| Background and objective:Lung cancer(LC)is the leading cause of cancer deaths worldwide.The number of Americans who die of lung cancer each year is almost the sum of those who die from prostate cancer,breast cancer and colon cancer.Due to the extensive development of basic research in recent years,the early diagnosis,minimally invasive surgery and targeted therapy of lung cancer have been greatly improved,but the number of deaths from lung cancer is still high.This is mainly due to the fact that there are no obvious clinical symptoms in early lung cancer.Most patients(approximately 75%)are at an advanced stage when initially diagnosed,and even have distant metastases.Studies have shown that deletion,mutation and amplification of related genes can significantly promote the occurrence and development of lung cancer.Therefore,exploring the key molecular targets that affect the regulation of lung cancer will further improve the understanding of the pathogenesis of lung cancer.At the same time,by exploring related molecules,powerful and effective diagnostic markers can be found for clinical preventive treatment.Although many studies have found that multiple factors play a role in the pathogenesis of lung cancer,including protein-coding genes and non-protein-coding genes,the occurrence and evolution of lung cancer is an intricate regulatory process,and there are still many unknown factors that need to be further studied.Long noncoding RNA(LncRNA)is a class of non-protein transcripts longer than 200 nucleotides,involved in a variety of major biological functions,such as proliferation,apoptosis,metastasis,and cell metabolism.LncRNA can be transcribed by RNA polymeraseⅡ and undergo splicing of multiple exons.According to reports,there are still a large number of abnormally expressed lncRNAs in lung cancer tissues.These lncRNAs can promote or inhibit the occurrence and development of lung cancer,and are expected to become biomarkers for early diagnosis of lung cancer.LncRNA ARAP1-AS1 is a new type of lncRNA located on chromosome 11.It is currently found to be highly expressed in cervical cancer,bladder cancer,breast cancer,gastric cancer,colorectal cancer and lung cancer and affects their occurrence and development.Therefore,this study comprehensively analyzed the expression of LncRNA ARAP1-AS 1 in lung cancer and its biological effects for the first time,and initially studied the molecular mechanism of its regulatory effect on lung cancer.This study consists of three parts:the first part is the abnormal expression of LncRNA ARAP1-AS 1 in lung cancer tissues and its clinical and pathological correlation;the second part is the preliminary study of the effect of LncRNA ARAP1-AS1 on the proliferation and invasion of lung adenocarcinoma and the molecular regulation mechanism;the third part is the molecular mechanism of LncRNA ARAP1-AS1 regulating lung adenocarcinoma proliferation,invasion and migration by targeting miR-149-3p/S100A4.Part One:Abnormal expression of LncRNA ARAP1-AS1 in lung cancer tissues and its clinical and pathological correlationObjective:To explore the abnormal expression of LncRNA ARAP1-AS1 in lung cancer tissues and its correlation with the clinical pathology and prognosis of patients.Methods:1.The non-coding RNA sequencing method was used to analyze the expression changes of LncRNA in lung cancer tissues,and the KEGG system was used to analyze the signal pathway of up-regulated/down-regulated LncRNA enrichment.2.Analyze the lung cancer-related transcriptome data in the TCGA database,and further investigate the expression of LncRNA ARAP1-AS 1 in lung cancer tissues.3.QRT-PCR was used to detect the expression of LncRNA ARAP1-AS1 in cancer tissues and adjacent tissues and its correlation with the clinicopathology of lung cancer patients.4.The Kaplan-Meier survival curve was used to analyze the correlation between the expression of LncRNA ARAP1-AS 1 and the prognosis of lung cancer patients.Results:1.Compared with adjacent tissues,there were 2890 lncRNA transcription levels in tumor tissues.The transcription level of 1189 lncRNAs increased significantly(P<0.05),such as:CLDN10,FEZF1-AS1,DPP10-AS1,etc.Among them,1701 lncRNAs transcription and expression levels decreased significantly(P<0.05),such as:ARAP1-AS1,MYO16-AS1,MED4-AS1,etc.It is suggested that these differentially expressed lncRNAs may be relevant to the occurrence,development,early diagnosis,prognosis and treatment of lung cancer.Among up-regulated lncRNAs,the most enriched pathways are neural activation ligand-receptor interaction,PI3K-Akt signaling pathway and MAPK signaling pathway;in down-regulated lncRNAs,the most enriched pathways are PI3K-Akt signaling pathway,cell cycle Interact with cytokine and cytokine receptor.Among them,ARAP1-AS1(ENSG00000256007.1,ARAP1 antisense RNA 1)is high in lung cancer tissues,and its average expression is about 7.0 times that of normal tissues adjacent to cancer.2.LncRNA ARAP1-AS1 has a certain tissue specificity and plays an important regulatory role in the occurrence and development of lung adenocarcinoma.Its expression can be used as a biomarker to determine the tumor stage of patient.3.Compared with adjacent tissues,the expression level of LncRNA ARAP1-AS1 in cancer tissues was significantly higher(P<0.001),and LncRNA ARAP1-AS1 was significantly related to TNM staging and lymph node-related metastasis.4.The prognosis of patients with high LncRNA ARAP1-AS1 expression is poor,and its expression is significantly correlated with the prognosis of lung cancer patients.Conclusion:LncRNA ARAP1-AS1 has important value in the development and prognosis of lung cancer.Part Two:The effect of LncRNA ARAP1-AS1 on the proliferation and invasion of lung adenocarcinoma and the preliminary study of its molecular regulatory mechanismObjective:To preliminarily explore the effect of LncRNA ARAP1-AS1 on the proliferation and invasion of lung adenocarcinoma and its molecular regulation mechanism.Methods:1.QRT-PCR was used to detect the expression level of LncRNA ARAP1-AS 1 in lung cancer cell lines(PC-9 cells,A549 cells,H226 cells,H1975 cells,H1299 cells)and normal human lung epithelial cells(BEAS-2B).2.QRT-PCR was used to detect the knockdown efficiency in lung adenocarcinoma cells after transfection of shRNA-1,shRNA-2 and shRNA-NC.3.CCK-8 was used to detect the effect of knockdown of LncRNA ARAP1-AS1 on the proliferation of lung cancer H1975 cells and H1299 cells.4.The clone formation experiment was used to detect the effect of the clone formation ability of lung cancer H1975 cells and H1299 cells after knocking down the LncRNA ARAP1-AS1.5.Use flow cytometry to detect the effects of knocking down LncRNA ARAP1-AS 1 on the cycle of lung cancer H1975 cells and H1299 cells.6.Detect the expression of cycle-related proteins by Western blot.7.The Transwell experiment was used to detect the effect of knockdown of LncRNA ARAP1-AS1 on the migration ability of lung cancer H1975 cells and H1299 cells.8.Transfect H1975 cells and H1299 cells with lentiviral interference vectors(shRNA1 and shRNA-2)and shRNA-Control(shRNA-NC)respectively.After 48 hours of transfection,collect total cell protein and detect by Western blot.Results:1.The expression level of LncRNA ARAP1-AS1 in various lung adenocarcinoma cell lines is quite different.The expression level is highest in H1975 cells and H1299 cells,followed by A549 cells,H266 cells and PC-9 cells,the expression of BEAS-2B is the least in normal human lung epithelial cells.2.Compared with shRNA-NC,transfection of shRNA-1 and shRNA-2 that knock down LncRNA ARAP1-AS 1 can significantly reduce the expression level of LncRNA ARAP1AS1 in H1975 cells and H1299 cells,and its knockdown efficiency in H1975 cells were respectively reached 56.7%and 72.4%(P<0.01);in H1299 cells were 51.4%and 67.5%(P<0.01).3.Compared with shRNA-group cells,shRNA-1 and shRNA-2 had no significant effect on the proliferation of H1975 cells and H1299 cells at 24h.With the extension of the culture time,they began to significantly inhibit cell proliferation at 48h(P<0.05),this inhibition is particularly significant at 72h(P<0.01).4.Compared with the shRNA-NC group,transfection of LncRNA ARAP1-AS1 shRNA-1 and shRNA-2 can significantly inhibit the formation of H1975 cells and H1299 cells clones(P<0.01).5.Compared with the shRNA-NC group,transfection of LncRNA ARAP1-AS1 shRNA-1 and shRNA-2 can significantly promote the arrest of H1975 cells and H1299 cells in the G0/G1 phase,and the corresponding G2/M phase ratio is significantly reduced,while the ratio of S phase cells decreased,but the statistical difference was not significant.6.In H1975 cells,compared with the blank control group,knocking down LncRNA ARAP1-AS1 can significantly inhibit the expression of the cell cycle-related protein Cyclin D1,and has no significant effect on the expression of other proteins such as CDK4,the expression of CDK6 has a slight decrease,but there is no significant statistical difference.7.Transwell migration experiments showed that the number of shRNA-NC cells migrating were 257±44(H1975 cells)and 263±39(H1299 cells).After transfection of LncRNA ARAP1-AS 1 shRNA-1 and shRNA-29 the numbers of H1975 cells migration were:165±35 and 134±39,the numbers of H1299 cells migration were:187±58 and 123±15.Tranwell invasion experiments showed that the number of shRNA-NC cells invasion were 247±52(H1975 cells)and 223±25(H1299 cells).After transfection of LncRNA ARAP1AS1 shRNA-1 and shRNA-2,the numbers of H1299 cells invasion were:115±25 and 94±19,the numbers of H1299 cells invasion were:127±28 and 90±23.The above results showed that knockdown of LncRNA ARAP1-AS 1 could significantly inhibit cell migration and invasion.8.In H1975 cells,compared with the blank control(shRNA-NC),knocking down LncRNA ARAP1-AS1 can significantly affect the expression of EMT-related proteins,that is,knocking down LncRNA ARAP1-AS 1 can significantly promote the expression of cell surface adhesion protein E-cadherin,inhibits the expression of N-cadherin and Vimentin,but has no significant effect on the expression of related transcription factors such as Slug and Twist.In H1299 cells,after using two interfering shRNAs targeting the LncRNA ARAP1-AS1 sequence,the expression of EMT-related proteins changed significantly,compared with the blank control(shRNA-NC),knocking down the two shRNAs of LncRNA ARAP1-AS1 can significantly affect the expression of EMT-related proteins.That is,knocking down LncRNA ARAP1-AS1 can significantly promote the expression of cell surface adhesion protein E-cadherin,inhibit the expression of N-cadherin and Vimentin,but has no significant effect on the expression of related transcription factors such as Slug and Twist.Conclusion:Knockdown of LncRNA ARAP1-AS1 can significantly inhibit lung adenocarcinoma cell proliferation,cloning,invasion and migration ability formation,and cause cell cycle arrest in G0/G1 phase.Part three:LncRNAARAP1-AS1 regulates the molecular mechanism of lung adenocarcinoma proliferation,invasion and migration by targeting miR-1493p/S100A4Objective:To study the molecular mechanism of LncRNA ARAP1-AS1 regulating lung adenocarcinoma proliferation,invasion and migration.Methods:1.Separate the nucleus and cytoplasm,extract total RNA for fluorescence quantitative PCR to detect the sub-localization of LncRNA ARAP1-AS1 in the cell.The cytoplasmic internal control GAPDH was used as a cytoplasmic positive control,and U6 was used as a nucleus positive control.2.Through bioinformatics analysis and detection of miRNA that LncRNA ARAP1-AS 1 may bind;by dual luciferase reporter gene to detect the binding activity of LncRNA ARAP1AS1 and miR-149-3p.3.Fluorescent quantitative PCR was used to detect the expression of knockdown miR149-3p in lung cancer tissues;fluorescent quantitative PCR was used to detect the expression of miR-149-3p in the above 70 lung adenocarcinoma tissues and adjacent tissues.4.Use bioinformatics software and open source databases to predict the downstream target genes of miR-149-3p;by Western blot to detect the relationship between S100A4 expression and miR-149-3p.5.The inhibitor and NC inhibitor of miR-149-3p were designed and co-transfected with shRNA-2 targeting LncRNAARAP1-AS1 into H1795 cells,and the expression of S1004A was detected by fluorescence quantitative PCR.6.The clone formation experiment detects the ability of LncRNA ARAP 1-AS 1 to regulate lung adenocarcinoma cell proliferation through miR-149-3p/S100A4 signaling pathway.7.Perform in vivo experiments,use a stable knockdown lentiviral vector(shRNA-2)to interfere with H1975 cells.After adding screening drugs,a stable knockdown cell line is obtained and inoculated into male BALB/c nu/nu thymus-deficient nude mice subcutaneously.8.Extract total mRNA from nude mouse tissues and detect it by fluorescence quantitative PCR;detect the expression of S100A4 in tumor-bearing tissues of shRNA-NC group and shRNA-2 group by immunohistochemistry.Results:1.LncRNA ARAP1-AS1 is mainly located in the cytoplasm,while the cytoplasmic internal reference GAPDH is mainly located in the cytoplasm,and U6 is located in the nucleus.2.Transfection of miR-149-3p mimics in 293T cells can significantly promote the expression of miR-149-3p.Compared with miR-NC and mutant(Mut)LncRNA ARAP1AS1 group,there was no significant change in the transcriptional activity of the luciferase reporter gene after mutant(Mut)LncRNA ARAP1-AS1 and miR-149-3p mimics were cotransfected into 293T cells.Compared with miR-NC and wild-type(Wt)LncRNA ARAP1AS1 group,the transcriptional activity of luciferase reporter gene was significantly reduced after wild-type(Wt)LncRNA ARAP1-AS1 and miR-149-3p mimics were co-transfected into 293T cells.3.Compared with the shRNA-NC group,knocking down LncRNA ARAP1-AS1 can significantly increase the expression of miR-149-3p;compared with adjacent tissues,miR149-3p has a low expression in tumor tissues;LncRNA ARAP1-AS1 and miR-149-3p have a negative correlation in lung adenocarcinoma tissues,and the P value is 0.0079,which has a significant statistical difference.4.S100A4 is a downstream target gene for miR-149-3p to function.5.In lung adenocarcinoma cells,LncRNA ARAP1-AS1 regulates the expression of S1004A by targeting miR-149-3p.6.Inhibiting the expression of miR-149-3p can significantly block the growth inhibitory effect caused by knocking down S100A4,S100A4 is an important downstream participant of miR-149-3p.7.Knockdown of LncRNA ARAP1-AS 1 can significantly inhibit the proliferation of lung adenocarcinoma carcinoma in nude mice.8.LncRNA ARAP1-AS1 may inhibit the proliferation of lung adenocarcinoma by negatively regulating the expression of miR-149-3p;knocking down LncRNA ARAP1-AS1 may inhibit the expression of S100A4 and thereby inhibit the proliferation of lung adenocarcinoma.Conclusion:The molecular mechanism of LncRNA ARAP1-AS1 regulating lung adenocarcinoma proliferation,invasion and migration is to target miR-149-3p/S100A4.This research is mainly divided into the following three parts.Firstly,qRT-PCR was used to detect the expression of LncRNA ARAP1-AS1 in cancer tissues and adjacent tissues and its correlation with the clinicopathology of lung cancer patients,and the correlation between the expression of LncRNA ARAP1-AS1 and the prognosis of lung cancer patients was analyzed by Kaplan-Meier survival curve.The results found that LncRNA ARAP1-AS1 has important value in the development and prognosis of lung cancer.Secondly,qRT-PCR detection method,CCK-8 detection method,clone formation experiment,Western blot detection method and Transwell experiment were used to preliminarily explore the effect of LncRNA ARAP1-AS1 on the proliferation and invasion of lung adenocarcinoma and its molecular regulation mechanism.The results found that knocking down LncRNA ARAP1AS1 can significantly inhibit the proliferation,cloning,invasion and migration of lung adenocarcinoma cells,and cause cell cycle arrest in G0/G1 phase.Finally,the molecular mechanism of LncRNA ARAP1-AS1 regulating lung adenocarcinoma proliferation,invasion and migration was studied through bioinformatics methods and in vivo experiments in nude mice.The results showed that the molecular mechanism of LncRNA ARAP1-AS1 regulating lung adenocarcinoma proliferation,invasion and migration is targeting miR-1493p/S100A4.In summary,LncRNA ARAP1-AS1 is expected to become a biomarker of lung adenocarcinoma and even a new gene target for clinical treatment.This study provides a theoretical basis,new research ideas and medical reference for the clinical diagnosis and treatment of lung adenocarcinoma. |
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