| Lung cancer is the highest incidence and mortality of malignant tumors worldwide,has become a global public health problem.According to the histomorphologic characteristics of lung cancer,lung cancer is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Although the methods of diagnosis and treatment of lung cancer have been constantly improved in recent years,the 5-year survival rate of lung cancer patients is still less than 20%,and the 5-year survival rate of metastatic lung cancer is only 5%,due to the biological characteristics of lung cancer with high invasion and metastasis,which poses a serious threat to human life and health.Therefore,it has important clinical significance to deeply explore the molecular mechanism of the development of NSCLC for the early diagnosis,prognosis and treatment of the disease.The protein-coding genes account for only about 2% of the entire human genome,and the rest are non-coding RNA(nc RNA),including long non-coding RNA(LncRNAs),Circ RNA(Circ RNA),and Micro RNAs(mi RNAs).LncRNA is a type of RNA molecule with transcription length over 200 nt,located in the nucleus or cytoplasm,but without protein-coding capacity.The interaction between LncRNA and DNA,RNA and protein shows a strong ability to regulate gene expression at the levels of epigenetic,transcriptional and post-transcriptional,and is participated in a variety of important biological processes in cells.Recent studies have shown that many LncRNAs are abnormally expressed in a variety of malignancies,which are involved in the development,progression,metastasis and drug resistance of tumors,and are potential molecular markers and therapeutic targets for tumor diagnosis.At present,studies on LncRNA in tumors are still in the initial stage,so it is necessary to continue to search for LncRNA differentially expressed in NSCLC,and explore its function and specific molecular mechanism,so as to improve the pathogenesis of NSCLC.Up to now,the screening of lung cancer is mainly by imaging,but the imaging has a certain hysteresis.40% of NSCLC patients are already in the local progressive stage or advanced stage when they are diagnosed,which greatly increases the difficulty of treatment and affects the therapeutic effect of patients.The lack of early screening methods for lung cancer has become a bottleneck limiting the treatment of lung cancer patients.Therefore,it is urgent to explore efficient,sensitive,accurate and economical diagnostic methods.LncRNA has stability,specificity and accessibility,and its abnormal expression in body fluids can be used as a biomarker for the diagnosis,efficacy evaluation and prognosis of NSCLC.In addition,some abnormal LncRNAs expressed in tumor tissues are closely related to the occurrence,metastasis and drug resistance of NSCLC,which are potential therapeutic targets for NSCLC.Therefore,LncRNAs with potential for NSCLC diagnosis and treatment have become hot spots in tumor diagnosis and treatment at home and abroad.At present,studies on LncRNA in NSCLC are still in the initial stage,so it is necessary to continue to search for LncRNA differentially expressed in NSCLC and explore its functions and specific molecular mechanisms,so as to improve the pathogenesis of NSCLC.Purpose:The purpose of this study is to screen LncRNA that may be involved in the development of NSCLC through high-throughput sequencing,and to explore their roles and possible mechanisms in tumor cell proliferation and migration through in vitro and in vivo experiments,so as to provide theoretical support for the development of new targets for NSCLC.Methods:(1)High-throughput sequencing was applied to analyze the LncRNA expression spectrum from 3 cases of NSCLC tissues and corresponding adjacent tissues,Then,differentially expressed LncRNA and m RNA were analyzed,and gene ontology(GO)analysis and pathway analysis were performed on differentially expressed genes.Through bioinformatics analysis,LncRNA-m RNA co-expression network was constructed to predict the target genes of differentially expressed LncRNA,and the predicted target genes were enriched by GO and KEGG pathway.Based on the existing literature,the target LncRNA was selected.(2)Combined with the literature and research results,LncRNA HOXC-AS2 was selected as the target LncRNA in this study.The expression of LncRNA HOXC-AS2 in 20 cases of NSCLC tissues and 10 cases of corresponding adjacent tissues was verified by real-time quantitative PCR assay.(3)HOXC-AS2 si RNA,pc DNA3.1 HOXC-AS2 and negative control plasmids were transfected into A549 and HCC827 cells to establish HOXC-AS2 silencing and overexpression cell lines.Transfection efficiency was verified by real-time quantitative PCR assay.The effects of HOXC-AS2 on cell biological behaviors such as proliferation,migration and apoptosis of NSCLC cells were verified by CCK-8,cloning formation,flow cytometry,and Transwell assays.Western blot was used to detect cell epithelial mesenchymal transformation(EMT)and cell migration markers E-cadherin,β-catenin,α-SMA,MMP-1,MMP-2.(4)Real-time quantitative PCR was applied to determine the expression of HOXC13 in 20 cases of NSCLC tissues and 10 cases of corresponding adjacent tissues.RNA immunoprecipitation(RIP)was used to verify the binding relationship between HOXC-AS2 and HOXC13.The effects of HOXC-AS2 on the expression of target gene HOXC13 were detected by real-time quantitative PCR and/or western-blot,as well as the effects of HOXC13 on the expression of HOXC-AS2.(5)The effects of HOXC13 on the regulation of HOXC-AS2 on the proliferation,migration,apoptosis and EMT of NSCLC cells were determined by CCK-8,cloning formation,flow cytometry,Transwell,and Western blot after transfected HOXC13-si RNA and pc DNA3.1 HOXC-AS2 plasmids in A549 and HCC827 cells.(6)The effect of HOXC-AS2 on tumor growth in nude mice was detected by subcutaneous tumor formation experiment,and the expression of ki-67 protein in tumor tissues was detected by immunohistochemical staining.Results:(1)Through high-throughput sequencing technology,the LncRNA with both |log2(FC)|≥1 and statistical significance(P<0.05)were found to be differentially expressed LncRNA.A total of 730 differentially expressed LncRNA were found,among which 449 LncRNA were significantly up-regulated and 281 LncRNA were significantly down-regulated.Through bioinformatics analysis of the target genes of differentially expressed LncRNA,a total of 5,110 target genes were found,of which 1269 had significant changes.GO and KEGG enrichment analysis was carried out on the above 1269 target genes,and found the target genes involved in biological functions including signal transduction,immune system process and the transcriptional regulation of DNA template.The main enrichment pathways of LncRNA target genes include Arginine biosynthesis and mineral absorption.Through the construction of the LncRNA-target gene co-expression network for the 10 most significantly different LncRNAs,it was found that the abnormal expression of LncRNA might be related to the immune function of the body.According to the previous study,only HOXC-AS2 and IGHV1-69 among the 9 LncRNAs with the most significant differences have been reported to be involved in the development of malignant tumors.However,HOXC-AS2 is more significantly different from IGHV1-69,and the expression and function of HOXC-AS2 in NSCLC have not been reported.Therefore,HOXC-AS2 was taken as the target LncRNA in this study.(2)The present study revealed that compare to paracancer tissues,the expression of HOXC-AS2 in NSCLC tissues was significantly higher.(3)Real-time quantitative PCR results showed that high transfection efficiency of HOXC-AS2 si RNA and pc DNA3.1 HOXC-AS2 plasmids in A549 and HCC827 cells.The results of CCK-8 and cloning formation showed that overexpression of HOXC-AS2 can promote the proliferation,and knockdown of HOXC-AS2 can inhibit the proliferation of A549 and HCC827 cells.The results of Transwell assay showed that overexpression of HOXC-AS2 can promote the migration,and HOXC-AS2 silencing can inhibit the migration of A549 and HCC827 cells.Flow cytometry showed that overexpression of HOXC-AS2 inhibited the apoptosis,while knockdown of HOXC-AS2 induced apoptosis of A549 and HCC827 cells.Western blot results showed that overexpression of HOXC-AS2 could promote the expression of β-catenin,α-SMA,MMP-1 and MMP-2,and down-regulate the expression of E-cadherin protein,while knockdown of HOXC-AS2 had the opposite effect.(4)Real-time quantitative PCR results confirmed that HOXC13 was highly expressed in NSCLC tissues compared with the corresponding paracancer tissues.RNA immunoprecipitation results showed that HOXC-AS2 could specifically bind to HOXC13.Overexpression of HOXC-AS2 can up-regulate the expression of HOXC13,while knockdown of HOXC-AS2 can significantly down-regulate the relative expression of HOXC13 in A549 and HCC827 cells.In addition,HOXC13 can positively regulate the expression of HOXC-AS2,indicated by the silencing of HOXC13 can significantly inhibit the relative expression of HOXC-AS2 in A549 and HCC827 cells.(5)The results of CCK-8 and cloning formation showed that the transfection of pc DNA3.1 HOXC-AS2 could promote the proliferation,the transfection of HOXC13 si RNA could significantly inhibit the proliferation,and the HOXC13 silencing impaired the promotion of HOXC-AS2 overexpression on the proliferation ability of NSCLC cells.The results of Transwell assay showed that overexpression of HOXC-AS2 can promote the migration,and HOXC13 silencing impaired the promotion of HOXC-AS2 overexpression on the migration ability of NSCLC cells.Flow cytometry results showed that overexpression of HOXC-AS2 could inhibit NSCLC cell apoptosis,and HOXC13 gene silencing significantly inhibited the inhibition of HOXC-AS2 overexpression on NSCLC cell apoptosis.Western blot results showed that overexpression of HOXC-AS2 could promote the expression of β-catenin,α-SMA,MMP-1 and MMP-2,and down-regulate the expression of E-cadherin protein,while gene silencing of HOXC13 could significantly inhibit the regulatory effect of overexpression of HOXC-AS2 on the above proteins in NSCLC cells.(6)HCC827 cells with stable transfection of sh RNA-HOXC-AS2 and sh RNA-NC were injected subcutaneously into the nude mice.After 30 days of normal feeding,it was found that compare to the sh RNA-NC group,the tumor volume of the sh RNA-HOXC-AS2 group was significantly reduced.Immunohistochemical staining results showed that the Ki67 staining optical density value of tumors in the sh RNA-HOXC-AS2 group was significantly lower than that in the sh RNA-NC group,suggesting that HOXC-AS2 gene silencing could inhibit the growth of non-small cell lung cancer in vivo.Conclusion:(1)LncRNA HOXC-AS2 and HOXC13 are highly expressed in non-small cell lung cancer tissues.HOXC13 is the target gene of LncRNA HOXC-AS2,and they can positively regulate each other.(2)Overexpression of HOXC-AS2 can promote the proliferation,migration,and EMT of NSCLC cells and inhibit apoptosis,while down-regulation of HOXC-AS2 had the opposite effect,suggesting that HOXC-AS2 may play a role mainly by promoting the malignant growth of tumors.(3)Down-regulation of LncRNA HOXC-AS2 inhibited the growth of NSCLC in nude mice.(4)LncRNA HOXC-AS2 may play a biological role in promoting NSCLC by positively regulating the expression of adjacent gene HOXC13. |
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