| Objective:Analyze the clinical significance and bioinformatics of miR-508-3p in lung adenocarcinoma,and study the biological behaviors of miR-508-3p on proliferation,migration,invasion,cell cycle and apoptosis in lung adenocarcinoma cells.Predict the potential targets of miR-508-3p and related signal pathways,and conduct preliminary verification of its potential target genes to provide a certain theoretical basis for the diagnosis and treatment of lung cancer.Methods:1.Download the general information and clinicopathological information of lung adenocarcinoma patients in the TCGA database,including:gender,age,smoking history,pathological type,tumor stage,history of targeted therapy,gene expression,survival time,prognosis,etc.2.The independent sample T test was used to statistically analyze the expression of miR-508-3p in 446 cases of cancer and 44 cases of adjacent tissues in the TCGA database,and the paired sample T test was used to analyze the miR-508-3p expression in 38 cases of paired cancer and adjacent tissues.508-3p expression level was statistically analyzed.3.According to the entry criteria,the data of 421 cases in the TCGA database were screened out,and the miR-508-3p expression profile was divided into a high expression group and a low expression group with the median of 3.073 miR-508-3p expression as the cut-off value.Chi-square test was used to analyze the relationship between miR-508-3p gene expression and clinical pathological characteristics of lung adenocarcinoma patients,such as gender,age,tumor stage,smoking history,etc.;Kaplan-Meier method was used to analyze the effects of miR-508-3p on lung adenocarcinoma patients The impact of prognosis;Cox regression analysis of miR-508-3p expression,gender,age,tumor stage,smoking history and other factors risk.4.According to the miR-508-3p gene expression,the lung adenocarcinoma cases in the TCGA database are divided into quartiles,and the R language limma package is used for differential gene analysis.Use DAVID to perform GO and KEGG analysis of the above-mentioned differential genes,and screen out signal pathways that may have regulatory relationships.Use online analysis tools(http://starbase.sysu.edu.cn/)to predict miR-508-3p target genes,and perform negative correlation analysis with the above-mentioned significantly low-expressed differential genes to screen out possible existence Regulatory target genes.5.Use mimics to transiently transfect the A549 cell line,and use RT-qPCR to detect the transfection efficiency:inoculate the well-growing lung adenocarcinoma cell line A549 into a 6-well plate,and when the cell density reaches 50-60%,Use Lipo3000 to transfect mimics miR-508-3p into A549 cells;use RT-qPCR to verify the transfection efficiency 48 hours after transfection;and calculate the relative expression of miR-508-3p according to the 2-ΔΔct formula.6.Cell function experiments:use CCK8 to detect cell proliferation ability;scratch test to detect cell migration ability;Transwell to detect cell invasion ability;flow cytometry to detect cell cycle and apoptosis.7.Using the lung adenocarcinoma cell line 48h after transfection as a sample,use RT-qPCR to preliminarily verify the potential target genes;and calculate the relative expression of each potential target gene according to the 2-ΔΔct formula.Results:1.The expression of miR-508-3p in 446 cases of cancer tissues and 44 cases of adjacent tissues in the TCGA database has no statistically significant difference(P>0.05);there is no statistically significant difference in expression of miR-508-3p in 38 cases of paired cancers and adjacent tissues.Academic difference(P>0.05).2.In the lung adenocarcinoma samples in the TCGA database,the expression level of miR-508-3p is significantly different from age and tumor T stage(P<0.05);the relative risk of high expression level of miR-508-3p is 0.679 And there is a significant statistical difference(P<0.05);TNM staging of tumor is a risk factor for OS in patients with lung adenocarcinoma and there is a significant statistical difference(P<0.05);after correcting for factors such as T,N,and M staging,miR The relative risk of high expression level of miR-508-3p was 0.665 and there was a statistically significant difference(P<0.05).3.Through target gene prediction analysis,8 target genes that may have regulatory relationships in lung adenocarcinoma were obtained:TPX2,KIF14,PFN2,CENPA,GREM1,CEP55,CKAP2L and HOXC11.4.RT-qPCR results showed that 48 hours after transfection of the A549 cell line,the expression of miR-508-3p overexpression group increased significantly,which was 10.68 times that of the NC control group,and there was a significant statistical difference(P<0.05).5.The high expression level of miR-508-3p can inhibit the proliferation,migration,invasion and cycle of the A549 cell line,and there is a statistical difference(P<0.05);but the high expression level of miR-508-3p cannot promote A549 cells There was no statistical difference in the apoptosis of the strains(P>0.05).6.RT-qPCR results showed that the relative expression levels of CEP55,CKAP2L,GREM1,and KIF14 in the miR-508-3p overexpression group were 0.2585±0.03285,0.6196±0.06806,0.3434±0.13426,0.7947±in the NC group,respectively.0.09365 times,both lower than the NC group,and statistically different(P<0.05);according to the negative correlation and intersection analysis during the screening of target genes,it is suggested that the two genes CEP55 and GREM1 may be potentially regulated by miR-508-3p The target gene of the relationship.Conclusion:1.There is no difference in the expression level of miR-508-3p in lung cancer and adjacent tissues,but the high expression level of miR-508-3p is a protective factor for the survival and prognosis of patients with lung adenocarcinoma;2.Overexpression of miR-508-3p can inhibit the proliferation,invasion and migration of lung adenocarcinoma cell A549,block the cell cycle,but has no effect on cell apoptosis;3.The two genes CEP55 and GREM1 may be miR-508-3p potentially regulatory target genes. |
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