Silence Of ERBB2 Induced By MiR-193a-5p On Inhibiting The Invasion Of Esophageal Squamous Cell Carcinoma

1 Background and ObjectiveEsophageal cancer(EC)is one of the highest incidence of morbidity and mortality in gastrointestinal cancer,the incidence of esophageal cancer ranked to the9 th in the world,the total mortality rate accounted for the 6th.In developed countries,the incidence of esophageal cancer ranked to the 20 th,but the mortality ranked to the11 th.In developing countries,the incidence of esophageal cancer ranked to the 8th,the mortality ranked to the 5th.Esophageal cancer has risen from the seventh largest cancer in 1990 to the sixth largest cancer in 2013,aging and growing populations are the increased factors for esophageal cancer.In esophageal cancer,squamous cell carcinoma and adenocarcinoma as the main pathological type,of which squamous cell carcinoma accounted for more than 90%.Around 400,000 people die from esophageal cancer in the world each year,and China is one of the highest incidence areas of esophageal cancer in the world.The average annual death is more than150,000 because of the disease.The early symptoms of esophageal cancer is not obvious and lack of early diagnosis,the majority of patients in the first diagnosis has been in the later stage,the survival rate of resection after 5 years is 9.5-45%,tumorinvasion and metastasis is the leading cause of esophageal cancer deaths,invasion and metastasis are mainly dependent on the activation of proto-oncogene and tumor inactivation of tumor suppressor genes,as well as cell proliferation and apoptosis and other factors co-operation of the common results.If we can study the development and metastasis of esophageal cancer from the perspective of genetics and epigenetics,it will provide new targets and strategies for the diagnosis and treatment of esophageal cancer.Human epidermal growth factor receptor 2(ERBB2)is a member of the human epidermal growth factor receptor family,located at chromosome 17q12-21.32,encoding the relative molecular weight of 185,000 transmembrane receptor protein,is a transmembrane tyrosine kinase receptor protein with tyrosine kinase activity,over expression in breast cancer,prostate cancer and other tumor have a poor prognosis.It has been shown that ERBB2 amplification has been shown to play an important role in the development and progression of invasive breast cancer.ERBB2 has become an important biomarker and therapeutic target in 30% breast cancer patients.ERBB2 plays an important role in cell growth,differentiation and migration,and its overexpression promotes tumor growth,metastasis and tumor angiogenesis.ERBB2 overexpression has been reported in esophageal squamous cell carcinoma,and ERBB2 is considered a potential therapeutic target for esophageal cancer.However,the molecular mechanism of ERBB2 regulates the occurrence of esophageal squamous cell carcinoma remain unclear.Micro RNA(mi RNA)is a small non-coding RNA molecule found in recent years,containing about 22 nucleotides,widely distributed in eukaryotes.Although most mi RNAs are located within the cell,a number of mi RNA have been found in the extracellular environment,commonly known as circulating mi RNA or extracellular mi RNA.The most common regulation of target genes is mi RNAs binding with protein,leads to complete or incomplete pairing with the 3’untranslated region(3’-UTR)of messenger RNA,which can be degraded or repressed by the recognition of a specific gene.Mi RNA play an important role in the process of cell proliferation,differentiation and apoptosis by regulating the expression of target genes.The strict regulation plays an important role in maintaining the normalfunctioning of cells.Researchers have found that mi RNA disorders are associated with many diseases,such as tumors.In this study,the expression of ERBB2 in esophageal squamous cell carcinoma and its relationship with clinicopathological features were investigated by immunohistochemical method.The molecular mechanism of mi R-193a-5p inhibition of ERBB2 translation was revealed the new regulation of ERBB2.The expression of mi R-193a-5p in esophageal cancer tissues and patients’ serum was measured and the value of mi R-193a-5p was evaluated as the prognostic marker of esophageal carcinoma.2 Methods and Results2.1 Expression and significance of ERBB2 in esophageal squamous cell carcinoma by immunohistochemistryWe used immunohistochemical method to investigate the expression of ERBB2 in esophageal squamous cell carcinoma and its clinical pathological features.The expression of ERBB2 in esophageal squamous cell carcinoma(35/68,51.5%)was higher than that in normal esophageal tissue 8.8%(6/68)(χ~2 =33.03 P<0.01).The expression of ERBB2 was correlated with tumor lymph node metastasis and tumor staging(P<0.01),and there was no significant difference in age and sex(P>0.05).2.2 mi R-193a-5p inhibits ERBB2 expression at translationThe mRNA and protein expression of ERBB2 in normal esophageal mucosal cells Het-1A and esophageal cancer cells KYSE150,KYSE410,ECA-109,TE-2 and TE-13 were detected by QRT-PCR and Western blot.The expression of ERBB2 m RNA and protein expression in esophageal cancer cell lines was different from that of normal cells.We conclude that ERBB2 in esophageal cancer has a transcriptional regulatory mechanism to inhibit its expression.The ERBB2 3’URT region was analyzed by micro RNA prediction software,through the combination of different prediction algorithm and ERBB2 3’URT area three main prediction software(Targetscan,mi Randa and Micro Inspector),it was found that mi R-193a-5p binding the ERBB2 3’URT located at 41 nt.Up-regulated the expression of mi R-193a-5p by transfection of mi R-193a-5p mimics,after detected the expression of ERBB2 protein and m RNA,found that mi R-193a-5p can significantly down-regulate ERBB2 protein level and have no effect on the expression of RNA.The results suggested that mi R-193a-5p can inhibit the translation of ERBB2 and down regulate the expression by binding with ERBB2 3’URT region.2.3 miR-193a-5p in tissue and serum expression and clinical indicatorsWe detected the expression of mi R-193a-5p in 35 cases by QRT-PCR,it was compared by paired T test in cancer tissue and adjacent normal tissue.The results showed that mi R-193a-5p expression level was significantly lower than that of adjacent normal tissue.According to the clinical data,we divided the specimens into lymph node metastasis group and non metastasis group.The Wilcoxon rank test showed that the expression level of mi R-193a-5p in lymph node metastasis group was significantly lower than that in non metastasis group(P<0.05).The results showed that mi R-193a-5p may be involved in the process of inhibiting esophageal cancer metastasis.We divided the expression of ERBB2 into low and high expression group,we found that the expression level of mi R-193a-5p in ERBB2 low expression group was significantly higher than that in ERBB2 high expression group by Wilcoxon rank sum test(P<0.05),suggesting that the expression of ERBB2 was negatively correlated to expression of mi R-193a-5p in esophageal squamous cell carcinoma.The survival of mi R-193a-5p high expression group and low expression group was compared by the log rank test,showed that the prognosis of mi R-193a-5p high expression group was significantly higher than that of mi R-193a-5p low expression group(P<0.05).We extracted micro RNA in 35 cases of frozen esophageal squamous cell carcinoma tissue and serum,the expression of mi R-193a-5p was detected by QRT-PCR and analysed the relationship between tissue and serum by Pearson correlation,the results showed that the expression level of mi R-193a-5p in serum was positively correlated with the expression in the tissue.3 Conclusion3.1 The increased expression of ERBB2 indicates the poor prognosis of esophageal cancer.ERBB2 may be involved in the mechanism of invasion and metastasis of tumor.ERBB2 is one of the risk factors of poor prognosis in esophageal cancer.3.2 In esophageal squamous cell carcinoma,the expression of ERBB2 after the transcriptional level was inhibited,we analyzed the possible regulation of mi RNA,mi R-193a-5p binding to ERBB2 3’URT region and inhibited ERBB2 translation and silencing its expression,revealing the new regulatory mechanism of ERBB2.3.3 The expression of ERBB2 in esophageal squamous cell carcinoma was negatively correlated with the expression of mi R-193a-5p,and the expression of mi R-193a-5p was negatively correlated with the metastasis and poor prognosis.The expression of mi R-193a-5p in serum can reflect the expression in the tissue,suggesting that the expression of mi R-193a-5p in serum can be used as a non-invasive marker for the prognosis of esophageal cancer.