRNA level was positively correlated with poor prognosis of patients.According to the median risk score of the model,patients were divided into high-risk and low-risk groups.Kaplan-Meier survival analysis showed that the overall survival time of patients in the high-risk group was shorter than that in the low-risk group.ROC curve analysis showed that the model could accurately predict the 1-year,3-year,and 5-year survival rates of patients,and the AUC values were 0.695,0.721,and 0.718,respectively.We divided patients into eight clinical subgroups based on age,BMI,tumor grade,and hypertension history.It suggested that the overall survival time of patients in the high-risk group was shorter than that in the low-risk group in all subgroups,indicating that the prognostic risk model was applicable to different patients.GSVA enrichment analysis was used to explore the correlation between the risk score and the biological pathways,and it was found that the risk score was positively correlated with the activation of tumor signaling pathways such as KEGG_MISMATCH_REPAIR,KEGG_DNA_REPLICATION,KEGG_HOMOLOGOUS_RECOMBINATION,and KEGG CELL CYCLE.In addition,this study constructed a nomogram based on the risk score and tumor stage to assess the 2-year,5-year,and 8-year survival rates of patients.Finally,we performed qRT-PCR,cell proliferation,colony formation,and nude mouse xenograft tumor experiments to explore the abnormal expression and function of the model genes PRXL2 A and GNG3 in EC.It showed that the transcription levels of GNG3 and PRXL2 A in tumors were higher compared to those in normal tissues.Knockdown of GNG3 or PRXL2A expression can significantly inhibit the proliferation activity and colony forming ability of EC cells,and knockout of PRXL2 A expression can inhibit the growth of xenografts in vivo.In summary,this study screened and established a prognostic risk model consisting of five estrogen/progesterone-related genes:CDC25B,PRXL2A,GNG3,ITIH3,and SDHB.The above model genes and prognostic models may be used as biomarkers and strategies for the evaluation of patient prognosis.In addition,downregulation of GNG3 or PRXL2A expression can inhibit the malignant proliferation of EC cells.The model genes GNG3 and PRXL2A are candidate drug therapeutic targets for individualized treatment of EC.