| Background and Objectives:According to the Cancer statistics,2018,lung cancer is the second most common cancers and the rank first in causes of cancer death,which is the great public health problem in the world.Lung squamous cell carcinoma(LUSC)is one of the main histological types of lung cancer,which occurs in men and is closely related to smoking.In clinical,the survival rate of patients is low because of low rate of early diagnosis and high rate of metastasis.Unfortunately,effective biomarkers to diagnose LUSC patients for reducing recurrence and improving survival rate are still lacking.Therefore,altogether,it is important to find new biomarkers for prompt diagnosis and effective treatment options of LUSC.Recently,numerous studies have reported that microRNA(miRNA)can be used as new tumor maker in the development and progression of LUSC.With the development of sequencing technology,increasing high-throughput sequencing databases have provided a good platform for cancer research.The Cancer Genome Atlas(TCGA)is the largest and most vital database of cancer gene information,and its data mining is conducive to the discovery of more reliable tumor biomarkers,it provides better clues for exploring the biological mechanisms of tumors development.In the present study,we obtained the LUSC-specific miRNAs and relate biomarker from TCGA database for further study,and then the results of TCGA data analysis were analyzed in a population sample.On this basis,we selected the miR-486-5p,which is down-regulation expression and related to diagnosis and prognosis in LUSC.Furthermore,we investigated the results in TCGA database,GEO database and qRT-PCR to analyze the expression of miR-486-5p in LUSC and its potential as a biomarker.Meanwhile,the related signal pathways of miR-486-5p were detected to elucidate the molecular,mechanism of miR-486-5p in the carcinogenesis and development of LUSC.Next,lentiviral transfection was used to construct a miR-486-5p overexpressing cell model and tumor-bearing nude mice,and the effects of miR-486-5p on the biological functions of proliferation,apoptosis,migration and invasion of lung squamous carcinoma cells were analyzed.Finally,we interested in determined the key protein and downstream molecules involved in important signaling pathways to explain the possible molecular mechanisms of miR-486-5p in development of LUSC.This study provides experimental and theoretical evidence for understanding the pathogenesis,early diagnosis,therapeutic targets and prognostic biomarkers of LUSC.Methods:1.The present study investigated LUSC-specific key microRNAs(miRNAs)from large-scale samples in TCGA.Most aberrantly expressed miRNAs were identified between tumor and normal samples from 332 LUSC patients.Subsequently,the correlation between key miRNAs and clinical features and prognosis of LUSC patients was analyzed by combining the clinical data and survival time of patients.Finally,the target genes of key miRNAs were predicted and their possible biological functions and functions were analyzed.2.According to the results of TCGA bioinformatics analysis,miR-486-5p was selected as the candidate gene for further studies.We obtained the expression and evaluated the diagnostic ability of miR-486-5p in LUSC by Meta analysis of TCGA database,GEO database and qRT-PCR results.Then,the target genes of miR-486-5p were predicted.Several softwares and multi-angle comprehensive analysis were used to analyze the core genes and possible pathways of the regulation of miR-486-5p.According to the Rich Factor scores and the analysis of the key target genes,important pathways were selected for further study.3.Firstly,the expression of miR-486-5p in different LUSC cell lines,benzopyrene(Bap)malignant transformation cell lines and the lung tissues of mice after long-term B(a)p exposure was verified.Moreover,miR-486-5p overexpression cell model was established by using lentiviral miR-486-5p overexpression stable transfection cell line,whose transfection efficiency was detected by qRT-PCR.We further used CCK8,flow cytometry and Transwell assays to detect the effects of miR--486-5p overexpression on the biological function of LUSC.Western Blot(WB)technique was used to explore the possible regulatory mechanism of miR-486-5p.At last,tumor-bearing nude mice was built to study the function of miR-486-5p in vivo.Results 1.Identification of microRNA associated with LUSCThe present study investigated LUSC-specific key microRNAs(miRNAs)from large-scale samples in TCGA.By analyzing 332 samples of LUSC patients,we successfully mapped the expression profiles of LUSC-related microRNAs and screened 42 miRNAs which may play a key role in the development of LUSC.Among 42 key miRNAs,two miRNAs(miR-629-3p and miR-511-5p)were aberrantly expressed in gender,four miRNAs(miR-30c-2-3p,miR-130b-5p,miR-629-3p and miR-130b-3p)were aberrantly expressed in lymphatic metastasis and one miRNA(miR-30a-3p)were aberrantly expressed in patient outcome assessment.Five of these miRNAs were significantly associated with the overall survival: three(miR-139-5p,miR-139-3p and miR-326)negatively(p<0.05)and two(miR-30d-3p and miR-486-5p)positively(p<0.05).The results of population sample analysis showed that the results of TCGA data analysis were highly reliable and accurate.2.Comprehensive analysis of miR-486-5p in diagnosis and prognosis of LUSCAccording to the screening results of LUSC miRNAs,we selected miR-486-5p,which were significantly down-regulated in LUSC and were associated with the diagnosis and prognosis of LUSC.Comprehensive analysis of TCGA database,GEO database and qRT-PCR test showed that the expression of miR-486-5p was significantly lower in LUSC(SMD = 2.25,95% CI:-3.47~-1.03,P = 0.0003),and the area under sROC curve(AUC)of was 0.9082.We obtained target genes of miR-486-5p using ten predicting software.Furthermore,functions of LUSC-specific intersection miRNAs were predicted.Target genes were mainly involved in melanoma,prostate cancer,FoxO and PI3K-AKT pathways.Among them,the number of enriched target genes in PI3K-AKT signaling pathway is the highest.By survival analysis and mutation analysis of core genes in PPI network,PTEN,TEK,PIK3R1 and PM1 B were the key target genes of miR-486-5p.PTEN,TEK and PIK3R1 were involved in PI3K-AKT signaling pathway.In conclusion,the PI3K-AKT pathway may be an important signal pathway for miR-486-5p to influence the LUSC development and progression.3.Study of the effect of miR-486-5p on biological behavior of LUSC and its mechanismMiR-486-5p expression levels detection results showed that miR-486-5p was significantly downregulated in NCI-H520,benzopyrene(Bap)malignant transformation cell lines and the lung tissues of mice after long-term B(a)p exposure,they were downregulated 7.87,6.36 and 2.16 times,respectively,whose results were 100% consistent with our previous study.CCK8 assay showed that the cell proliferation ability of miR-486-5p overexpression group was significantly lower than that of negative control group at 36 h and 72h(p<0.05),suggesting that overexpression of miR-486-5p could inhibit cell growth.The flow cytometry results showed the apoptosis of miR-486-5p overexpressing cells increased significantly(P<0.05).However,transwell assay suggested that the cell invasion ability of miR-486-5p overexpression group had no significant changes.The key proteins of PI3K-AKT pathway were detected.The results showed that the expressions of AKT,p-GSK-3?,PI3 K,p-4ebp,mTOR and p-mTOR were significantly decreased,while the expressions of ERK,GSK-3? and caspase-3 protein were significantly increased.The tumor bearing nude mice assay results showed that the tumor growth was on the decline with no significant difference(P>0.05),and the expressions of AKT,PI3 K,mTOR and p-mTOR were significantly decreased,while the expressions of caspase-3 protein were significantly increased.Conclusions1.Bioinformatics analysis based on TCGA database revealed that six miRNAs were closely related to clinical characteristics of LUSC patients(miR-629-3p,miR-511-5p,miR-30c-2-3p,miR-130b-5p,miR-30a-3p and miR-130b-3p)and five miRNAs were closely related to patient prognosis(miR-139-5p?miR-139-3p ?miR-326?miR-30d-3p and miR-486-5p).These 11 LUSC-specific miRNAs have potential as biomarkers of LUSC.The results of population sample analysis showed that the TCGA data analysis results were highly reliable and accurate.The key miRNAs screened from TCGA data have potential as LUSC biomarkers and can be used as targets for further study.2.The comprehensive analysis of TCGA database,GEO database and qRT-PCR test showed that the expression of miR-486-5p was abnormally low LUSC,which had high diagnostic value.Using predicting target genes,pathway analysis,Kaplan-Meier Plotter and PPI network analysis,PI3K-AKT pathway may be an important signal pathway for miR-486-5p to influence the development of LUSC.3.MiR-486-5p was significantly downregulated in NCI-H520,benzopyrene(Bap)malignant transformation cell line and the lung tissues of mice after long-term B(a)p exposure.MiR-486-5p overexpression may significantly affect biological function of LUSC by regulating PI3K-AKT signaling pathway.In conclusion,miR-486-5p is a LUSC anti-oncogene and plays an important regulatory role in LUSC. |
PDF Full Text Request