| Background and Objective:Esophageal cancer is one of the most common human malignancies,with its mortality ranking sixth amongst cancer-related deaths worldwide.Esophageal squamous cell carcinoma(ESCC)is the major histological type.It is the leading cause of death from esophageal cancer in Asian countries,especially in China.Because of the lack of early detection,the majority of patients with ESCC are diagnosed at advanced stages with high risk of metastasis and recurrence.The 5-year overall survival rate of ESCC is less than 20%.Therefore,further investigation of molecular mechanism involved in ESCC is urgent and essential for developing early diagnostic and therapeutic strategies.The development and progression of ESCC involve synergic effects of various pathogenic factors,including particular dietary factors(chemical and physical),human papillomavirus infection,and genetic susceptibility.To develop personalized targeted therapy for ESCC,high-throughput techniques were used to further investigate genetic susceptibility.Genetic landscapes of ESCC with whole genome and exome sequencing illustrated that genomic alterations in ESCC included single nucleotide variants,copy number alterations,and alteration in multiple signaling pathways,such as cell cycle regulation,DNA damage control,and RTK-Ras-MAPK-PI3K-Akt,Notch and Wnt.Many researchers have recentlyreported the significance of both canonical and non-canonical Wnt signaling pathways in ESCC,thereby indicating the potential of Wnt signaling pathway markers as prognostic and therapeutic targets.The regulation of Wnt signaling pathway in ESCC,however,remains largely unknown.microRNA is a class of small(21-23nt),single-stranded noncoding RNAs that regulate gene expression post-transcriptionally by binding to the 3’-untranslated region(UTR)of target m RNAs.This typically causes m RNA degradation or translation repression.Highly conserved across species,microRNAs not only participate in biological processes,but also in the pathogenesis of human cancers.microRNA-30(miR-30)family is evolutionarily conserved,and is comprised of5 members,miR-30 a through miR-30 e.miR-30 family plays different roles as oncogenes or tumor suppressor genes in different kinds of cancer.For instance,miR-30 family members inhibited non-small-cell lung cancer,breast cancer,and colorectal cancer,but promote glioma,gasctic cancer,and pancreatic cancer.The miR-30 family was involved in the regulation of cancer cell apoptosis,proliferation,invasiveness,and metastasis,as well as epithelial-mesenchymal transition.miR-30 does this through targeting oncogenes and tumor suppressor genes under different circumstances,the detailed/complete mechanism of which remains to be explored.Emerging evidence indicated that the two strands of miR-30a(miR-30a-3p and miR-30a-5p)play roles in various kinds of cancer.Recent studies revealed that miR-30a-3p/5p were closely associated with WNT signaling pathway in breast cancer,multiple myeloma,and glioma;however little has been reported about the expression and roles of miR-30a-3p/5p in ESCC progression.Analysis of public microarray data along with our previous experiment results showed that miR-30a-3p/5p was down-regulated in ESCC in comparison with matched adjacent normal tissues.Additionally,bioinformatics analyses indicated that the target genes of miR-30a-3p/5p were significantly enriched in Wnt signaling pathway.Based off of these findings we sought to investigate the relationship between miR-30a-3p/5p and ESCC prognosis and the underlying mechanism involving the Wnt signaling pathway.Methods:Methods:1.Microarray data of ESCC from the public database GEO(https://www.ncbi.nlm.nih.gov/geo/)was used to analyze the expression pattern of miR-30a-3p/5p in ESCC tissue and the matched adjacent normal tissue.2.q RT-PCR assay was performed to detect the expression pattern of miR-30a-3p/5p in ESCC tissue and their matched adjacent normal tissue.Furthermore,analyze the relationships between miR-30a-3p/5p expression levels and clinicopathological parameters of ESCC as well as prognosis of patients with ESCC.3.q RT-PCR assay was used to examine the expression levels of miR-30a-3p/5p in normal esophageal squamous epithelial cell line Het-1A and ESCC cell lines KYSE30 and KYSE150.Lentivirus system was used to establish ESCC cell line KYSE30 with stable over-expression of miR-30a-3p/5p.By transient transfection technology,miR-30a-3p/5p inhibitors were used to down-regulated the endogenic expression of miR-30a-3p/5p in ESCC cell line KYSE150.4.MTT assay,plate colony formation assay and soft-agar colony formation assay to investigate the effects of miR-30a-3p/5p on ESCC cell proliferation in vitro.Subcutaneous tumor formation assay in node mice was performed to further confirm the effects of miR-30a-3p/5p on ESCC cell proliferation in vivo.5.Target genes of miR-30a-3p/5p were predicted in public database and algorithm miRWalk2.0.Pathway enrichment analysis was performed through public database KEGG.6.Luciferase reporter gene assay,western blot and q RT-PCR assay were carried out to verify the regulation effects of miR-30a-3p/5p on WNT signaling pathway in ESCC cell,as well as to screen and confirm the target genes of miR-30a-3p/5p in ESCC cell.Results:1 Expression pattern of miR-30a-3p/5p in ESCC tissue1.1 Expression pattern of miR-30a-3p/5p in ESCC tissue indicated by analysis of ESCC microarray data from public database GEOTo investigate the role of miR-30a-3p/5p in ESCC,the expression pattern of miR-30a-3p/5p in ESCC tissues and normal tissues using public microarray data(GSE43732,n=338)were analyzed firstly from GEO database.Results showed that both miR-30a-3p and miR-30a-5p were down-regulated in ESCC tissues when compared with adjacent normal tissues(P<0.05).1.2 Expression pattern of miR-30a-3p/5p in ESCC tissue indicated by analysis of 99 pairs of ESCC tissue and matched adjacent normal tissue through q RT-PCR assayqRT-PCR assay was performed to detected the expression of miR-30a-3p/5p in99 cases in fresh ESCC biopsies and their paired adjacent normal tissues.Consistent with the public microarray data,miR-30a-3p/5p was down-regulated in 81.82%(81/99)of ESCC tissues compared to their matched adjacent normal tissues.1.3 Relationships between miR-30a-3p/5p expression levels and clinicopathological parameters of ESCC as well as prognosis of patients with ESCCStatistical analyses further revealed that miR-30a-3p/5p expression between different differentiation status of ESCC showed no difference,but miR-30a-3p/5p expression inversely correlated with classifications of primary tumor and lymphatic metastasis(P<0.05).Importantly,survival analysis indicated that the group of ESCC patients with lower miR-30a-3p/5p expression had shorter overall survival time(P<0.05).2 Effects of miR-30a-3p/5p on the proliferation ability of ESCC cell2.1 Over-expression and down-regulation of miR-30a-3p/5p expression in ESCC cell linesqRT-PCR assay results showed that expression of miR-30a-3p/5p was significantly lower in ESCC cell lines KYSE30 and KYSE150 than that in normal esophageal epithelial cell line Het-1A(P<0.05).Besides,expression of miR-30a-3p/5p in KYSE30 was lower than that in KYSE150.Molecular clone technology was used to establish lentivirus vector containing miR-30a-3p/5p successfully,without any kinds of variant as verified by sequencing examination.ESCC cell line KYSE30 with stable over-expression of miR-30a-3p/5p was successfully established through lentivirus system and down-regulation of miR-30a-3p/5p in ESCC cell line KYSE150 was achieved by transient transfection of miR-30a-3p/5p.Over-expression and down-regulation of miR-30a-3p/5p in ESCC cell lines were confirmed by q RT-PCR assay(P<0.05).2.2 Effects of miR-30a-3p/5p on ESCC cell proliferation in vitroMTT and colony formation assays indicated that over-expression of miR-30a-3p/5p significantly repressed the proliferation of KYSE30 cells(P <0.05)while down-regulation of miR-30a-3p/5p evidently promoted the proliferation of KYSE150 cells(P<0.05),in comparison with control groups respectively.Besides,soft-agar assay showed that the anchorage-independent growth ability of ESCC cell was attenuated by over-expression of miR-30a-3p/5p(P <0.05)but was enhanced by down-regulation of miR-30a-3p/5p(P<0.05),in comparison with control groups respectively.2.3 Effects of miR-30a-3p/5p on ESCC cell proliferation in vivoSubcutaneous tumor formation assay showed that miR-30a-3p/5p over-expression group exhibited remarkably smaller tumor volume(P<0.05)and slower tumor growth rate(P<0.05)while miR-30a-3p/5p down-regulation group had significantly larger tumor volume(P<0.05)and faster tumor growth rate(P<0.05),in comparison with the control groups respectively.3 Molecular mechanism of the regulation effects of miR-30a-3p/5p on ESCC development3.1 Prediction of miR-30a-3p/5p’s target genes and pathway enrichment analysis of target genesPrediction of miR-30a-3p/5p target genes through miRWalk2.0 indicated that miR-30a-3p might target CCND2,PRKACB,MAPK10,WNT2,FZD10 and DVL3 while miR-30a-5p might target LRP6,FZD3,CSNK1A1,FZD2,MAP3K7 and PRKCA.Pathway enrichment analysis of these two groups target genes in KEGG showed that the related and potential pathways included Pathway in cancer,Ras signaling pathway,MAPK signaling pathway and Wnt signaling pathway.3.2 miR-30a-3p/5p inhibited the activity of WNT signaling pathway in ESCC cellThe WNT signaling luciferase reporter assay showed that over-expression of miR-30 a significantly attenuated the activity of WNT signaling pathway,while inhibition of miR-30 a remarkably enhanced it(P<0.05).Analyses of WNT signaling downstream genes further indicated that over-expression of miR-30 a decreased the expression of Cyclin D1(P<0.05)and increased the expression of p27 and p21(P<0.05),while inhibition of miR-30 a increased the expression of Cyclin D1(P<0.05)and decreased the expression of p27 and p21(P<0.05),in both protein level and m RNA level,as respectively indicated by the results of western blot and q RT-PCR.3.3 miR-30a-3p and miR-30a-5p directly targeted WNT signaling pathway components,WNT2 and FZD2 respectively in ESCC cellQRT-PCR assay was performed to screen the potential targeting genes related to WNT signaling of miR-30a-3p/5p.Results showed that m RNA expression of WNT2 was repressed by transfecting miR-30a-3p-minics(P<0.05),and FZD2 was inhibited by miR-30a-5p-mimics(P<0.05).Further confirmation through q RT-PCR assay and western blot assay demonstrated that over-expression of miR-30a-3p decreased the expression of WNT2(P<0.05)while inhibition of miR-30a-3p increased it(P<0.05),in both protein level and m RNA level.The miR-30a-5p showed the same effects on FZD2 expression.Over-expression of miR-30a-5p decreased the expression of FZD2(P<0.05)while inhibition of miR-30a-5p increased it(P<0.05),in both protein level and m RNA level.Luciferase reporter assays also demonstrated that over-expression of miR-30a-3p significantly reduced the luciferase activity of wild-type WNT2-3’-UTR in a dose-dependent pattern(P<0.05),while it had no effects on the mutant one.Likewise,over-expression of miR-30a-5p evidently reduced the luciferase activity of wild-type FZD2-3’-UTR in a dose-dependent pattern(P<0.05),but had no influence on the mutant one(P>0.05).Conclusion:1.ESCC tissue had lower expression of miR-30a-3p/5p than normal esophageal squamous epithelial tissue.Low expression of miR-30a-3p/5p was closely associated with advanced ESCC progression and poor prognosis of ESCC patients.2.Down-regulation of miR-30a-3p/5p promoted ESCC cell proliferation while over-expression of miR-30a-3p/5p suppressed it,both in vitro and in vivo.3.Mi R-30a-3p and miR-30a-5p could inhibit the activity of Wnt signaling pathway by respectively targeting the 3’-UTRs of WNT2 and FZD2. |
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