MiR-146a-5p Mediates Epithelial-mesenchymal Transition Of Esophageal Squamous Cell Cancer Via Targeting Notch2

Part ? The expression level and clinical value of miR-146a-5p in esophageal squamous cell cancerBackground:Esophageal cancer is one of the deadliest cancers worldwide.Esophageal squamous cell cancer(ESCC)accounts for the majority of esophageal cancer cases in China.Recently,many advanced techniques have been used for its diagnosis and treatment,however,the prognoses of ESCC patients remain to be poor.The overall 5-year survival of esophageal cancer patients ranges from 15%to 25%,mainly due to the fact that patients are diagnosed at advanced stages.Therefore,early diagnosis is of great value to improve prognosis of patients.Prognostic biomarkers can be used to guide the clinical treatment.Current diagnostic and prognostic biomarkers,however,are of low sensitivity and specificity.It is necessary to search biomarkers with great sensitivity and specificity.MicroRNAs(miRNAs)are small non-coding regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins.Accumulating evidence has shown that miRNAs can participate in tumor genesis,progression and metastasis either as oncogenes or tumor suppressors.Measurement of miRNA expression in tumors may have diagnostic and prognostic implications and aberrant miRNA expression is associated with the development and progression of various types of human cancer.Down-regulation of miR-146a-5p is found in breast,lung,pancreatic and gastric carcinomas.Exogenous miR-146a-5p expression could decrease the proliferation of several kinds of cancer cells.This contrasts with the fact that miR-146a-5p is upregulated in melanoma,squamous cell carcinoma of the cervix and thyroid carcinoma,which has led to controversy as to its role in carcinogenesis.Taking the above findings as a whole,miR-146a-5p seems to play a wide variety of roles in the regulation of different genes in various cancers.We aimed to evaluate the expression level of miR-146a-5p at ESCC and its value in the prognosis and diagnosis of ESCC.Methods:We examined miR-146a-5p expression in 62 pairs of ESCC cancerous and matched paracancerous tissue,115 formalin-fixed paraffin-embedded(FFPE)tissue samples,and serum samples from 154 ESCC patients and 154 healthy volunteers using quantitative reverse transcription polymerase chain reaction(qRT-PCR).Kaplan-Meier method,Cox regression and Receiver operating characteristic(ROC)curve analysis were applied to analyze its prognostic and diagnostic value.Results:miR-146a-5p expression level was significantly decreased in ESCC tissue compared with paracancerous tissue(0.50±0.36 vs 1.06±0.09,P<0.001).Its regulation level was negatively associated with T factor and TNM stage.Kaplan-Meier curve revealed that its down-regulation level predicted worse overall survival(OS)(P= 0.017)and progression-free survival(PFS)(P=0.004).Both univariate and multivariate analyses identified miR-146a-5p expression as independent prognostic factor for OS(P=0.022)and PFS(P=0.004).Serum miR-146a-5p was significantly reduced in ESCC patients than in healthy controls(P<0.001).Area under the curve of ROC(AUC),sensitivity and specificity for this marker were 0.863 ± 0.033,85.7%and 68.6%in Discovery Group,and 0.891±0.027,82.1%,83.3%in Validation Group.Conclusion:miR-146a-5p is significantly reduced in cancerous tissue and serum samples of ESCC patients.It is an ideal biomarker for the prognosis and diagnosis of ESCC with promising sensitivity and specificity.Part II The expression level and prognostic value of Notch2 in esophageal squamous cell cancerBackground:Targeted therapy is one breakthrough method for cancer.Targeted medicine can bind to specific site and kill tumor cells,which reduces the side effects on normal tissues.Researches of ESCC targeted therapy are unfolding,however,the results of in vitro mechanisms and clinical researches are far from ideal.To investigate new targeted medicines or target sites is of great value.Notch signal pathway regulates embryo growth,cell immigration and apoptosis,and stem cell differentiation.It plays important roles in esophageal cancer,breast cancer,colorectal cancer,pancreatic cancer,non-small-cell lung cancer and cervical cancer.The aberrant expression levels of Notch signal pathway are found in esophageal cancer.High expression level of Notchl is correlated with differentiation,staging and lymph nodes metastasis of esophageal adenocarcinoma(EAC).Gamma-secretase inhibitor(GSI)enhances chemotherapy sensitivity of EAC via inhibiting Notch pathway.Notch2 is upregulated in lung cancer,glioma,cervical cancer,liver cancer and gastric cancer and acts as oncogene in these cancers.It contrasts with the fact that Notch2 is down-regulated in bladder cancer,breast cancer and colorectal cancer.This phenomenon indicates various regulation roles of Notch2 in cancer.Whole-genome sequencing and exon sequencing revealed Notch signal pathway mutation in esophageal cancer,especially Notch2.However,the expression level of Notch2 and its clinical values have never been reported in ESCC.We aim to investigate Notch2 expression lever and the relation between Notch2 and clinical characteristics.Besides,we analyzed its prognostic values and roles in ESCC proliferation and colon survival fraction.Methods:qPCR,immunohistochemical staining(IHC)and western blot were used to detect mRNA and protein expression levels of Notch2 in ESCC tissues.Log-rank test and multivariate analysis were used for survival analysis.Moreover,CCK8 and clonogenic assay were applied to validate whether Notch2 downregulation could decrease proliferation and colon formation abilities of ESCC cells.Results:A notably higher Notch2 expression level was found in ESCC tissues at the mRNA(P<0.0001)and protein levels(IHC:P = 0.004;WB:P = 0.021).Log-rank analysis demonstrated that Notch2 overexpression was significantly associated with worse OS(29.1%vs.49.1%;P = 0.013)and PFS(15.3%vs.34.4%;P = 0.006)rates in ESCC patients.The multivariate analysis revealed Notch2 as an independent prognostic factor for OS and PFS(P = 0.002 and 0.006,resp.).Besides,in vitro assays showed that OD450 values and colony formations were significantly reduced in shRNA-Notch2 group(all P<0.0001).Conclusion:These results showed that Notch2 is up-regulated in ESCC tissues and could serve as a promising biomarker for identifying individuals with poor prognosis.Notch2 inhibition could decrease the ability of ESCC proliferation and colony formation,which provide a new target for ESCC therapy.Part ? miR-146a-5p mediates epithelial-mesenchymal transition of esophageal squamous cell cancer via targeting Notch2Background:Metastasis is one barrier to improve the prognosis of ESCC.It is necessary to explore the mechanisms of metastasis and investigate new targets.Epithelial-mesenchymal transition(EMT)plays important roles in embryos development.Recent researches revealed the roles of EMT in primary tumors.EMT is an evolutionarily conserved development process during which epithelial cells lose polarity and develop a mesenchymal phenotype.It promotes invasion and metastasis of tumor.miRNAs are small non-coding regulatory RNA molecules.They could bind to and cleave their messenger RNAs or inhibit their translation into proteins.In this way,miRNAs inhibit the expression of specific target genes.Accumulating evidence has shown that miRNAs can participate in tumourigenesis,progression and metastasis either as oncogenes or tumor suppressors.miRNAs also participate in tumor EMT regulation.miR-630 inhibits EMT of gastric cancer via targeting Wnt/betacatenin.miR-514a-3p inhibits cell proliferation and epithelial-mesenchymal transition by targeting EGFR in clear cell renal cell carcinoma.miR-146a-5p also plays important roles in EMT.It inhibits EMT in non-small cell lung cancer by targeting insulin receptor substrate 2.It is down-regulated in metastatic colorectal tumor tissues compared with primary tumor tissues.Our previous study found that miR-146a-5p is down-regulated in ESCC tissues and serum,and it could act as a promising diagnostic and prognostic biomarker.Notch2 is increased in mesenchymal phenotype cells compared with epithelial cells which indicates that Notch2 is correlated with EMT.Inhibition of Notch2 could reverse EMT and decrease Vimentin,Zinc Finger E-box binding homeobox-box1(ZEB1),Slug and Snail,as well as the invasion ability.Our previous study revealed Notch2 overexpression level and oncogene roles in ESCC.miRNA databases indicate that Notch2 exhibited miR-146a-5p binding sequences in its 3'-UTR regions.We aimed to investigate the mechanisms of miR-146a-5p and Notch2 in ESCC metastasis to provide new targets for ESCC treatment.Methods:Invasion assay,qRT-PCR and western blot were used to validate the roles of miR-146a-5p and Notch2 in EMT progression.miRNA target gene prediction databases and dual-luciferase reporter assay were used to validate the target gene.Results:miR-146a-5p mimics inhibited invasion ability of ESCC cells(EC9706:86.00±3.74 vs 198.33±5.44,P<0.001;Eca109:73.33±2.87 vs 153.00±5.72,P<0.001),miR-146a-5p inhibitor led to increase of invaded ESCC cells(EC9706:417.00±12.83 vs 198.33±5.44,P<0.001;Ecal09:358.67± 10.66 vs 153.00±5.729 P<0.001).Protein level of E-cadherin decreased(EC 9706:P = 0.007;Eca 109:P<0.001),while Snail(EC 9706:P= 0.005;Eca 109:P=0.017)and Vimentin(EC 9706:P<0.001;Eca 109:P<0.001)increased in anti-miR-146a-5p group,which demonstrated that miR-146a-5p inhibits EMT progression of ESCC cells.MiRNA target gene prediction databases indicated the potential of Notch2 as a direct target gene of miR-146a-5p and dual-luciferase reporter assay validated it.Importantly,shRNA-Notch2 restrained EMT and partially abrogated the promoting effects of miR-146a-5p inhibitor on EMT progression.Conclusions:miR-146a-5p functions as a tumor suppressive miRNA targeting Notch2 and inhibits EMT progression of ESCC.