| BackgroundEsophageal cancer(EC)is the 11th most common malignant tumors worldwide and the sixth leading cause of death.Esophageal squamous cell carcinoma(ESCC)remains a predominant subtype of esophageal tumors in China and it is the 4th most frequent cause of death of malignant tumors in China.With the improvement of social and economic status,lifestyle and medical level,the incidence and mortality of esophageal cancer in China showed a trend of decline year by year,but the 5-year survival rate of patients was still very low.Therefore,it is particularly important to deepen the understanding of esophageal malignant tumors.The occurrence of esophageal cancer is a multi-factor and multi-step process,and the discovery of multiple oncogenes and tumor suppressor genes have partially elucidated the mechanism of tumor occurrence.And these results will be expected to become a new direction of cancer treatment.MYBL2 gene,also known as b-myb,isa highly conserved member of Myb transcription factor family and an important regulator of cell cycle progression,cell survival and differentiation.More and more evidences show that the abnormal expression of MYBL2 in tumor cells is involved in the formation of malignant phenotype of tumor cells and associated with the prognosis of multiple tumor patients which indicated that MYBL2 plays an important role in the genesis and progression of tumors.In recent years,the studies have found that epithelial-mesenchymal transition(EMT)plays an important role in the biological processes in invasion and metastasis of epithelial cell-derived malignant tumor cells.Recent studies have shown that MYBL2 is involved in the tumor EMT process.So far,there are few studies to illustrate the role of MYBL2 in ESCC,the effectof the oncogenic function of MYBL2 in ESCC remains unexplored.In this study,we will explore The oncogenic role and mechanism of MYBL2 expression in ESCC.This study will be divided into five parts:Part I Analysis of MYBL2 mRNA expression and its clinical significance in Esophageal cancer based on high-throughput multi-omics databasesObjectiveTo analyze the expression and clinical significance of MYBL2 mRNA in ESCC based on high-throughput multi-omics databases.Methods1.CBioPortal,Oncomine and Linked Omics were used to analyze the expression of MYBL2 mRNA in esophageal cancer and its relationship with the clinical prognosis of patients.2.Kaplan-meier method was used to analyze the relationship between the mutation of MYBL2 gene and the survival of patients with esophageal cancer.Non-parametric test was used to analyze the differences of MYBL2 mRNA level between esophageal cancer tissues and normal tissues.Spearman non-parametric test was used to analyze the relationship between MYBL2mRNA level and clinicalpathologyical characteri stics.Results1.The results of CBioPortal showed that 19.5%(36 cases/185 cases)of the 185 cases of esophageal cancer had MYBL2 gene changes(including mutation,alteration of copy number and mRNA level up-regulation),among which 14%of the MYBL2 mRNA level up-regulation exceeded the default threshold(EXP>2).Patients with MYBL2 gene changes had lower disease-free survival and overall survival than those without MYBL2 changes,but the difference was not statistically significant(P=0.179,P=0.273)2.The results of Oncomine showed that the mRNA level of MYBL2 in esophageal squamous cell carcinoma and esophageal adenocarcinoma was significantly higher than that in adj acent tissues(P<0.001,P<0.001)3.The results of Linked Omics showed that patients with distant metastasis had higher MYBL2 mRNA level than those without distant metastasis(P<0.01).In addition,MYBL2 mRNA expression was negatively correlated with the patients age(P<0.01).However,high MYBL2 expression was found not to be significantly associated with tumor invasion,lymph node metastasis and clinical stagePart ? Expression of MYBL2 protein in esophageal squamous cell carcinoma and its relationship with prognosisObj ectiveTo detect the expression of MYBL2 protein in ESCC patients,and to explore the relationship with the clinicopathological characteristics and prognosis in patients with ESCC.MethodsThe expression of MYBL2 protein was examined in cancer tissues and adj acent non-cancerous tissues from 107 patients by immunohistochemistry.The chi-square test was used to analyze the relationship between MYBL2 and clinicopathological features.Kaplan-meier method and Log rank method was used to estimate the survival curve.Multivariate Cox regression model were used to explore the associations of patient outcome and characteristicsResultsOur results indicate that MYBL2 staining was localized to the nuclei and cytoplasm in 71%of the ESCC tissues which was significantly higher than the staining observed in the corresponding non-tumor tissues(31.8%positivity)(?2=7.171,P=0.007).Furthermore,high MYBL2 expression was found to be significantly associated with histological differentiation,tumor invasion,lymph node metastasis and clinical stage.Kaplan-Meier analysis showed that elevated MYBL2 expression in ESCC tissues was negatively associated with post-operative overall survival in ESCC patients(P<0.001).The result further confirmed that MYBL2 expression(P=0.003),Tumor size(P=0.001),Lymph-node metastasis(P<0.001)were independent prognostic markers in ESCC patients.Part ? Effects of MYBL2 gene silencing and overexpression on proliferation,migration and invasion of esophageal squamous cell carcinomaObjectiveTo investigate the effect of MYBL2 on proliferation,epithelial-mesenchymal transition,invasion and migration of esophageal squamous cell carcinoma and its possible mechanismMethods1.Screening of esophageal squamous cell carcinoma cell lines:we analyzed MYBL2 mRNA and protein levels in TE7,EC9706,EC 109,and KYSE510 cell lines by RT-PCR and Western blot.To examine the function of MYBL2 in ESCC cells,we transduced the EC9706 cell lines with Lv-shNC and Lv-shMYBL2 to suppress MYBL2 expression.KYSE510 cell lines were transfected with MYBL2-expression vector to overexpress MYBL22.Effects of MYBL2 gene silencing and overexpression on proliferation of ESCC:The effect of MYBL2 on ESCC cell proliferation was measured by CCK-8 and EdU retention assay.The effect of MYBL2 on ESCC cell cycle progression was measured by flow cytometry.The expression of CDK1,CyclinBl and P21 were detected by Western blot.Finally,LinkedOmics was used to analysis the correlation between MYBL2mRNA and CDK1,CyclinB1 and P21 mRNA in ESCC3.Effects of MYBL2 gene silencing and overexpression on the migration and invasion of ESCC:cell motility and invasiveness were detected by wound healing assay and transwell invasion assay.The expression of E-cadherin,Vimentin,STAT3,p-STAT3 were detected by Western blot.Finally,LinkedOmics was used to analysis the correlation between MYBL2mRNA and E-cadherin and Vimentin mRNA in ESCC.4.Effects of AG490(JAK2-STAT3 pathway inhibitor)on MYBL2-mediated EMT in ESCC:the cell line KYSE510 was divided into three groups:KYSE510-control group,KYSE510-MYBL2 stable overexpression group,KYSE510-MYBL2 stable overexpression group+AG490 treatment group.The expression of MYBL2,E-cadherin,Vimentin,STAT3,p-STAT3 of the three groups were detected by Western blot.Results1.Screening of esophageal squamous cell carcinoma cell lines:At the level of MYBL2 mRNA,the expression levels of the four cell lines were EC9706>Eca109>TE7>KYSE510.At the level of MYBL2 protein,the expression level of four cell lines was EC9706>Eca109>TE7>KYSE510.According to the expression level of MYBL2 in each cell line,EC9706 was selected as the cell line with high expression of MYBL2,and KYSE510 was selected as the cell line with low expression of MYBL2.2.Effects of MYBL2 gene silencing and overexpression on proliferation of ESCC:cell growth was inhibited in cells transfected with Lv-shMYBL2 shRNA in comparison to the cells transfected with control shRNA of EC9706 cell lines(P<0.05).In contrast,overexpression of MYBL2 accelerated the cell growth in KYSE510 cells(P<0.05).We found that the proportion of cells in G2/M phase was increased in EC9706 cells knocked down for MYBL2(P<0.05).In contrast,the proportion of cells in S phase was increased in ESCC cells overexpressing MYBL2(P<0.05).Furthermore,Loss of MYBL2 caused a reduction in the levels of CDK1,and Cyclin B1 in EC9706 cells whereas these proteins were upregulated in KYSE510 cells overexpressing MYBL2(all P<0.05).We also analyzed the protein expression of cell cycle-related gene cyclin-dependent kinase inhibitor 1A(p21).We found that p21 was upregulated in cells with suppressed MYBL2 in EC9706 and downregulated in cells overexpressing MYBL2 in KYSE510(both P<0.05).Analysis of the LinkedOmics showed that MYBL2 mRNA was positively correlated with CycliB 1 mRNA and CDK1 mRNA,negatively correlated with P21 mRNA.3.Effects of MYBL2 gene silencing and overexpression on the migration and invasion of ESCC:the wound healing rates of EC9706 cells were inhibited after MYBL2 knockdown(P<0.05),while exogenous MYBL2 accelerated the wound healing rates of KYSE510 cells(P<0.05).In addition,transwell assays revealed that the degree of EC9706 cell invasion was significantly decreased after transduction of Lv-shMYBL2(P<0.05).In contrast,MYBL2-transfected KYSE510 cells exhibited increased invasiveness compared to the controls(P<0.05).E-cadherin and Vimentin were respectively upregulated and downregulated at the mRNA level indicating that MYBL2 is crucial for maintaining the mesenchymal characteristics of EC9706 cells.On the other hand,LV-MYBL2 transfected KYSE510 cells exhibited decreased E-cadherin and increased Vimentin levels indicating that MYBL2-overexpression induced EMT in these cells.Furthermore,STAT3 and p-STAT3 was significantly decreased in shMYBL2 cell lines and increased in MYBL2-overexpressing cell lines indicating that MYBL2-induced EMT in ESCC.Analysis of the LinkedOmics showed that MYBL2 mRNA was negatively correlated with E-cadherin mRNA.4.Effects of AG490 on MYBL2-mediated EMT in ESCC:treatment of the stable MYBL2-overexpressing KYSE510 cells with the JAK/Stat-3 inhibitor AG490 inhibited the MYBL2-induced decrease in E-cadherin and increase in Vimentin levels compared with that in the MYBL2-overexpressing cells.These results indicated that the activation of the JAK2-STAT3 pathway was critical for maintaining MYBL2-induced EMT in ESCC cells.Part IV The effect of MYBL2 blockade on cell proliferation and EMT of xenograft tumors with esophageal squamous cell carcinoma in nude miceObj ectiveTo study the effect of MYBL2 shRNA on cell proliferation and EMT of ESCC in vivo.Methods1.We established a xenograft mouse model where EC9706 cells transfected with Lv-shNC or Lv-shMYBL2 were injected subcutaneously into the dorsal region of the mice.The tumor volume was measured and the growth curve was generated.2.The tumors were removed after the experiment.The volume and weight of the tumors were measured.HE staining was used to observe the histological morphology of xenograft tumors.Immunohistochemistry were used to detect the expression of MYBL2,Ki67,CDK1,CyclinBl,E-cadherin and Vimentin protein in different groups of transplanted tumors.Results1.The xenograft tumors model of ESCC in the nude mouse was established successfully.2.We found that in mice that received MYBL2-depleted cells,the xenograft tumors were significantly reduced in terms of weight(g)(0.92±0.16 vs 1.79±0.56)and volume(mm3)(826.63±266.22 vs 1626.99±651.11)when compared to the control xenografts(P<0.05).3.IHCanalysis indicated that MYBL2-depleted tumor tissues had a marked decrease in Ki-67 expression(P<0.05)suggesting that the loss of MYBL2 inhibits tumor proliferation in vivo.We also found that MYBL2-depleted tumor tissues had a marked decrease in cyclinBl and CDK1 expression(both P<0.05),indicating that the loss of MYBL2 inhibits the expression of cell cycle related proteins in vivo.Compared to negative control group,the expression E-cadherin protein were significantly upregulated and Vimentin protein were significantly downregulated in the MYBL2-depleted tumor tissues(both P<0.05),indicating that the loss of MYBL2 inhibits the EMT process of ESCC in vivo.Part V MiR-29c-3p suppresses cell proliferation,migration and invasion in esophageal squamous cell carcinoma by inhibiting MYBL2Obj ectiveTo investigate the role of miR-29c-3p on cell proliferation,migration and invasion in ESCC by inhibiting MYBL2.Methods1.Prediction and screening of target miRNAs regulating MYBL2:firstly,Targetscan was used to predict miRNAs regulating MYBL2 expression.Furthermore,the gene expression database GEO was used to analyze the expression level of target miRNAs in ESCC tissues by using the miRNAs expression profile data sets GSE43732 and GSE114110.LinkedOmics was used to analyze the effect of the target miRNAs on the prognosis of patients with esophageal cancer and its correlation with MYBL2 mRNA.2.Effects of miR-29c-3p expression changes on MYBL2mRNA and protein expression:miR-29c-3p Mimics and miR-29c-3p Inhibitor were used to activate and inhibit the expression of mir-29c-3p respectively.The experiment was divided into four groups:mimis-control group,miR-29c-3p Mimics group,Inhibitor-control group and miR-29c-3p Inhibitor group.RT-PCR was used to detect the expression changes of miR-29c-3p in each group to verify the activation and inhibition effect of miR-29c-3p.The expression of MYBL2 in the four groups were detected by RT-PCR and Western blot which was designed to verify the effect of miR-29c-3p activation and inhibition on MYBL2 expression.3.Luciferase expression assay verified the targeted regulation of miR-29c-3p on MYBL2:the Mimics-control group and the miR-29c-3p Mimics group were transfected with MYBL2 wild-type(MYBL2 3'UTR,WT)and MYBL2 mutant(MYBL2 3'UTR,MUT)plasmids,respectively.The luciferase reporting system was used to verify the targeting relationship between miR-29c-3p and MYBL2.4.Effect of miR-29c-3p expression on proliferation,migration and invasion in ESCC:cells were divided into four groups:Mimics-control group,miR-29c-3p Mimics group,Inhibitor-control group and mir-29c-3p Inhibitor group.The effect of miR-29c-3p on ESCC cell proliferation was measured by CCK-8 and EdU retention assay.The effect of miR-29c-3p on ESCC cell migration and invasion was detected by Transwell migration and invasion assays.5.MiR-29c-3p suppresses cell proliferation,migration and invasion in esophageal squamous cell carcinoma by inhibiting MYBL2:the inhibition of miR-29c-3p Mimics on MYBL2 expression was restored by MYBL2-overexpression lentviral plasmids.The cells were divided into Mimics-control+NC plasmids,miR-29c-3p Mimics+NC plasmids and miR-29c-3p Mimics+MYBL2 plasmids.Firstly,the expression of MYBL2 in each group was detected by Western blot.Then the proliferation of cells in each group was detected by CCK8 and EdU retention assay,and the migration and invasion ability of cells in each group was detected by Transwell migration and invasion test.Results1.Prediction and screening of the target miRNAs regulating MYBL2:the prediction results of Targetscan indicated that the miR-29 family had the regulatory sites binding to the 3 'UTR of MYBL2.The miRNAs expression profile data sets GSE43732 and GSE114110 of ESCC were used to analyze the expression of miR-29 family members.The results showed that the expression of miR-29c-3p in the ESCC tissues was significantly lower than that in the adjacent tissues(P<0.001).The analysis by LinkedOmics found that miR-29c-3p was negatively correlated with MYBL2 expression in esophageal cancer patients(P<0.001).In summary,miR-29c-3p was finally selected for further experiments.2.Effects of miR-29c-3p expression changes on MYBL2 mRNA and protein expression:the RT-PCR results confirmed that miR-29c-3p Mimics group was significantly up-regulated compared with the control group(P<0.001).MiR-29c-3p expression was down-regulated in the mir-29c-3p inhibitor group compared with the control group(P<0.05).After the activation of miR-29c-3p,the mRNA level and protein level of MYBL2 were significantly down-regulated(all P<0.01).After inhibiting the expression of miR-29c-3p,the mRNA level and protein level of MYBL2 were up-regulated(all P<0.05).3.Experimental results of luciferase expression:compared with the Mimics-control+MYBL2 WT group,the luciferase activity of miR-29c-3p Mimics+MYBL2 WT group was significantly decreased,with statistically significant difference(P<0.05).Compared with Mimics-control+MYBL2 MUT group,the luciferase activity of miR-29c-3p Mimics-control+MYBL2 MUT group showed no significant change,and the difference was not statistically significant(P>0.05).4.Effect of miR-29c-3p expression on proliferation,migration and invasion in ESCC:the activation of miR-29c-3p can significantly inhibit the proliferation,migration and invasion of ESCC(all P<0.05);The inhibition of miR-29c-3p expression can promote the proliferation,migration and invasion of ESCC(all P<0.05).5.MiR-29c-3p suppresses cell proliferation,migration and invasion in esophageal squamous cell carcinoma by inhibiting MYBL2:MYBL2-overexpression lentviral plasmid was co-transfected with miR-29c-3p Mimics.The results showed that the MYBL2-overexpression lentviral plasmid significantly reduced the inhibitory effect of miR-29c-3p Mimics on MYBL2 expression(P<0.05),and significantly reduced the inhibitory effect of mir-29c-3p Mimics on cell proliferation,migration and invasion ability(all P<0.05).Conclusions1.The expression of MYBL2 protein in ESCC tissues was much higher than that in normal esophageal epithelial tissues and was an independent prognostic factor of ESCC patients.2.MYBL2 may promote the cell proliferation of ESCC by regulating cell cycle progression.3.MYBL2 is involved in EMT of ESCC.4.MiR-29c-3p suppresses cell proliferation,migration and invasion in ESCC by inhibiting MYBL2. |
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