The Effects And Molecular Mechanism Of MiR-21 Targeted Regulating PTEN On Biological Behavior Of Wilms'tumor

Background and objectiveWilms' tumor(WT)is one of the most common pediatric malignant solid tumor.According to statistics,it comprises 6%of all pediatric tumors,and affects approximately 1 child per 10,000 younger than 15 years.The average age at diagnosis is 36 months.Patients of WT older than 10 years or younger than 6 months are infrequence.WT is etiology unknown at present,it is generally considered that WT may be derived from metanephric blastemal of abnormal hyperplasia,which fails to differentiate to nephrone normally.The pathological classification of WT includes epitheliated type,mesenchymal type,embryonal type and mixed type.Another frequently-used classification method according to the relation between tissue typing and prognosis is to classify WT into FH(favorable histology)type and UH(unfavorable histology)type.The presence of anaplasia is currently the only criterion for "UH type" in a Wilms' tumor.The clinical presentation of WT includes asymptomatic abdominal mass,indolent hematuria,bellyache,hypertension and tumor compression symptoms.Wilms' tumor shows a strong association with certain congenital anomalies,for example,WAGR syndrome,BWS syndrome,DDS syndrome,The occurrence of WT,which is related to a number of abnormal expression genes and possible genetic factors,is a complex process of multi-factors,stages,and steps.The activation of oncogenes and the loss and expression disorder of tumor suppressor genes are its important characteristics during the occurrence and development of WT.The current tendency of WT treatment is to avoid unnecessary radical high-intensity therapy,and to reduce treatment-related complications,which is based on the assumption that the survival rate will remain steady or that it will increase.Because of this,WT is generally acknowledged as one of the most treatable tumors because of modern comprehensive therapy,of which to analyze sensitive molecular biomarkers to guide treatment and follow-up is playing an important role.MicroRNAs(miRNAs)are small non-coding and single-chain RNAs of 20-22 nucleotides.MiRNAs have functions on regulation of gene expression and are involved in crucial biological processes,including cell development,differentiation,apoptosis and proliferation.More and more reports have shown that miRNAs have been proposed to participate in the critical stage of tumor genesis and development regulation,and to contribute to oncogenesis because they can function either as tumor suppressor or oncogenes.As one of the most representative oncogenes found in miRNAs,miR-21 is highly expressed in almost all the reported solid tumors.MiR-21 exerts its effects as oncogene by incomplete complementary pairing with the 3'-UTR region of downstream target genes,which suppresses the function of the target genes.It is confirmed in various adult tumors that miR-21 is able to negatively regulate multiple target genes such as PDCD4,RECK,TIMP-3,TPM-1,and PTEN,and plays an important role in promoting the proliferation and differentiation of tumor cells.However,the expression and mechanisms of miR-21 in pediatric Wilms' tumor have not yet been clarified.The PTEN,which is the first discovered tumor suppressor gene with phosphatase activity,plays a significant role not only in the induction of cell cycle arrest and apoptosis but also in other aspects of cell physiology,including the regulation of cell adhesion,migration,and differentiation.Many studies have confirmed that in a variety of advanced tumors including WT,deletions or inactivating mutations in the PTEN gene result in cancer susceptibility and progression.Concerning about the PI3K/AKT signaling pathway,which is a classical signaling pathway with oncogene activity.It is reported to be abnormally activated in various types of tumor cells,and plays an important role in promoting malignant proliferation and invasion,inhibiting apoptosis of tumor cells,and mediating resistance of radiotherapy or chemotherapy.PTEN is the key molecular of negatively regulating the PI3K/AKT signaling pathway.PTEN can reduce the activation of AKT and block the downstream signaling pathway regulated by AKT,thus make the oncogenic signaling pathway deactivated.According to various reports on miR-21 and PTEN expressions in different types of tumor tissues,we obtained the predictable conclusion that PTEN is a latent target gene of miR-21 in Wilms' tumor,and miR-21 regulates the biological behavior of WT via targeting PTEN expression.Based on the theory above,the purpose of this study is:to examine the expression levels of miR-21 and PTEN protein in WT,and to investigate the relationships among miR-21,PTEN expression,clinicopathological parameters,including gender,age,clinical stage,histopathological tumor type and lymphatic metastasis,and the prognosis of patients with WT.And on this basis,to investigate the effect of miR-21 inhibition on the biological behaviors including cell proliferation,invasion and apoptosis,and on the expression of PTEN for verifying their targeting regulatory relations,and to analyze the potential mechanisms of miR-21 and PTEN in the occurrence and development of WT.Methods1.Clinicopathological parameters and prognostic relevance of miR-21 and PTEN expression in Wilms' tumor:The expression levels of miR-21 and PTEN protein in WT(89 cases)and corresponding para-tumoral tissues(30 cases)were investigated by qRT-PCR and Western Blot,respectively.The independent sample t-test was used to evaluate the differences in the miR-21 and PTEN expression levels in categorical variables.The ?2 test was used to show differences in categorical variables.Differences in patient survival were determined using the Kaplan-Meier method and the log-rank test.A Cox proportional hazards regression analysis was used for univariate and multivariate analyses of prognostic values.A difference was considered statistically significant when P<0.05.2.MiR-21 promotes proliferation and inhibits apoptosis of primary Wilms' tumor cells via negatively targeting PTEN:MiR-21 inhibitor was transferred into primary WT cells by transient transfection,to inhibit the miR-21 expression in primary cells.Subsequently,qRT-PCR and WB were applied to detect the changes in miR-21,PTEN mRNA and protein levels in primary cells,respectively;dual luciferase reporter gene assay was utilized to measure the change in PTEN activity and its effect on the expression of key proteins in the PTEN/PI3K/AKT signaling pathway;and CCK-8 assay and flow cytometry were used to observe the effect of miR-21 inhibition on cell proliferation and apoptosis.The expression of each gene and related protein was expressed as the mean±the standard deviation(x±s).The comparison of two sample means was carried out by the two independent samples t-test,and the comparison of multiple sample means was performed using analysis of variance(ANOVA).A difference was considered statistically significant when P<0.05.All experiments were performed in triplicates.Results 1.Clinicopathological parameters and prognostic relevance of miR-21 and PTENexpression in Wilms' tumor:Compared with para-tumoral renal tissues,the expression levels of miR-21 were significantly upregulated in WT tissues,while the PTEN protein were significantly downregulated(P<0.05).Analyses of the clinicopathological parameters showed that the miR-21 expression level was significantly associated with age,late clinical stage,histopathological tumor type and lymphatic metastasis(P<0.05).PTEN protein expression was significantly associated with age,late clinical stage and histopathological tumor type(P<0.05).The univariate linear regression analysis illustrated a significant negative correlation between miR-21 and PTEN expression(r =-0.687,P<0.05).The Kaplan-Meier curve showed that patients with high miR-21 and low PTEN protein expression survived significantly longer(P<0.05).However,a multivariate analysis suggested that neither the expression level of miR-21 nor that of PTEN is an independent prognostic factor for overall survival.2.MiR-21 promotes proliferation and inhibits apoptosis of primary Wilms' tumor cells via negatively targeting PTEN:In primary WT cells,the down-regulation of miR-21 caused by miR-21 inhibition:(1)had an obvious inhibitory effect on cell proliferation,and meanwhile increased the apoptosis significantly(P<0.05);(2)caused an increase in PTEN protein level,while the change in PTEN mRNA level was not obvious,which confirmed that miR-21 silences the expression of PTEN at the post-transcriptional level;(3)caused an increase in the fluorescence activity of wild-type PTEN,while had no effect on the fluorescence activity of mutant PTEN,verifying that PTEN is one of the direct target genes of miR-21;(4)increased the expression of PTEN protein level,while decreased the protein expression levels of key molecules in its downstream PI3K/p-AKT signal pathways,confirming that miR-21 positively regulates PI3K/p-AKT signal pathways through PTEN,and thereby exerting its carcinogenic effect.Conclusion1.Both upregulated miR-21 and downregulated PTEN expression have a possible correlation with the aggressive progression and poor prognosis of WT,which suggests that upregulated miR-21 and downregulated PTEN expression may be valuable markers of tumor progression and indicators of the prognosis of WT.2.In primary WT cells,miR-21 negatively regulates the expression of PTEN at the post-transcriptional level,and positively regulates PI3K/p-AKT signal pathways through PTEN,exerting its carcinogenic effect:promoting cell proliferation and invasion,and suppressing cell apoptosis.miR-21 may be a potential target for new treatments of WT,and this provides some theoretical and experimental basis for targeted drug therapy of WT.