| BackgroundWorldwide,lung cancer has the highest incidence of human cancers and is also the leading cause of malignancy-related death.In recent years,the incidence of lung cancer in China has been increasing,ranking second only to breast cancer among female malignant tumors,and ranking first among male cancers.Lung cancer mainly includes two pathological types,namely small cell lung cancer and non-small cell lung cancer(NSCLC),among which NSCLC accounts for about 85%of the diagnosed cases of lung cancer,mainly including lung adenocarcinoma(LUAD),lung squamous cell carcinoma and lung large cell carcinoma.LUAD is the most common type of NSCLC.At present,the therapeutic methods for lung cancer mainly include minimally invasive surgical resection,adjuvant chemotherapy,targeted therapy and immunotherapy.With the development of sequencing technology and the deepening of research on lung cancer-related driver genes,molecular typing,such as ALK gene fusion,BRAF mutation,KRAS mutation,and EGFR mutation,has been applied to the detection of clinical specimens of lung cancer to guide targeted therapy for patients.However,about 50%of NSCLC patients developed drug resistance during targeted drug therapy.In addition,the current stage of lung cancer immunotherapy is mainly based on PD-1/PD-L1 immune checkpoint inhibitor drugs,including pembrolizumab and atezolizumab,and its clinical indications are mainly for locally advanced or metastatic NSCLC patients without EGFR/ALK mutations.These factors severely limit the further improvement of overall survival in NSCLC patients.Therefore,identifying new target related to the malignant progression of NSCLC and clarifying its potential mechanism are the key issues that need to be solved urgently in the clinical targeted therapy of NSCLC.Long noncoding RNA(IncRNA)is a class of noncoding RNA molecules,consisted of more than 200 nucleotides.Although lncRNAs do not encode proteins,they have been shown to be important in cellular processes such as regulation of gene expression,cell biology,developmental biology,and disease pathogenesis.In recent years,the role of lncRNA in many common human diseases,including cancer,neurodegeneration,and cardiovascular dysfunction,has attracted extensive attention and research.At present,a variety of lncRNAs have been reported to be closely related to NSCLC and play the role of tumor suppressor or oncogene in malignant progression.For example,the expression level of LINC00961,SPRY4-IT1,GAS6-AS1 or MEG3 in NSCLC tissues was significantly down-regulated compared with that in adjacent normal tissues,and restoring their expression obviously inhibited the proliferation,invasion and drug resistance of tumor cells.On the other hand,the expressions of AGAP2-AS1,LINC00673,ANRIL,HOTTIP,or SNHG1 was up-regulated in NSCLC tissues and positively correlated with tumor size,lymph node metastasis,TNM stage,and poor clinical prognosis.These findings indicate that lncRNAs play an important role in the occurrence and development of NSCLC.However,the research on the biological function and molecular mechanism of lncRNAs in NSCLC is still far from enough.Therefore,discovering new lncRNAs related to the malignant biological behavior of NSCLC and revealing its relevant mechanism will provide an important theoretical basis for further exploring the complex biological characteristics of lung cancer and promoting the development of Targeted therapy strategies.Objectives1.Revealing the LncRNA expression pattern of LUAD tumor tissue and adjacent normal lung tissue,and screening out CLDN10-AS1 with the highest fold change among upregulatd factors in LUAD tumor tissue;2.Verifying the expression level of CLDN10-AS1 in LUAD clinical samples and cell lines;3.To clarify the effect of CLDN10-AS1 expression level on the prognosis of LUAD patients;4.Elucidating the role of CLDN10-AS1 in LUAD cell proliferation,migration,invasion and anchorage-independent growth;5.To explore the molecular mechanism underlying CLDN10-AS1-regulated malignant phenotype of LUAD.Methods1.Seven pairs of LUAD clinical tissues were selected for RNA sequencing,and profiles of lncRNA differential expression were obtained through bioinformatics analysis.CLDN10-AS1 has the highest fold change among up-regulated lncRNAs in LUAD cancer tissues,which was selected for our research target.2.Analyze the sequencing data of LUAD in TCGA database to verify the expression level of CLDN10-AS1 in LUAD cancer tissue and adjacent normal lung tissue.3.The Kaplan-Meier Plotter database was used to analyze the effects of CLDN10-AS1 expression levels on Overall Survival(OS),First-progression Survival(FPS)and Post-progression Survival(PPS)of LUAD patients.4.One normal bronchial epithelial cell(BEAS-2B)and three LUAD cell lines(A549,H1975,H1650)were selected to extract RNA,and the expression level of CLDN10-AS1 was detected by RT-qPCR.5.CLDN10-AS1 knockdown plasmid(sh-CLDN10-AS1)and no-load control(sh-NC)were constructed.HEK293T cells were applied for the construction of lentiviruses containing sh-CLDN10-AS1 or sh-NC.A549 cell was infected with indicated lentiviruses,and stable CLDN10-AS1 knockdown cell lines(A549-sh-CLDN 10-AS1,A549-sh-NC)were established.6.CCK8,Transwell and soft agar assays were used to evaluate cell proliferation,migration,invasion and anchorage-independent growth in A549-sh-CLDN10-AS1,A549-sh-NC cells.7.Western blot was applied to detect the protein level of c-Myc and E-Cadherin in A549-sh-CLDN10-AS1 and A549-sh-NC cells.8.RNA-Seq was used to detect the transcription patterns of A549-sh-CLDN10-AS1,A549-sh-NC cells.Bioinformatics analysis was performed to obtain differentially expressed genes,while KEGG pathway enrichment analysis was applied to determine the downstream signaling pathway of CLDN10-AS 1.Results1.RNA-Seq was applied to test transcriptome of 7 pairs of LUAD tumor tissues.Following to the cateria(|Log2 Fold Change |>2,P value<0.05),275 differentially expressed lncRNAs in LUAD tumor tissue were increased compared to normal lung tissues,while 90 lncRNA significantly down-regulated in LUAD.Among all differentially expressed lncRNAs,CLDN10-AS1 had the highest fold change of upregulation in LUAD tumor tissues.2.The analysis of LUAD sequencing data in the TCGA database showed that CLDN 10-AS1 in tumor tissues of LUAD was significantly higher than that in adjacent normal lung tissues.3.Kaplan-Meier Plotter database was used to analyze the influence of CLDN10-AS1 expression level on the prognosis of LUAD patients,and the results showed that the OS,FPS and PPS of LUAD patients with high CLDN10-AS1 expression were significantly shortened.4.The expression level of CLDN10-AS1 in LUAD cell lines(A549,H1975,H1650)was significantly up-regulated compared with that of normal human bronchial epithelial cells(BEAS-2B).5.Knockdown of CLDN 10-AS 1 significantly inhibited proliferation,migration,invasion and anchored independent growth of A549 cell.6.Knocking down CLDN10-AS1 significantly decreased c-Myc protein level in A549 cells,but increased E-Cadherin protein level.7.RNA-Seq was applied to test transcriptome of 7 pairs of LUAD tumor tissues.Following to the criteria(| Log2 Fold Change |>1,P value<0.05),1037 expressed mRNAs in A549-sh-CLDN10-AS1 cells were increased compared to A549-sh-NC cells,while 535 mRNAs down-regulated.KEGG pathway enrichment analysis of the selected differentially expressed genes revealed that knocking out CLDN10-AS1 affects several pathways related to LUAD,such as the Hippo signaling pathway,AMPK signaling pathway,DNA replication,RNA degradation,RNA transport,and some classic metabolic pathways.Conclusion1.CLDN10-AS1 is abnormally up-regulated in LUAD and is closely associated with poor prognosis of patients.2.Knockdown of CLDN10-AS1 expression significantly inhibited proliferation,migration,invasion and anchored independent growth of LUAD cells.3.The molecular mechanism underlying CLDN10-AS1-mediated malignant phenotype of LUAD may be related to changes in classic intracellular pathways such as Hippo and AMPK. |
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