The Effect Of MiR-218 In Maligant Transformation Of Het-1A Cells Induced By HPV Combined With MNNG

Background and objective:The occurrence of esophageal cancer is the result of the combination of internal and external factors.Environmental risk factors related studies have shown that nitrosamine compounds exposure and human papillomavirus(HPV)infection are closely related to the occurrence of esophageal cancer.Among the studies on various carcinogenic mechanisms of esophageal cancer,microRNA(miRNA),as a way of epigenetic regulation,has become a research hotspot in recent years.Our previous study results in the high incidence area of esophageal cancer in Huai 'an area showed that there was a group of disorganized miRNA in the cancer tissues and adjacent tissues of patients with esophageal cancer,in which the expression of miR-218 was significantly down-regulated.In this study,the malignant transformation model of esophageal cells caused by HPV combined with MNNG constructed by the research group was applied,to observe the effect of miR-218 on the biological behavior of malignant cells,conduct transcriptome analysis on malignant cells,explore the target gene of miR-218 and its involved signal transduction pathway,and preliminarily analyze the possible role of miR-218 in the occurrence of esophageal cancer caused by HPV combined with MNNG.Methods and results: 1.Effect of miR-218 on the biological behavior of esophageal cancer cells and esophageal malignant cellsRT-qPCR was used to detect the expression levels of miR-218 in esophageal cancer cells(EC109/EC9706/KYSE510),immortalized esophageal epithelial cells(Het-1A)and four groups of cells in different algebras during the malignant transformationin.EC109 and malignant cells were taken as target cells,mimic of miR-218 was transfected and corresponding biological behavior was detected;CCK-8 assay and cell plate clonal formation assay were used to detect cell proliferation;Transwell migration and invasion assay was used to detect the migration and invasion ability of cells;Flow cytometry was used to detect cell cycle and apoptosis.In this study,the malignant transformation model(malignant cells)of esophageal cells caused by HPV combined with MNNG established by the research group was applied,and the cells were cultured for 35 generations.The malignant transformation was confirmed by the tumorigenesis experiment in nude mice.(1)Effect of miR-218 on the biological behavior of esophageal carcinoma cell EC1091)The expression of miR-218 in the three types of esophageal cancer cells(EC109/EC9706/KYSE510)was lower than that in the immortalized esophageal epithelial cells(Het-1A)(P<0.05).2)CCK-8 results showed that the proliferation activity of EC109 cells transfected with miR-218 mimic(miR-218 group)was lower than that of the negative control group(miR-NC group)(P<0.05).The results of cell plate cloning formation experiment showed that the cell cloning rate of miR-NC group and miR-218 group were(20.57±0.59)% and(9.5± 0.95)%(P<0.05),respectively.3)The result of transwell migration assay showed the number of cells penetrated by miR-NC group and miR-218 group were 156.67±7.64 and 77.67±6.81(P<0.05),while the result of transwell invasion assay showed the number of cells penetrated by miR-NC group and miR-218 group were 268.33±7.64 and 79.00±7.94(P<0.05).4)AnnexinV-PI double staining results showed that the apoptosis rate of miR-218 group(12.06±0.46)% was higher than that of miR-NC group(10.40±0.97)%(P<0.05).(2)Effect of miR-218 on the biological behavior of esophageal malignant cells1)The expression of miR-218 in the malignant transformation was as follows: before generation of 25,the expression level of miR-218 in the four groups of cells was basically unchanged compared with that in generation 0;after generation of 25,the expression level of miR-218 in the HPV+MNNG group and MNNG group increased rapidly;after generation of 40,the expression of miR-218 in the four groups showed a downward trend.2)CCK-8 results showed that 24 h after transfection with miR-218 mimic,the malignant cell activity of miR-NC group and miR-218 group were 1.45±0.02 and 1.42±0.14,the difference was not statistically significant(P>0.05).At 48 h after transfection,the cell activity of miR-218 group(1.99±0.18)was lower than that of miR-NC group(2.33±0.05)(P<0.05).3)The result of transwell migration assay showed that the number of cells penetrated by miR-NC group and miR-218 group were 85.33±5.03 and 54.33±5.13,respectively(P<0.05);the result of transwell invasion assay showed the number of cells penetrated by miR-NC group and miR-218 group were 207.67±7.77 and 151.67±7.64,respectively(P<0.05).4)Cell cycle analysis results showed that the proportion of S phase cells in the miR-218 group and miR-NC group were(34.15±1.09)% and(24.57±1.07)%(P<0.05),and that in the G2 phase were(27.01±1.51)% and(36.24±2.17)%(P<0.05),respectively.AnnexinV-PI double staining results showed that the apoptosis rates of miR-NC group and miR-218 were(15.23±0.41)% and(14.29±0.58)%,the difference was not statistically significant(P>0.05).2.The mechanism of miR-218 in malignant transformation of esophageal cellsIllumina sequencing technology was used for transcriptome sequencing of malignant cells,GO functional annotation and Pathway analysis of differentially expressed genes were conducted,and the interaction relationship between differentially expressed genes was analyzed using STRING database,Cytoscape software was used to realize visualization of the interaction network.miR-218 functional target genes were predicted by miRNAs target gene prediction software(TargetScan/Pictar/miRDB),and verified by RT-qPCR,Western Blot and dual-luciferase reporter gene experiments.(1)Transcriptome analysis of malignant cells1)The results of DEGs analysis showed that compared with the control group,there were 604 differentially expressed genes in the HPV+MNNG group,2462 genes in the MNNG group and 247 genes in the HPV group.2)GO analysis results show that,compared with control group,the differentially expressed genes in the HPV+MNNG group were mainly enriched in the defense response to the virus,cytochemotaxis,and so on,the differentially expressed genes in MNNG group were mainly enriched in tumor formation processes such as angiogenesis,while the differentially expressed genes in HPV group were enriched in inflammatory reaction processes,such as neutrophil chemotaxis,response to type I interferon,and cytokine activity.3)KEGG signaling pathway analysis showed that,compared with the control group,the differentially expressed genes in the HPV+MNNG group were mainly enriched in the TNF signaling pathway,Influenza A,NF-kappa B signaling pathway,and the differentially expressed genes in the MNNG group were mainly enriched in the pathways directly related to tumorigenesis,such as Pathways in cancer,and the differentially expressed genes in the HPV group were enriched in the IL-17 signaling pathway.4)The node size of the gene in the protein-protein interaction network diagram of the differentially expressed gene was positively correlated with its product activity.Compared with the control group,the nodes of EGR1,JUN,FOS,IL8 and TNF in the HPV+MNNG group were larger,the nodes of JUN,FOS,FYN and SMAD3 in the MNNG group were larger,and the nodes of EGR1 and JUN in the HPV+MNNG group were larger,suggesting that the above gene products had a greater ability to act.(2)Screening and validation of miR-218 target genes1)miRNA target gene prediction software(TargetScanHuman7.2/miRDB/Pictar)were used for online prediction of miR-218 target genes.Taking the intersection of the three predicted results,134 target genes were obtained.2)There were 604 differentially expressed genes in the HPV+MNNG group,which were again intersected with 134 target genes above,and finally 7 genes were obtained(ELMO1,NPAS2,ITM2 C,GAB2,CNTNAP2,MDGA1,SOCS3).3)GeneAnalytics,a biological information database,was used to carry out GO functional annotation and Pathway analysis for the above 7 genes.Four genes(ELMO1,NPAS2,GAB2 and SOCS3)were found to be involved in multiple links of tumor occurrence and development.After further review of relevant literature on tumor,the two genes most closely related to tumor,namely GAB2 and SOCS3,were finally determined.4)RT-qPCR and Western Blot showed that transfection with miR-218 mimic reduced the expression of GAB2 and SOCS3 in mRNA and protein levels.The results of dual-luciferase reporter gene assay showed that luciferase activity in the transfection group with GAB2-WT+miR-218 mimic decreased by 52.44% and 48.68%,respectively,compared with the transfection group with GAB2-WT+miR-NC and the transfection group with GAB2-MUT+miR-218 mimic,indicating that GAB2 was a direct target gene of miR-218 in malignant cells.(3)miR-218 regulates the SHP2/ERK and Akt/mTOR pathways by targeting GAB21)CCK-8 results showed that the proliferation activity of cells transfected with si-GAB2(si-GAB2 group)at 72 h and 96 h was lower than that of the negative control group(P<0.05).2)The result of transwell migration assay showed that the number of cells penetrating the microporous membrane in the si-NC group and the si-GAB2 group was 47.33±1.53 and 16.67±1.52,respectively(P<0.05),the result of transwell invasion assay showed the number of cells penetrating the microporous membrane in the si-NC group and the si-GAB2 group was 38.00±1.00 and 16.00±1.00,respectively(P<0.05).3)Western Blot was used to detect the expression levels of key proteins in the SHP2/ERK and Akt/mTOR pathways.The results showed that SHP2,ERK and p-ERK protein expression levels in the SHP2/ERK pathway decreased after the transfection of si-GAB2 by malignant cells,and Akt,mTOR and p-mTOR protein expression levels in the Akt/mTOR pathway decreased.3.Human HPV infection and miR-218 expression were analyzedQualitative detection of HPV infection in plasma was performed by ELISA.Total plasma RNA was extracted using the miRNeasy Serum /Plasma Kit,and the expression levels of miR-218 and GAB2 in plasma were detected by RT-qPCR.(1)The result of ELISA showed that 3 out of 50 cases were found to be HPV positive,with a positive rate of 6%.All 50 controls were negative for HPV(P>0.05).(2)The results of RT-qPCR showed that compared with the healthy control group,the expression level of miR-218 in the plasma of patients with esophageal cancer showed a downward trend(P>0.05),while the relatively high expression level of GAB2 was 2.86 times that of the control group(P<0.05).Conclusions:1.miR-218 was low expressed in esophageal cancer cells.Overexpression of miR-218 inhibits the proliferation,migration and invasion of esophageal carcinoma cells EC109,and promote apoptosis.2.Overexpression of miR-218 can inhibit the proliferation,migration and invasion of esophageal malignant cells,and block the cells in S phase.3.The differentially expressed genes in the HPV+MNNG group were mainly enriched in the TNF signaling pathway,Influenza A,NF-kappa B signaling pathway,the MNNG group was enriched in Pathways in cancer,Basal cell carcinoma and other signaling pathways directly related to tumorigenesis,and the HPV group was enriched in the IL-17 signaling pathway.4.GAB2 is one of the direct target genes of miR-218 in esophageal malignant cells.5.In the malignant transformation of esophageal epithelial cells caused by HPV combined with MNNG,miR-218 may be involved in the regulation of malignant transformation of cells by targeting the expression of GAB2 and thereby activating the downstream GAB2-related signal transduction pathways SHP2/ERK and Akt/mTOR.