The Function And Mechanism Research Of MiR612 Associated With Esophageal Squamous Cell Carcinoma Metastasis Mediated Through P53

Esophageal squamous cell carcinomas(ESCC)is one of the most common cancer in the world with the clinical features of insidious onset,invasive fast-growing,high recurrence rate and fatality.Most patients were diagnosed at middle-late stage.At present,operation and chemoradiotherapy are the main methods of treatment,but the surgical resection rate of esophageal cancer was only 58%-92%.Although surgery is thus far the most efficient for treatment,strongly potential invasion and high frequent metastasis of cancer cells may result in bad prognosis.Recurrence and metastasis were found in many patients after resection of esophageal cancer.The prognosis depends on early diagnosis.microRNAs are small,non-coding RNAs that negatively regulate gene expression,play an important role in the occurrence and development of tumors.miRNAs can be used not only as poncogenes to promote tumor growth by regulating tumor-suppressor genes,but also in posttranscriptional level as a tumor suppressor gene to inhibit the expression of proto-oncogene by combining with it.Recent researchs show that several human cancer are more closely related to abnormal expression of miRNAs.So miRNAs are the most popular biological molecules in tumor pathology.At present,the hotspot in the field of cancer research include miRNAs gene expression profiles,the mechanism for genetic regulation and miRNAs targets prediction.With the development and metastasis of esophageal squamous carcinoma,miRNAs are thought to be more and more important.Those miRNAs participated in the development of tumor may be served as an important marker for early diagnosis,disease progresses and evaluation of prognosis.However,the mechanism of differentially expressed genes remains unknown.So we study miRNAs gene expression profiles by microarray technique to screen out the differentially expressed miRNAs and analyze its main function.We would screen out specific target genes of miRNAs with the theories and methods of bioinformatics.Moreover,we validated the relations between the miRNAs and their targets at the gene level.Finally,we concluded that miRNAs are the important promoting factors in the infiltration and transition of esophageal squamous carcinoma by controlling target genes.Objective1.We aimed to identify miRNAs differentially expressed in esophageal squamous carcinoma with lymph node metastasis and those without metastasis by whole genome miRNAs microarray.2.miR-612 was found highly expressed in the tumors,we detected this differentially expressed gene by RT-PCR to prove the reliability of our results.3.We screened out specific target genes of miRNA-612 with the theories and methods of bioinformatics.4.The functions of miRNA-612 were verified by scratch assay?Transwell cell migration test and invasion test with human esophageal squamous carcinoma cell line.5.Egfp fluorescent reporter carrier experiment proved that wt-P53 was the direct target of miR-612.6.wt-P53 protein expression in tumor and normal tissues were detected by Western blot.7.The gene and protein expression of miR-612 and wt-P53 in EC 109 cells were detected by RT-PCR and Western Blot.Materials and methods1.We conducted miRNAs expression profiling for 10 esophageal squamous carcinoma and normal tissue,using miRCURYTM LNA Array.Total RNA was isolated using Trizol,acquired the fluorescence intensity by the chip scanner of Axon GenePix 4000B.Scanned images were then imported into GenePix Pro 6.0 software for data analysis.Differentially expressed miRNAs were identified by combination of t test combined with Fold Change.Cluster analysis was performed with screened miRNAs using MEV software(v4.6,TIGR,Rockville)to find the transfer related specific miRNAs of ESCC.2.We selected up-regulated miRNAs of miRNA-612 to validate by RT-PCR.U6 was used as reference for normalization.CT value was first obtained from comparing the amplification curves with threshold value.The fold change between miRNA-612 and U6 was obtained by 2-??CT.3.We applied TargetScans?Pictar?miRanda and MirTarget 2 to predict the miRNA targets.Go and KEGE analysis were used to screen out the miRNA-612 possible targets.They have more biological functions including cell growth?differentiation?apoptosis?transcription and signal transduction,which co-regulate the occurrence and development of tumors.4.Human esophageal carcinoma cell line EC109 was selected to transfect silence plasmid of miRNA-612.To validate the transfection efficiency by RT-PCR.The functions of miRNA-612 were verified by scratch assay?Transwell cell migration test and invasion test with different groups.5.pSilencer/miR-612 or pSilencer/NC and the p53 3'UTR mutant(pcDNA3-Egfp-P53-3'UTR mut)or non mutant(pcDNA3-Egfp-P53-3'UTR(0.3?g))were cotransfected to EC 109 cells with Lipofectamine 2000.After 48h,the expressing level of green fluorescent proteins were measured with fluorescence spectrophotometer F-4500 at 484nm for excitation and 510nm for emission.6.The fresh frozen tissues of 70 primary esophageal squamous cell carcinomas,34 with lymph node metastasis,36 without the metastasis and 20 nomal esophageal mucosa tissues were selected from the tissue collection bank from 4/2002 to 2/2005 in the Department of Pathology,Qianfoshan Hospital of Shandong Province.P53 protein expression in tumor and normal were measured by Western blot.7.EC 109 cells were transfected with pSilencer/miRNA-612 which expresses miRNA-612 and the control plasmid pSileneer/NC respectively.P53 protein expression in EC 109 cells transfected with miRNA-612 by RT-PCR and Western blot.Rusult1.16 miRNAs were differentially expressed between lymph node metastasis group and non-metastasis group,9 of them were upregulated,and 7 of them were downregulated.Further conducte differences multiple analysis and non-parametric PAM(prediction analysis for microarrays)analysis revealed miRNA-612 as a meaningful miRNAs that is upregulated in ESCC development and metastasis.2.We validated that the expression of miRNA-612 increased in tumors by RT-PCR,and the lymph node metastasis group was higher than that non-metastasis group,which was consistent with miRNAs expression profiling.3.After miRNA-612 expression was repressed and without impacting the cell proliferation remarkable,the group with miRNA-612 inhibitor had significantly reduced expression of miRNA-612 while the two control groups essentially had no change miRNA-612 inhibitors.This suggested that miRNA-612 is positively correlated with tumor aggressiveness.4.Gene Ontology analysis on these genes suggest these genes are involved in several important cancer related functions/pathways such as cell growth regulation,differentiation,apoptosis,cell-cell adhesion and cell-matrix adhesion,and cell invasion and migration.We selected the target gene of wt-P53 that participate in cell apoptosis proliferation.In our "blast" analysis,the miRNA-612 seed sequence at 5'end had matched with the 3' UTR of wt-P53 blast.5.Compared to the control group,the cells infected with miRNA-612 expression plasmid had reduced wt-P53 3'UTR reporter plasmid green fluorescent protein(GFP)with 29.3%inhibition rate(p<0.05),which demonstrated that miRNA-612 could act on the wt-P53 3'UTR,that is to say wt-P53 is the direct target of miR-612.6.Western blot analysis was conducted to measure wt-P53 protein expression in ESCC.Both the tumors with metastasis and the tumors without metastasis had significantly reduced wt-P53 protein expression compared to the normal tissues(P<0.05).As result of analysis of western blot,the tumors with metastasis had further reduced wt-P53 expression compared to the tumors without metastasis(P<0.05).7.The wt-P53 mRNA as measured by RT-PCR was significantly reduced in the pSilencer/miRNA-612 infected cells compared to the negative controls(P<0.05).We further measured the protein expression of P53 by Western blot,comparing to the control group,the P53 expression in pSilencer/miRNA-612 group was significantly reduced(P<0.05).The expression of miRNA-612 is inversely correlated with P53 mRNA and protein expression.Conclusion1.With genome miRNAs microarray and the methods of bioinformatics,we screened out one of the specific mRNAs—miRNA-612,which is associated with esophageal squamous cell carcinoma development and metastasis.2.miRNA-612 is positively correlated with esophageal squamous cell carcinoma development and metastasis.3.miRNA-612 promoted esophageal cancer's invasion and metastasis through down regulating of wt-P53 mRNA and protein expression.