Expression And Effect Of MiR-451and MiR-21on The Proliferation, Metastasis And Apoptosis In Esophageal Squamous Cell Carcinomas

Esophageal squamous cell carcinoma (ESCC) is one of the most commonmalignant diseases with poor prognosis, metastasis and high mortality, and themajority of recurrence after resection occurs due to invasion-related spreading. Therehas been the highest esophageal cancer rate in Linzhou city of Henan Province.Although the different available multi-modalities of therapies, including radicaltherapy, surgical operation, chemical therapy, intervention therapy and so on, havecurrently been used in the treatment for esophageal squamous cell carcinoma, theprognosis of esophageal cancer has not been markedly improved. Thus it isimperative to investigate the mechanism of ESCC metastasis and invasiveness and toexplore the effective therapeutic strategies.microRNAs (miRNAs) are small, non-coding RNAs approximately20~25nucleotides in length that negatively regulate gene expression at thepost-transcriptional and/or translational level by binding to complimentary sequencesin the3′-untranslated regions (UTRs) of target mRNAs. MiRNAs affect the stabilityand translational efficiency of their target mRNAs. They have been implicated in anincreasing number of biological processes, including carcinogenesis. The up-ordown-regulation of specific miRNAs during carcinogenesis has been documentedwidely, and the molecular pathways that mediate the effects of miRNAs on different aspects of cancer development, including proliferation, angiogenesis and metastasis,have been identified. Dysregulation of miRNAs plays roles in the pathogenesis ofhuman diseases, including malignancy. miRNAs may function as both oncogenes andtumor suppressors.miR-451was identified using bioinformatics methods and was first reported byY. Altuvia in2005. It maps to the chromosomal region17qll.2. Several reports havedescribed the functions of miR-451in the differentiation of the hematopoietic system,embryo maturation, epithelial cell polarity and nervous system development. Recentstudies showed that miR-451plays a role in a variety of tumors, and not only theabnormal expression of this miRNA in tumor tissue, but also its involvement in thebiological behavior of certain tumors and expression of drug resistance genes havebeen demonstrated. miR-21, a well-known oncogenic miRNA, is most frequentlyoverexpressed in several different types of human cancers, and it has been shown tobe implicated in multiple malignancy-related processes including cell proliferation,apoptosis, invasion, and metastasis. The present study examined the effect andmechanism of miR-451and miR-21on the proliferation, apoptosis and invasion of theesophageal carcinoma cell line EC9706.The research includes three parts. The first part is altered miRNA expression inesophageal cancers and correlation with clinicopathological characteristics of ESCCpatients. The second part is effect of miR-451and miR-21on the biological behaviorof the esophageal carcinoma cell line EC9706. The last part is identification andmechanism of miR-451and miR-21target genes.Part One Altered miRNA expression in esophageal cancers and correlationwith clinicopathological characteristics of ESCC patients.Methods：1. Fifty-three pairs of primary esophageal squamous cell cancer tissues andcorresponding adjacent normal esophageal tissues were used.2. Agilent miRNA microarray was used to analyze the miRNA expression profile inESCC compared to normal tissues. Then we made a preliminary analysis ofbiological function for the most differentially expressed miRNAs and their potentially target genes regulated.3. Twelve microarray results were validated by performing quantitativeRT-PCR.,including three miRNAs upregulated (miR-21, miR-125a-3p andmiR-503) and nine miRNAs downregulated(miR-429, miR-133b, miR-451,miR-139-5p, miR-133a, miR-127-3p, miR-210, miR-199a-5p and miR-199b-5p).4. miR-451and miR-21expression levels in ESCC tissues were associated withclinicopathological characteristics of ESCC patients.5. Statistical analysis: All the dates were analyzed by SPSS17.0statistical package,the enumeration data was calculated the positive rate, the comparison of positiverates uses the Chi-square; measurement data were expressed by standard deviation,the mean of two groups uses the student’s t-test. The mean among more groupsuses the ANVOA. The relation of two variables was analyzed by the Spearmancorrelation analysis. α=0.05is considered as significance.Results：1. Compared with the normal tissues, tumor tissues exhibited199differentiallyexpressed miRNAs, including103miRNAs upregulated and96miRNAsdownregulated. miR-451is downregulated and miR-21is upregulated inesophageal tissues.2. Using corresponding adjacent normal esophageal tissues as reference, miR-429,miR-133b, miR-451, miR-139-5p, miR-133a, miR-127-3p, miR-210,miR-199a-5p and miR-199b-5p expression in ESCC tissues were found to besignificantly reduced (P<0.05), miR-615-3p, miR-125a-3p significantly increased(P<0.05). We found quantitative RT-PCR and Agilent microarray miRNA profilescorrelate strongly，Among the12miRNAs examined, only miR-503was notmatched(P>0.05), the rest of the miRNAs assayed were matched by bothmethods.3. miR-21and miR-451expression levels in ESCC tissues were associated with theoccurrence of lymph node metastases, differentiation status and TNM stage. Therewas no statistically significant difference between miR-21, miR-451expressionand either gender, age or tumor location.Part Two Effect of miR-451and miR-21on the biological behavior of the esophageal carcinoma cell line EC9706.Methods：1. Synthetic miR-451mimics, miR-21inhibitor and miRNA scramble weretransfected into EC9706cells using LipofectamineTM2000.2. The expressions of Bcl-2、AKT、CDKN2D、FasL and TIMP3were analyzed byRT-PCR and Western blotting.3. Transwell assay was used to assess the effect of miR-451on EC9706cell invasionand metastasis.4. CCK-8assay was used to assess the effect of miR-451on EC9706cellproliferation.5. FACS was used to assess the effect of miR-451on EC9706cell cycle andapoptosis.6. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/cnude mice.7. Statistical analysis: All the dates were analyzed by SPSS17.0statistical package,The mean among more groups uses the ANVOA. α=0.05is considered assignificance.Results：1. Compared with control group，the expression of miR-451was significantlyincreased（P<0.01）, and the expression of miR-21was significantly decreased（P<0.01）.2. The expression of Bcl-2、AKT and CDKN2D were significantly decreased inmiR-451mimics group (P <0.05). The expression of FasL and TIMP3weresignificantly increased in miR-21inhibitor group (P <0.05).3. The average amount of cells penetrating Matrigel in miR-451mimics group was47.4±7.4, which was significantly decreased (P <0.01). The average amount ofcells penetrating Matrigel in miR-21inhibitor group was24.1±3.1, which wassignificantly decreased (P <0.01).4. The growth was significantly suppressed in2days later（P <0.05）,which wasmore obvious with time passing. 5. Compared to blank control, liposome control and scramble treated cells, thefractions of EC9706cells in the G0/G1, S and G2/M phase of the cell cycle inmiR-451mimics group were74.89%,15.84%and9.27%, respectively.6. The apoptosis rate in miR-451mimics group is (12.07±1.12)%, which wassignificantly increased (P <0.01). The apoptosis rate in miR-21inhibitor group is(12.89±1.28)%,which was significantly increased (P <0.01).7. A significant decrease in tumor volume was observed in the miR-451mimics-treated and miR-21inhibitor-treated group.Part Three Identification and mechanism of miR-451and miR-21target genes.Methods：1. Using bioinformatics analysis, the target genes of differentially expressed miRNAwere predicted.2. Western blot and dual luciferase report experiments were used to identify thetarget genes of miR-451and miR-21.3. Restore experiment were used to analyze the mechanism of target genes.Results：1. Using bioinformatics analysis, TIMP3and FASL are target genes of miR-21，CDKN2D is target gene of miR-451.2. Relative luciferase activity was significantly suppressed in EC9706afterco-transfection miR-451mimics and wild type pmirGLO-CDKN2D（P <0.05）,which indicated miR-451regulated CDKN2D negatively by binding3’UTR seedregion.3. Relative luciferase activity was significantly suppressed in EC9706afterco-transfection miR-21mimics and wild type pmirGLO-TIMP3, significantlyincreased after co-transfection miR-21inhibitor and wild type pmirGLO-TIMP3（P <0.05）, which indicated miR-21regulated TIMP3negatively by binding3’UTR seed region.4. Relative luciferase activity was significantly suppressed in EC9706afterco-transfection miR-21mimics and wild type pmirGLO-FASL, significantlyincreased after co-transfection miR-21inhibitor and wild type pmirGLO-FASL （P <0.05）, which indicated miR-21regulated FASL negatively by binding3’UTR seed region.5. Cell cycle experiment results showed that G0/G1proportion of cells issignificantly lower and S phase proportion is significantly higher after transfectionwith pcDNA3.1-CDKN2D expression vector without3’UTR region（P <0.05）.There was no statistically significant difference between transfections with miR-451mimics, pcDNA3.1-CDKN2D and only pcDNA3.1-CDKN2D without3’UTR region (P>0.05).6. Cells invasion results showed that cells through the basement membrane weresignificantly reduced after transfection with pcDNA3.1-TIMP3expression vectorwithout3’UTR region（P <0.05）. There was no statistically significant differencebetween transfections with miR-21mimics, pcDNA3.1-TIMP3and onlypcDNA3.1-TIMP3without3’UTR region (P>0.05).7. Cells apoptosis results showed that apoptosis cells were significantly increasedafter transfection with pcDNA3.1-FASL expression vector without3’UTRregion（P <0.05）. There was no statistically significant difference betweentransfections with miR-21mimics, pcDNA3.1-FASL and only with pcDNA3.1-FASL without3’UTR region (P>0.05).Conclusion1. Compared with the normal tissues, tumor tissues exhibited199differentiallyexpressed miRNAs, including103miRNAs upregulated and96miRNAsdownregulated. miR-451is downregulated and miR-21is upregulated inesophageal tissues.miR-21and miR-451expression levels in ESCC tissues wereassociated with the occurrence of lymph node metastases, differentiation statusand TNM stage. No significant differences were observed between miR-21,miR-451expression and either gender, age or tumor location.2. Upregulated expression of miR-451and downregulated expression of miR-21induced apoptosis and suppressed cell proliferation, invasion and metastasis in theesophageal carcinoma cell line EC9706.3. TIMP3and FASL are target genes of miR-21，CDKN2D is target gene of miR-451.miR-451can bind to putative binding sites within theCDKN2D mRNA3’ untranslated regions (UTR) to regulate cell cycle. miR-21can bind to putativebinding sites within the TIMP3and FASL mRNA3’ untranslated regions (UTR)to regulate cell invasion and apoptosis.