Effects Of Hsa-let-7b-5p Targeted Regulation Of RNA-binding Protein MEX3A On Malignant Biological Behavior Of Nephroblastoma

Objective:Wilms tumor(WT)is the most common renal malignant tumor in children with a high degree of malignancy.Although surgery combined with chemotherapy plays a role in improving the prognosis of WT,the treatment of children with postoperative recurrence and chemotherapy resistance is still a difficult problem.Deepening the knowledge and understanding of WT at the molecular biological level and exploring the regulatory molecules and mechanisms of the malignant biological behavior of WT cells can not only provide a reliable basis for the prognosis of patients,but also provide new strategies for the precise treatment of WT.RNA binding protein(RBP)is an important class of proteins in cells.They are widely involved in many post-transcriptional regulatory processes such as RNA shearing,transport,sequence editing,intracellular localization and translation control by recognizing specific RNA domains and interacting with RNA.The abnormal expression of RBP in various malignant tumors is closely related to the occurrence and development of tumors.MEX3 A,a member of the human MEX3 family,is an E3 ubiquitin binding enzyme that mediates m RNA degradation through ubiquitination and regulates the proliferation,migration and apoptosis of many tumor cells.Some scholars found the DNA copy number of MEX3 A was significantly up-regulated in patients with WT,suggesting that MEX3 A may be involved in regulating the occurrence of WT.Micro RNAs(mi RNAs)are a class of small(19-25 nucleotides)single-stranded noncoding RNAs discovered in recent years,which play a regulatory role by sequence-specific binding to the coding region or non-coding region of messenger RNA(m RNA).There are about 2,500 mi RNAs that control thousands of genes for physiological and pathological processes,including the malignant biological behavior of tumor cells.MEX3 A expression has been confirmed to be regulated by mi RNAs in some tumors,but mi RNAs that play a major role in WT regulation of MEX3 A remain unclear and their mechanisms of action are unknown.Hsa-let-7b-5p is one of the most studied mi RNAs in human cancer and plays a role of tumor suppressor gene in a variety of malignant tumors.TCGA and TARGET database were used to predict the existence of targeted binding sites between Hsa-let-7b-5p and MEX3 A.However,the expression and mechanism of Hsa-let-7b-5p in WT remain unclear.Therefore,this study aimed to explore the effect of MEX3 A expression in RBP on malignant biological behavior of TW cells and the regulatory role of Hsa-let-7b-5p on MEX3 A in WT.Methods:Part I: Therapeutic Applied Research to Generate Effective therapies(TARGET)and Gene Expression Omnibus(GEO)were utilized.GEO was used to obtain gene expression data and relevant clinical data.Differentially expressed genes(DEGs)of RNA-binding protein(RBP)were identified in GEO database,and prognostic genes of RBP were obtained from TARGET training cohort dataset.Then,univariate,LASSO and multivariate Cox regression analysis were used to establish the correlation characteristics of RBP.Finally,the prognostic value of the signature was validated in TARGET test cohort dataset.Part II: 1)Paraffin sections of WT tissues operated in Shengjing hospital from 2010 to 2018 and clinical information of children were collected,and the expression level of MEX3 A was detected by immunohistochemistry to analyze the impact of different MEX3 A expressions on clinical characteristics of patients with WT.2)Three MEX3 A si RNA fragments and one negative control(NC)were synthesized and purified,transfected into WT cells by transient transfection method.Cell proliferation and apoptosis were detected by CCK-8 method and FITC Annexin V/ PI apoptosis kit.3)WT cells with stable and low MEX3 A expression were transfected with lentivirus plasmid and screened for cell cycle detection compared with the control group.Part III: 1)Combined with databases and websites,Hsa-let-7b-5p was predicted to have targeted binding sites with MEX3 A,and the targeting relationship between Hsa-let-7b-5p and MEX3 A was further confirmed by dual luciferase reports.2)Lentiviral plasmids with MEX3 A overexpression,Hsa-let-7b-5p overexpression,and control group were constructed and transfected into WT cells,respectively.Cells with stable MEX3 A overexpression,Hsa-let-7b-5p and MEX3 A overexpression were screened.MTT assay,TUNEL cell apoptosis assay and RI cell cycle assay were used to observe the effects of MEX3 A and HSA-let-7b-5p on proliferation,apoptosis and cell cycle of WT cells.Results:Part I: 49 genes were predicted to be prognostic RBP-associated DEGs.Subsequently,we identified 19 RBP-related genes as prognostic markers for WT patients.Prognostic models(CPEB2,AFF3,MEX3 B,EZH2,RBPMS,CPEB4,HNRNPA0,MRPS6,CRYZ,BARD1,BRCA1,NOP56,TCERG1,TLR3,RBM47,PURG,SRSF12,SPATS2,MEX3A)were established,the survival time of high-risk group was significantly shorter than that of low-risk group(P < 0.01).Then,the Kaplan-Meier(K-M)curve,the AUC value of ROC,and Cox regression analysis verified that the 19 RBP-related gene model were a new and substantial prognostic marker independent of other clinical traits.Poor survival results appeared in WT patients with a high-risk score.The area under the time-dependent ROC curve of the prognostic model was 0.855 in TARGET training cohorts and 0.668 in TARGET test cohorts,indicating a good prognostic model.Moreover,the nomogram on the foundation of risk score and other clinical traits was established for predicting the survival rate of WT patients.The nomogram showed a great discriminating capacity for WT.Biological function analyses displayed that tumor related pathways were affluent.Part II: Immunohistochemical analysis of tumor sections confirmed that the survival rate of children with high MEX3 A expression was significantly lower than that of children with low MEX3 A expression(P < 0.05).Subsequently,CCK-8 assay and FITC Annexin V/ PI apoptosis assay were used to confirm the decreased proliferation and significantly increased apoptosis of MEX3 A knockdown cells.The mechanism may be related to the increase of the protein levels of cyclin Securin,cyclin B1 and cyclin A.Stable and low expression MEX3 A cells were obtained by transfection with lentivirus.Subsequent RI cell cycle tests confirmed that inhibition of MEX3 A reduced the proportion of S phase cells.In summary,MEX3 A achieves its oncogenic effect by promoting the malignant biological behavior of WT.Part III: Double luciferase assay confirmed the targeted binding of Hsa-let-7b-5p to MEX3 A.We successfully transfected WT cells with overexpression of Hsa-let-7b-5p,Hsalet-7b-5p and MEX3 A,and the control group by using lentivirus transfection method,and screened out stably transfected cells.Subsequent experiments confirmed that overexpression of Hsa-let-7b-5p could inhibit the expression of MEX3 A,inhibit the proliferation of WT cells,promote cell apoptosis,and affect the tumor cell cycle by reducing S phase cells.Co-transfection MEX3 A in cells of overexpression of Hsa-let-7b-5p could counteract the effect of overexpression of Hsa-let-7b-5p on WT.Conclusion:The expression level of RNA-binding protein-related genes may provide a new prognostic marker and potential therapeutic target for the treatment and prevention of WT.As an RBP,MEX3 A expression affects survival.MEX3 A also promotes malignant biological behavior in WT cells.MEX3 A is therefore a tumor promoter and may serve as a target for future tumor molecular therapy.Hsa-let-7b-5p has a targeted binding and regulatory relationship with MEX3 A m RNA.Hsa-let-7b-5p affects tumor cell biological behavior by modulating MEX3 A.To inhibit MEX3 A with Hsa-let-7b-5p may be a potential approach for prevention and treatment of WT.