P53/hsa-miR-30a-5p/SOX4 Feedback Loop Mediates The Development Of Non-small Cell Lung Cancer

Objective: The malignancy is the primary reason caused death worldwide.Among all kinds of malignancies,the morbidity and mortality of lung cancer are both in the first place,which means that lung cancer poses a significant hazard to human's health and life.80%-85% of the lung cancer cases were non-small cell lung cancer(NSCLC)cases.Despite of the development of diagnosis and treatment,the five years survival rate remains very low.Currently,smoking is considered as the biggest environmental risk factor of lung cancer.However,incidence rate of lung cancer in the population of Chinese female was higher than the incidence rate in European females,although Chinese women have a lower smoking prevalence,which means that some other factors play a vital role in the tumorigenesis and tumor development except for smoking,such as genetic factors.As a complex disease,the mechanisms of tumorigenesis and development in lung caner need to be further investigated.Micro RNA is a sort of endogenous and non-coding RNA in a length of 20-24 nucleotides.According to principle of complementary base pairing,micro RNAs could target binding to the 3'-UTR to inhibit the translation of the m RNA or decrease its stabilization.In the tumorigenesis and tumor development,the ectopic expression mi RNAs are very common,which could lead to the changes of target genes expression and signal pathways activity.micro RNAs which might play critical roles in cancers could be screened out via comparisons of the expression of micro RNA between tumor tissue and adjacent normal tissue.Therefore,the comparison of the expression of micro RNAs between NSCLC tissue and adjacent normal tissue was conducted to screen out the important differentially expressed micro RNAs.The influence of the micro RNA on NSCLC and the regulation pathway of it in NSCLC were investigated.Methods: In this study,differentially expressed mi RNAs were screened by analyses of the mi RNA expression profiles in GEO database.The functions of the differentially expressed mi RNAs were explored and the regulation pathway of the differentially expressed mi RNAs were studied for the better understanding of the mechanisms.In the first section of this study,GSE56036 was obtained from GEO database and was analyzed by using R packages.The differentially expressed mi RNAs were identified as mi RNAs with fold change ?2 or fold change ?0.5.The volcano plot was drawn in this study.The expression of the chose mi RNA was verified in the data from TCGA and the expression profiles of tissue.Meanwhile,the association between the expression of hsa-mi R-30a-5p and the clinical characteristics was analyzed.The risk factors of NSCLC survival were also explored.In the second section of this study,the functions of hsa-mi R-30a-5p in NSCLC were further studied.The level of hsa-mi R-30a-5p was altered by transfecting cells with lentivirus of LV-hsa-mir-30 a and LV-hsa-mi R-30a-5p-inhibition.The proliferation ability of cells was determined with CCK-8 kit.The apoptosis was measured with flow cytometry after cells were dyed by 7-AAD and Annexin V-APC.The migration ability was determined with transwell assay.In the third section of this study,the regulation pathway of hsa-mi R-30a-5p was explored and investigated.The upstream regulator was predicted using PROMO and Jaspar databases and the regulation was verified with PCR and Ch IP.The downstream target genes of hsa-mi R-30a-5p were predicted using Target Scan and star Base and verified with luciferase reporter gene assay and Western Blotting.All the statistical analyses were two-tailed and performed by using SPSS software.P<0.05 was identified as the significant difference.Comparisons between two groups of continuous variables were conducted by using t test.Comparisons among three or more groups of continuous variables were conducted by using ANOVA.Comparisons of categorical variables were performed with chi-square test.The correlation between two continuous variables was determined with Pearson correlation.Results: 1.miRNA expression profiles of GSE56036 were analyzed using bioinformatic methods and 130 differentially expressed mi RNAs in NSCLC were identified,including 38 overexpressing mi RNAs and 92 downregulating mi RNAs.Given the fold change and expression abundance,hsa-mi R-30a-5p was chosen for the further study.The downregulation of hsa-mi R-30a-5p was verified in mi RNA expression profiles of TCGA and 60 pairs of tissues.Moreover,the expression of hsa-mi R-30a-5p was significantly different in different genders,T classifications,N classifications and clinical stages.In male NSCLC patients,the expression of hsa-mi R-30a-5p was associated with the survival.The higher expression of hsa-mi R-30a-5p meant the better prognosis.2.The expression levels of hsa-mi R-30a-5p in NSCLC cell lines were altered by transfected with lentivirus.And compared to the negative control,overexpression of hsa-mi R-30a-5p could inhibit the cellular proliferation ability,promote the apoptosis and inhibit the migration of NSCLC.Conversely,inhibition of hsa-mi R-30a-5p could promote the proliferation and migration but inhibit the apoptosis.3.According to the data from TCGA,the expression of hsa-mi R-30a-5p was decreased in individuals with p53 mutants.Bonded to the MIR30 A promoter and increased the expression of hsa-mi R-30a-5p by p53 was predicted with PROMO and Jaspar databases and verified with Ch IP and PCR.SOX4 was predicted as a potential target of hsa-mi R-30a-5p according to the Target Scan and star Base databases.The expression of hsa-mi R-30a-5p was negatively correlated with the expression of SOX4 according to the data from TCGA.It was verified with luciferase reporter gene assay and Western Blotting that hsa-mi R-30a-5p could directly binding to the 3'-UTR of SOX4 and inhibit the expression of SOX4.As the results of rescue assays showing,SOX4 could partly rescue the influence of hsa-mi R-30a-5p on the cellular proliferation,apoptosis and migration.Ultimately,the overexpression of SOX4 in cells could increase the level of p53,which demonstrated that SOX4 could induce the expression of p53.Conclusions: 1.hsa-mi R-30a-5p was significantly downregulated in NSCLC.The expression of hsa-mi R-30a-5p was associated with gender,T classification,N classification and clinical stage.And a higher level of hsa-mi R-30a-5p was associated with a longer survival in male patients.2.hsa-mi R-30a-5p functioned as a tumor suppressor in NSCLC and could inhibit the proliferation and migration but promote the apoptosis.3.hsa-mi R-30a-5p was directly targeted by p53 and it had effects on the cellular proliferation,apoptosis and migration via targeting SOX4.Meanwhile,the expression of p53 was induced by SOX4.Thus,p53/hsa-mi R-30a-5p/SOX4 feedback loop could mediate the proliferation,apoptosis and migration of NSCLC.