| Objective:Lung cancer is the leading cause of cancer-related death worldwide,and is associated with high mortality.In China,incidence and mortality rates of lung cancer ranked the first among all the cancers for 2020 year.The two main subtypes of lung cancer are small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC is the most common type of lung cancer and accounts for about 85% of all lung cancers.Lung adenocarcinoma is the most common histological subtype,accounting for about 50%of NSCLC.At present,although great progress has been made in the lung adenocarcinoma aspects of pathogenesis,clinical diagnosis,treatment and prognosis,the overall prognosis of patients with lung adenocarcinoma is still poor,due to its strong invasiveness and susceptibility to metastasis.Therefore,exploring the pathogenesis mechanism and novel specific diagnostic marker may directly affect clinicians,early diagnosis of lung adenocarcinoma,the selection of operative plans,the research and development of targeted new drugs and the construction and evaluation of prognosis indexes.Long non-coding RNAs(lnc RNAs)are RNA transcripts more than 200 nucleotides in length,which have no open reading frames,lack the ability to encode proteins but only code for shorter polypeptides.Meanwhile,they can participate in gene regulation through a variety of signaling pathways,and are associated with malignant phenotype of many tumor.Many studies have shown that lnc RNAs were dysregulated in a variety of human tumors,and their abnormal expression would lead to dysregulated expression of downstream effector genes.Lnc RNAs play an important regulatory role in the tumor genesis,development,proliferation,cycle,apoptosis,cell invasion and metastasis of a variety of tumors,including lung adenocarcinoma,which are even closely related to patient’s prognosis,chemotherapy response and drug resistance of targeted drugs.Therefore,exploring the detailed molecular mechanisms and regulating signaling pathway of lnc RNAs in malignant tumors,which will provide novel targets and new approaches.Meanwhile,it will provide a reliable theoretical basis for research and development of targeted drugs and the prognosis estimation of patients of LUAD.Materials and Methods:Firstly,in-deep bioinformatics analysis results showed that the molecular location,the secondary structure of LINC00115 through the bio-molecular databases.Seventy-two cases fresh clinical lung adenocarcinoma tissues and para-carcinoma normal tissue samples were collected.We simultaneously verified the expression level of LINC00115 in lung adenocarcinoma tissues and para-carcinoma normal tissues by using RT-PCR and Real-time Quantitative PCR(RT-q PCR).Subsequently,we recorded the clinical pathological characteristics of the patients,and revealed its correlation to expression level.Finally,we detected the clinical value of LINC00115 as diagnostic marker in LUAD by using Receiver Operator Characteristic(ROC)curve.Then,we screened human lung adenocarcinoma cell lines from CCLE,and detected expression level of LINC00115 in human lung adenocarcinoma cell lines(A549,H1299,H1975 and H1437)and normal bronchial mucosa epithelial cell lines(16HBE).The H1437 cells with relatively high expression level and the A549 cell lines with relatively low expression level were subjected to subsequent cell function experiments.Small interfering RNA(si RNA)and overexpression plasmids were transient transfected into cell lines H1437 and A549,and transfection efficiency were detected by RT-PCR and RT-q PCR.Cell proliferation ability was measured by CCK-8 and clone formation assay was used to explore cell proliferation;cell apoptosis and cell cycle were measured by flow cytometry;Cell invasion and migration was measured by scratches healing and transwell assay.To achieve the repeatability and stability of the transient transfection experimental results,lentivirus of knockdown and overexpression were stabilized transfection lung adenocarcinoma cell lines H1437 and A549.The results of stable transfection experiment were consistent with those of transient transfection.Eventually,the tumor-bearing model by subcutaneous injection and the lung metastasis model by caudal vein injection were established in nude mice.The effects of LINC00115 on the proliferation,invasion and metastasis of LUAD cells in vivo were detected by HE and IHC test.Finally,the subcellular localization of LINC00115 was detected by FISH assay,which prepare for the following research of molecular mechanism.Then,RNA pull-down assay + mass spectrometry(MS)was used to screen protein that could directly interact with LINC00115.The targeted protein KHSRP and its directly binding sites to LINC00115 was used to analyze and predict via cat RAPID and ENCORI database.Silver stain show that LINC00115 directly bind to KHSRP proteins.Western blot assay was used to verify the result of silver stain assay.KEGG signaling pathway and GO function enrichment analysis of down-regulated genes showed LINC00115 may participated in many biological processes and diseases,such as MAPK signaling pathway,AKT signaling pathway and disorders of transcription factor(TF).PPI network was constructed through STRING database,and LINC00115 were further verify that regulating of invasion and metastasis via IL6/JAK1/STAT1 signaling pathway in LUAD.Statistical analysis was performed using Graph Pad Prism8.0(Graph Pad Software Inc).The statistical methods used in this paper were: t-test,chi-square test,ordinary one-way ANOVA,etc.Each group of experiments was repeated three times.P<0.05 was considered to indicate a statistically significant difference.Results:1.LINC00115 was located in the chromosome region1p36.33,with two transcripts,one exon and uniquely conserved secondary structure.LINC00115 was significantly higher in lung adenocarcinoma tissues.LINC00115 was closely related to the patient’s TNM staging and lymph node metastasis(P<0.05),but had no significant correlation with age,gender,tumor size,degree of differentiation and smoking status of patients(P>0.05).Area under the curve(AUC)of the receiver-operating characteristics(ROC)cure was 0.7571,indicating that it has better diagnostic value for lung adenocarcinoma vs normal.2.The results of CCK-8 and clone formation assay showed that LINC00115 has no effect on the proliferation of lung adenocarcinoma cells.Flow cytometry assay results showed that LINC00115 inhibits cell apoptosis in lung adenocarcinoma.Flow cytometry assay results showed that LINC00115 has no effect on cell cycle in lung adenocarcinoma.Cell scratch healing,and transwell assay results showed that LINC00115 promotes cell invasion and metastasis of lung adenocarcinoma.3.The results of subcutaneous tumor-bearing in nude mice showed that LINC00115 has no effect on cell proliferation of lung adenocarcinoma in nude mice in vivo.The results of lung metastatic model by caudal venous injection in nude mice showed that LINC00115 has a significant effect on cell invasion and migration of lung adenocarcinoma in nude mice in vivo.4.The results of lnc ATLAS database and FISH assay showed that LINC00115 has located in the cytoplasm.The results of RNA Pull-down and MS assay showed that LINC00115 might directly bind to 1439 proteins.KHSRP was highly likely to bind to LINC00115 and there were two possible binding sites via cat RAPID and ENCORI database.The results of silver staining assay showed that LINC00115 may bind to KHSRP protein.Western blot assay further confirmed that LINC00115 can directly bind to KHSRP.5.The PPI network of KHSRP was constructed via STRING database.KEGG signaling pathway and GO function enrichment analysis were conducted by DAVID database.Results showed that LINC00115 promotes cell invasion and metastasis of lung adenocarcinoma through regulating IL6/JAK1/STAT1 signaling pathway.Conclusion:LINC00115 is an oncogene in lung adenocarcinoma,and has a significant effect on cell apoptosis,cell invasion and migration in vivo and in vitro.LINC00115 can directly bind to KHSRP protein and promote the cell invasion and migration of lung adenocarcinoma through regulating IL6/JAK1/STAT1 signaling pathway. |
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