Expression And Mechanism Of MiR-22-3p And Its Potential Target Gene ENO1 In Lung Adenocarcinoma

MicroRNAs(miRNAs)are a class of small,non-coding,evolutionarily conserved RNAs.Most of miRNAs binds with 3’ untranslated region of mRNAs to induce translational inhibition.miRNAs regulate almost 60% mRNAs of human.In addition,they play important roles in various cancers,such as carcinogenic or tumor suppressive factors to influence the occurrence,development,and even recurrence and metastasis of malignancies.A growing number of studies have shown that miRNAs are involved in the development of lung adenocarcinoma(LUAD).Therefore,dysregulated miRNAs may be one of the critical reasons for the occurrence and progression of LUAD.Our group keeps focusing on the molecular targeting research of malignant tumors.In recent years,the clinical significance and molecular mechanism of non-coding RNA in LUAD have been extensively discovered based on the combination of computational big data mining and experimental verification.In the early stage,a RT-qPCR text for a small sample of resected LUAD tissue specimens showed that miR-22-3p level is lower compared with non-cancerous samples.Studies have found that miR-22-3p differentially expressed in a variety of malignant tumors,showed lower expression in tumors such as retinoblastoma,esophageal squamous cell carcinoma,breast cancer,and colorectal cancer.But up-regulated in diffuse large B lymphoma.The studies of miR-22-3p in other diseases have offered some information to our research.It is suggested that the expression levels and functions of miR-22-3p may be different in various tumor types.miR-22-3p has also been studied in lung cancer.It has been reported that the expression of miR-22-3p in tissues of NSCLC(Non-Small Cell Lung Cancer)is lower than that of normal tissues.However,other studies found that miR-22-3p expression are higher in NSCLC patients compared with healthy controls,and studies have showed that miR-22-3p is highly expressed in the serum of NSCLC patients.What caused the above different expression ? Is not distinguishing the pathological type of NSCLC in detail ? Or is it due to the type of specimen? Or is it the error caused by the difference in the number of samples? The above information suggests that it is necessary to uncover the hypotypes in NSCLC separately.Blood and tissue specimens showed been studied separately.To answer the expression of miR-22-3p in LUAD and its clinicopathological significance.At the same time,different experimental methods can detect the expression of miRNA,such as miRNA sequencing,miRNA microarray,RT-qPCR and so on.Besides,the sensitivity and specificity of each method are different,and it is necessary to compare and balance these methods.The specific role of miRNAs is closely related to their target genes.Target genes of miR-22-3p in different diseases have been reported.miR-22-3p may inhibited proliferation,metastasis and invasion of liver cancer cells by down-regulating the target gene Sp1.Mi R-22-3p can promote neovascularization by targeting CD151 in gastric cancer.After overexpression of miR-22-3p,the content of VASP protein in gastric cancer cell line BGC-823 was detected,and it was found that miR-22-3p may have a targeting relationship with VASP.In LUAD,there are few target genes for miR-22-3p,and it has been reported that miR-22-3p may inhibit the proliferation of NSCLC cells by targeting AEG-1.Because one miRNA can target many different targets,the computational predictive tools that built with the sequence complementation and expression differences could provide a theoretical basis and convenient operation for studying the potential mechanism of miRNA.Meanwhile,the predicted target genes are all required to be verified by biological validation.Based on the above previous findings and the background,this project aims to validate(miRNA-sequencing,miRNA microarray,miRNA microarray,using a large sample of "experimental biology","computational biology" using a variety of detection methods.Independent clinical samples and cell samples RT-qPCR and multi-level data integration analysis)A variety of experimental assays are further performed(in vitro cell function experiments and clinical sample tests)to explore the expression of miR-22-3p in blood and tissues of LUAD patients,and its underlying molecular mechanisms.Based on the predictive computational biology results,ENO1 may be a potential target gene of miR-22-3p.The multi-level data analyzed integratedly and biological experimental assays were used to verify the targeting relationship between the miR-22-3p and ENO1.Provide a new perspective for clinical and basic research in LUAD.METHODS: 1.Expression level and biological function of miR-22-3p in LUAD.1.1 Expression level of miR-22-3p in LUAD.1)Tissue samples were used to detect the expression level by real-time RE-qPCR assay,and the clinicopathological significance of miR-22-3p were analyzed.A total of 20 LUAD tissues were achieved from our hospital.2)Verification of the expression level of miR-22-3p using miRNA sequencing data.3)Validation of expression levels of miR-22-3p using miRNA microarray data from GEO and Array Express.4)Keep to the concept of evidence-based medicine,calculate and analyze the different detection methods above and the multi-center miR-22-3p expression data.1.2 Biological function of miR-22-3p in LUAD.1)Construction of the LV-hsa-miR-22 lentiviral vector to obtain a stable A549 cell line highly expressed LV-hsa-miR-22.Or miR-22-3p mimic,miR-22-3p inhibitor was transfected into A549 cell line,which interfered with miR-22-3p expression.2)The effects of miR-22-3p over-expression on proliferation,migration and invasion and apoptosis of LUAD cells were detected by CCK8,MTS,scratch,Transwell migration and invasion,and Caspase-3/7 assay.2.Preliminary study on the molecular mechanism of miR-22-3p.2.1 miRwalk2.0 database predicts miR-22-3p target genes 1)Potential miR-22-3p target genes were performed GO enrichment and KEGG pathway analysis.2)Differentially expressed genes in LUAD were screened by TCGA and GTEx data.Collected the genes that are highly expressed in the predicted results.3)Analysis of protein-protein interaction network by potential miR-22-3p target genes.3.miR-22-3p may play a role in LUAD by targeting ENO1.3.1 Potential targeting relationship between miR-22-3p and ENO1.1)The complementary sequences between miR-22-3p and ENO1 are matched.2)Correlation between miR-22-3p and ENO1 expression in RNA-sequencing and miRNA-sequencing data.3)q RT-PCR was used to detect the expression of ENO1 in A549 cells after over-expression of miR-22-3p,and its correlation with miR-22-3p expression.3.2 Multiple assays verify the expression of ENO1 in LUAD.1)Real-time RT-qPCR was used to detect the mRNA expression level of ENO1 in 20 LUAD tissues and the adjacent noncancerous tissues.2)Verification of expression of ENO1 using RNA sequencing data.3)Verification of ENO1 expression using microarray data from GEO,Array Express and Oncomine databases.4)The different detection methods and multi-center ENO1 expression data were analyzed and analyzed by evidence-based medicine.5)Immunohistochemistry was utilized to discover the protein level of ENO1 in LUAD.RESULTS: 1.Expression level and biological function of miR-22-3p in LUAD.1.1 Expression level of miR-22-3p in LUAD.1)Real-time RT-qPCR was used to detect 20 LUAD tissues in this section.The expression of miR-22-3p was lower in patients than in adjacent control tissues(p = 0.1663).There were no significant differences in the clinical parameters of patients with LUAD.2)The expression level of miR-22-3p in miRNA sequencing data was higher than that in non-cancerous tissues,and was statistically significant(p < 0.0001).3)The expression level of miR-22-3p in LUAD was lower than that in normal tissues in 12 microarrays in GEO microarray data.The expression level of miR-22-3p in LUAD was higher than that in normal tissues in 4 chips.The expression of miR-22-3p in LUAD was lower in EAD than in normal tissues in E-MEXP-231,E-MTAB-5231 and E-TABM-15.The difference was insignificant(p = 0.2353,p = 0.6175,p = 0.7659).4)Keep to the concept of evidence-based medicine,and comprehensively calculate and analyze the different detection methods obtained above and the multi-center miR-22-3p expression data.The expression of miR-22-3p in peripheral blood and tissue samples of LUAD was lower than that of non-cancer group.1.2 Biological function of miR-22-3p in LUAD.1)Construction of the LV-hsa-miR-22-3p lentiviral vector to obtain a stable A549 cell line with high expression of LV-hsa-miR-22-3p(p = 0.014).2)The cells in the overexpressed miR-22-3p group were significantly lower than the control group at the 60 th hour by the CCK-8 cell proliferation kit(p=0.031).The cell proliferative ability of the experimental group of miR-22-3p mimics detected by MTS kit gradually decreased on the fifth and tenth days compared with the control group,and the difference was statistically significant(p=0.04,p<0.0001).).Cells overexpressing miR-22-3p showed significantly lower scratch area at 18 hours after scratching,with statistical difference(p < 0.05).After overexpression of miR-22-3p in LUAD cells,the migration ability of cells decreased(p<0.01).After overexpression of miR-22-3p in LUAD cells,the invasive ability of cells decreased significantly.The difference was statistically significant(p=0.033).No significant differences were observed in the Caspase-3/7 experiment.2.Preliminary study on the molecular mechanism of miR-22-3p.2.1 miRwalk2.0 website predicts miR-22-3p target gene 1)GO gene enrichment and KEGG pathway enrichment analysis,proteoglycans in cancer,endocytosi,Adherens junction,Erb B signaling pathway,m TOR Signal pathway(m TOR signaling pathway),insulin signaling pathway(Insulin signaling pathway)and other pathways are enriched.2)Combined with TCGA and GTEx database,the differentially expressed genes in LUAD were obtained.1024 genes were upregulated and 3230 genes were down-regulated.3)A total of 82 genes with high expression in the predicted results were screened.4)To construct a PPI protein interaction network for 82 potential target genes,and select ENO1 as a target gene for further study.3.miR-22-3p may play a role in LUAD by targeting ENO1.3.1 Potential targeting relationship between miR-22-3p and ENO1.1)It is predicted that there is a complementary sequence between miR-22-3p and ENO1.2)In RNA-sequencing and miRNA-sequencing data,miR-22-3p was negatively correlated with ENO1 expression(r=-0.045,p = 0.341).3)The expression of ENO1 in cells after over-expressed of miR-22-3p was lower than that of the negative control group(LVCON137/no load/Control),but the difference was not significant(p = 0.178).3.2 Multiple assays verify the expression of ENO1 in LUAD.1)Real time RT-qPCR results showed that the expression of ENO1 was significantly higher in LUAD than in normal control tissues(p = 0.021).2)RNA sequencing data The expression level of ENO1 in LUAD was higher than that in non-cancerous tissues,and was statistically significant(p < 0.0001).3)The expression level of ENO1 in the LUAD from the 12 datasets in the GEO database was significantly higher than that in the non-cancerous tissues.ENO1 expression in E-MEXP-231 and E-MTAB-5231 showed higher in LUAD,Low expressed in LUAD in E-MTAB-15.ENO1 in the studies of Stearman and Beer in the Oncomine database showed significantly high expression in LUAD and low expression in Bhattacharjee Lung.4)The different detection methods above,ENO1 expression data were analyzed by evidence-based medicine multi-center.The expression level of ENO1 was higher in LUAD than in normal group.5)Immunohistochemistry detected the higher expression of ENO1 protein in LUAD tissues.And ENO1 was located in the cytoplasm.CONCLUSION: 1.Lower expression of miR-22-3p or miR-22-3p deletion may play a role in the development of LUAD.2.The lower expression of miR-22-3p in LUAD may promote cell growth,migration and invasion.3.Higher expression of ENO1 may promote the occurrence and development of LUAD,but it needs to be verified in vitro and in vivo.4.miR-22-3p may exert a tumor suppressor effect in LUAD by targeting ENO1.5.Using the evidence-based concept,a variety of detection methods,multi-center sample detection results obtained miRNA and mRNA expression values are more objective and credible.