Different Expression And Clinical Significances Of MiRNA In Esophageal Squamous Cell Carcinoma And Precancerosis

Background:90%of esophageal cancer cases are squamous cell carcinomas(ESCC)in northern and central China.Esophageal squamous cell intraepithelial neoplasia is considered the precancerosis of ESCC and substantially elevates the risk of tumorigenesis of ESCC.Without specially medical therapeutic measures,approximately 65%of high grade intraepithelial neoplasia(HGIN)cases progress to ESCC within 3.5 years,and 74%progress to ESCC within 13.5 years.Early ESCC and precancerous lesions with the endoscopic minimally invasive treatment has become a trend in recent years.It’s reported that early ESCC with endoscopic minimally invasive treatment has a five-year survival rate of 85%-95%.So early diagnosis and treatment is crucial to improve the effect of treatment.MicroRNAs(miRNAs)are small non-coding RNAs,which are 21-25 nucleotides in length.The biological function of miRNAs is achieved by base pairing to complementary sequences in the 3’-untranslated region of their target messenger RNAs and involves in the occurrence and progress of cancer.We have known that tissues and serum samples of ESCC patients had distinctive expression of miRNAs,which could be used for diagnosis,prognosis evaluation and therapy of the diease.However,the miRNA expression profile of esophageal squamous cell intraepithelial neoplasia tissue and serum samples has not been reported until now.Objectives:To detect and analyse the deregulated expressed miRNAs in esophageal squamous cell intraepithelial neoplasia tissue and serum samples and exosomal miRNA in ESCC surum samples and to explore the feasibility of early diagnosis molecular markers for ESCC.Methods:The study examined the expression levels of the selected miRNAs in 30 pairs of esophageal squamous cell neoplasia lesion tissues and corresponding adjacent normal tissues.The patients were definitely diagnosed by the endoscopy and biopsy.The eight miRNAs that were reported to be deregulated in ESCC,miR-25,let-7a,miR-100,miR-133a,miR-223,miR-375,miR-483-5p and miR-1322,were examined in lesion tissues and corresponding adjacent normal tissues used qRT-PCR.Differently expressed miRNAs were examined by in situ hybridization(MISH)and analyzed the relationship with the disease.Furthermore,the deregulated miRNAs were measured in serum and serum exosome samples of patients and matched normal healthy controlsby the qRT-PCR method.We also predict the potential target genes of the possible miRNAs that would function in the ESCC.A total of 47 serum samples of ESCC patient and 46 serum samples with the information of age and gender matched healthy normal controls were included in the study.qRT-PCR method was used to detect the expression level of exosomal miR-25,miR-223,miR-100 and miR-483-5p.Results:According to qRT-PCR results of tissue samples,statistically significantly amplified expression of miR-25 was found in lesion tissues compared to adjacent normal tissues(0.290±0.448 vs 0.120±0.184,P=0.031)with the percentage of 66.7%(20/30)samples of esophageal squamous cell intraepithelial neoplasia lesions.There was no significant difference between the HGIN and LGIN groups(0.037±0.051 vs 0.024±0.041,P=0.459).The other seven miRNAs presented no significant difference.MISH demonstrated that miR-25 expression level was higher in the plaques of esophageal squamous cell intraepithelial neoplasia tissues than that in normal control tissues.Computational analyses revealed that several of the predicted target genes participate in the regulation of the cell signal pathway,gene transcription,cell-cycle arrest,apoptosis,tumorigenicity,cell metabolism and so on.There were no siginificantly differences between the age-and sex-matched healthy controls and the dignosed patients in the expression levels of both serum and serum exosomal miR-25.According to qRT-PCR results of the serum exosome samples of ESCC patients,miR-25 and miR-223 were upregulated compared with normal controls(0.234 ± 0.180 vs 0.115±0.079,P=0.0003;10.920± 13.113 vs 5.460± 5.386,P=0.034,respectively)with a significant difference but not the miR-100 or miR-483-5p.The areas under the ROC curve of serum exosomal miR-25 and miR-223 were AUC=0.716,95%CI:0.612～0.820;AUC=0.627,95%CI:0.513～0.741,respectively with specific value of 0.739 and 0.783,respectively.Conclusion:miR-25 was upregulated in esophageal squamous cell intraepithelial neoplasia tissues and serum exosome samples of ESCC patients.Exosomal miR-223 was also upregulated in ESCC patients.These indicate that miR-25 and miR-223 exhibited feasibility as early diagnosis molecular markers for ESCC.