The Up-regulation Of MiR-146a Inhibited Biological Behaviors Of ESCC Through Inhibition Of IRS2

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor of digestive tract in our country, which has the characteristics of high mortality and poor treatment effect. At present, the incidence of ESCC is only second to lung cancer, stomach cancer and liver cancer. ESCC etiology is complex, and mainly related to smoking, drinking, heated and hot food, esophageal reflux and genetic factors. Therefore, it is of great significance for the diagnosis and treatment of ESCC to screen the biological markers and to clarify the signaling pathways of ESCC. It is well known that cell signaling pathway requires cytokine stimulation, and then moves to the nucleus through the cell membrane or intracellular receptors to regulate the expression of genes involved in various physiological and pathological activities of cells. Cell proliferation plays an important role in the process of ESCC. When ESCC develops, the cell proliferation will become strong, accompanied by the disorder of all kinds of proliferation-related pathways. In vivo multiple signaling molecules have been demonstrated to be involved in the process of tumor cell proliferation. Therefore, the study of the key molecules in the cell signaling pathway has become a hot spot in the research of molecular mechanisms and treatment of ESCC.MicroRNA (miRNAs) is a class of non coding RNA with the length of 21-25 nucleotides, regulated by genome transcription and post transcriptional control of gene expression. miRNAs is involved in the inflammation, immune response, cell proliferation and apoptosis etc.. With the development of cDNA library, molecular cloning technology, RNA group and bioinformatics, thee role of miRNA in gene regulation is becoming more and more prominent; many hot miRNA have been recommended in clinical disease diagnosis, treatment and prognosis analysis. More and more evidence also shows that miRNAs are closely related to the occurrence and development of tumor, among which miR-146a is one of the hot spots in the research of many scientists. The study found that LPS and lipoprotein TOLL like receptor ligands, such as TLR2, TLR4, TLR5 ligands and inflammatory cytokines, such as TNF-a and IL-1βcan promote the expression of miR-146a in a NF-KB-dependent way, and then overexpression of miR-146a inhibits NF-κB pathway activity through inhibition of the expression of TNF receptor-associated factor 6 (TRAF6) linking TOLL like receptor (TLRs) and NF-κB, interleukin receptor associated kinase-1 (IRAK1), IFN regulatory regulator factor 5 (IRF5) protein, thereby reduced the expression of NF-κB pathway downstream inflammatory factors, including TNF-a, IL-1β and IL-6, by which miR-146a prevents excessive immune reaction.In addition, insulin receptor substrate (IRS) is an important protein in insulin signaling pathway in recent years. So far, studies have confirmed that IRS has four subtypes, of which, IRS1 and IRS2 are particularly concerned. IRS1 is widely distributed in all peripheral tissues, such as skeletal muscle, fat and liver, which are mainly expressed in skeletal muscle. At present, the amino acid sequence of human and mouse IRS1 has been proved to have high species and tissue conservation. The N ends of IRS 1 have two conserved domains, including PH (homology pleckstrin) and PTB (binding phosphotyrosine). PH domain mediates the interaction between phospholipids and proteins with acidic structure. PTB domain can identify tyrosine residues of phosphorylated NPXY, so that IRS1 can be coupled with Tyr950 or Tyr960 in the proximal membrane region of IGF-1R and insulin receptor IR. In conclusion, IRS1 plays an important role in the metabolism, cell proliferation and differentiation in the peripheral tissues of the body. In recent years, the role of IRS2 in tumor growth, proliferation and migration has also become more and more important. In this study, we detected the expression of miR-146a in esophageal squamous cell carcinoma tissue and adjacent normal tissue, and analyzed the relationship between miR-146a and clinical pathological parameters of esophageal squamous cell carcinoma. At the same time, the expression of miR-146a and IRS2 in esophageal squamous cell carcinoma cell line was also detected, and then the gene transfection was used to observe the influence of miR-146a on post transcriptional expression of IRS2 gene. The clear significance of miR-146a/IRS2 target gene networks in the diagnosis and treatment of esophageal squamous cell carcinoma will be elucidated. Methods and results:1. Real-time PCR detected the expression of miR-146a in esophageal squamous cell carcinoma and its relationship with clinical indicatorsIn the present study, the expression of miR-146a in 60 cases of esophageal squamous cell carcinoma was detected by PCR Real-time technique. In 60 cases of esophageal squamous cell carcinoma of the surrounding normal tissues, the expression level of miR-146a was 10.10±2.30; However, the expression of miR-146a in esophageal cancer tissue was 3.46±1.22, compared with normal esophageal tissue, the expression of miR-146a was significantly down regulated, and the difference is statistically significant. Through the analysis of miR-146a and multiple clinical pathological index, we found that miR-146a in human esophageal squamous cell carcinoma was significantly correlated with the degree of differentiation (P= 0.021), lymph node metastases (P= 0.004) and clinical stage (P= 0.039), while has no statistical correlation with the other age, gender and other clinical indicators. The results suggested that the expression level of miR-146a in esophageal squamous cell carcinoma was significantly lower than that in normal tissues, meanwihle miR-146a might be involved in the occurrence, development and metastasis of esophageal squamous cell carcinoma.2. Expression of miR-146a in human esophageal cancer cell lineThe total RNA in ESCC cells was extracted, and then we used the specific miR-146a reverse transcription primer to reverse miR-146a into cDNA, and then the real time PCR was used for detection of miR-146a expression in three kinds of different degree of differentiation of ESCC cell lines, and then the normal esophageal epithelium HEEC cells were used for control. The 2-△△CT method was used to calculate the expression level of miR-146a, with U6 as control. We found that miR-146a was highly expressed in HEEC cells, and the expression level was significantly higher than that of the three ESCC cell lines. In the highly differentiated EC 109 cells, the expression of miR-146a was significantly decreased, compared with HEEC (p<0.05). The expression of miR-146a was further reduced in the differentiated TE8 cells. In poorly differentiated TE2 cells, the expression of miR-146a was the lowest.3. The expression of IRS2 in human esophageal cancer cell line was regulated by m iR-146aIn this work, Real-time PCR and Western blot were used to detect the expression of mRNA and protein in EC 109, TE8 and IRS2 in normal HEEC cells and 3 esophageal cancer cells, respectively. The results showed that the mRNA and protein expression level of IRS2 in esophageal squamous cell carcinoma cell line were gradually increased with the degree of differentiation. By three prediction software Microinspector, miRanda and targetscan, we predicted the miR-146a has a regulatory role in the expression of IRS2, thus miR-146a may be involved in the post transcriptional regulation of IRS2 expression. Next, the dual luciferase reporter gene system was designed to verify the effect of miR-146a on IRS2 3’URT. MiR-146a was transfected by mimics or NC miRNA to regulate the expression level of miR-146a, and then to detect the level of IRS2 protein expression. The study showed that miR-146a could significantly down-regulate the expression level of IRS2 protein. The results suggest that miR-146a can bind the 3’URT region of IRS2 to inhibit the expression of IRS2.4. MiR-146a inhibited the proliferation, migration and invasion of esophageal squamous cell carcinomaThe ESCC cell lines EC 109 was selected to regulate the expression level of miR-146a by transfection of miR-146a mimics. CCK-8 method, scratch repair test and invasion test were used to detect the proliferation, migration and invasion of miR-146a in EC 109 cells. Compared with the control group, upregulation expression of endogenous miR-146a in EC109 and TE8 cells significantly decreased the proliferation, migration ability and invasion ability, while the control group has no significant change in the proliferation, migration and invasion ability. The results confirmed that down-regulation of miR-146a could significantly promote the metastasis and invasion of ESCC.5. The over-expression of IRS2 can reverse miR-146a inhibited ESCC biology behaviorBy wound healing assay, we found that in EC109 cells co-transfected with pc-IRS2 and IRS2 expression was upregulated, and the cell motility become significantly enhaced in the transwell migration assay. In EC109 cells co-transfected with pc-DNAs in the control group, the cell motilitycapacity became significantly decreased. Compared with treatment group, the difference was significant. The results indicated that miR-146a-inhibited migration ability was significantly improved after the over-expression of IRS2.By the invasion assay, our findings showed that in EC 109 cells cotransfected with pc-IRS2, IRS2 expression level increased, and the cells invading Matrigel can significantly increased. However, in EC 109 cells co-transfected with pc-DNA in control group, EC109 cells invading through Matrigel obviously decreased.the difference in two group was significant. The results indicated that over-expression of IRS2 could significantly relieve miR-146a-inhibited invasion capacity.Conclusion:For the first time, we detected the expression of miR-146a in ESCC, and found that miR-146a in ESCC cell lines, primary carcinoma and lymph metastasis tissue expression levels were significantly decreased, and the down-regulated expression of miR-146a was negatively correlated with ESCC metastasis, suggesting miR-146a plays an important role in ESCC development, progression and metastasis. In this work, the ectopic miR-146a expression inhibited the expression of IRS2 by inhibiting the post-transcription events of IRS2 gene, by which we revealed the role of miR-146a-IRS2 system in tumor differentiation, proliferation, invasion and migration and elucidated a new regulation mode. At the same time, our study provided a new biomarker for the diagnosis and treatment of ESCC.