Function And Mechanism Of Collagen COL10A1 In Regulating Invasion And Metastasis Of Lung Adenocarcinoma

BackgroundAccording to the latest data from the National Cancer Center,lung cancer is the highest incidence in China,and non-small cell lung cancer is the main pathological type of lung cancer,accounting for about 85% of the lung cancer population.In recent years,the incidence of lung cancer has presented new characteristic,among which the incidence and mortality of lung adenocarcinoma continue to rise.Recurrence and metastasis are the main factors affecting the treatment and prognosis of patients with lung adenocarcinoma.Therefore,it is important to actively screen lung metastasis-related genes and elucidate their regulatory mechanisms.MethodsThe TCGA lung adenocarcinoma database,GEO lung adenocarcinoma dataset(GSE19804,60 pairs of lung adenocarcinoma samples),and 5 pairs of female non-smoking women with lung adenocarcinoma data were collected earlier in the study and performed on the transcriptomes of the above dataset.Combined analysis showed that four genes were highly expressed in lung adenocarcinoma tumor tissues,and two were differentially expressed in lung adenocarcinoma tissues.The key gene of lung adenocarcinoma-COL10A1 was further obtained by weighted gene co-expression analysis(WGCNA).Immunohistochemical of lung adenocarcinoma tissue microarray was performed using 92,and the correlation between the expression level and clinicopathological characteristic was verified on the protein expression level;40 pairs of lung adenocarcinoma specimens were further selected in the lung cancer biological sample bank of our hospital and applied qRT-The PCR technique verifies the relationship between its expression level and clinicopathological characteristic at the m RNA level.The biological function of COL10A1 was verified by in vitro and in vivo functional assays.The possible binding proteins of COL10A1 were explored by literature review and protein co-immunoprecipitation(Co-IP);bioinformatics analysis and Westen blot were used to detect key pathway molecules that may be regulated by COL10A1;cross rescue experiments further confirmed COL10A1 Regulated signaling pathways.ResultsThrough qRT-PCR and immunohistochemistry(IHC)experiments revealed that COL10A1 was highly expressed in tumor tissues in patients with lung adenocarcinoma and was closely related to lymph node metastasis.Prognostic analysis showed that the expression of COL10A1 was closely related to the prognosis of patients.Correlation,the higher the expression,the worse the prognosis,and high expression of COL10A1 is an independent prognostic factor for lung adenocarcinoma.The expression of COL10A1 in common cell lines of lung adenocarcinoma was detected by qRT-PCR and Western blot.The high expression cell line H1299 and the low expression cell line A549 were screened out.Small interfering RNAs(si RNA)that specifically interfered with COL10A1 were design and constructed a plasmid overexpressing COL10A1.In vitro functional experiments Transwell,Matrigel,Wound healing and RTCA experiments showed that knockdown COL10A1 can significantly inhibit the invasion and metastasis capacity of lung adenocarcinoma cell lines,and overexpression is the opposite;we found that knocking down COL10A1 by constructing tail vein metastasis model in nude mice.Afterwards,the metastatic ability of lung adenocarcinoma cells was significantly inhibited.In the mechanism study,through review of the literature,we found that COL10A1 may interact biologically with integrin ?2?1 and DDR2;it was confirmed by Co-IP that COL10A1 only interacts with DDR2 in lung adenocarcinoma cells.To search for possible downstream signaling pathways that COL10A1 may regulate,WGCNA was used in the TCGA database to search for genes related to COL10A1 expression,and KEGG PATYWAY enrichment analysis revealed that the Focal Adhesion signaling pathway was significantly enriched and ranked first in the signaling pathway.The enriched gene FAK is closely related to the metastasis of tumors;Western blot showed that silencing COL10A1 in the H1299 cell line,the expression of phosphorylated FAK protein was significantly reduced,and in the A549 cell line,after the expression of COL10A1 protein,phosphorylation of FAK Protein expression increased significantly.Subsequent cross-rejection experiments after knockdown of COL10A1 in H1299 cells,transfection of FAK partially rescued the invasion and migration ability of H1299 cells;after overexpression of COL10A1 in A549 cells,phosphorylation of FAK inhibitor partially inhibited the invasion and migration ability of A549 cells.Taken together,these results suggest that the combination of C0L10A1 and DDR2 receptors activates FAK signaling pathway to regulate invasion and metastasis of lung adenocarcinoma.ConclusionThis study fully applied the public database and combined with the pre-expression microarray of the research group,and applied bioinformatics screening to obtain the gene COL10A1 closely related to lymph node metastasis and prognosis of patients.Immunohistochemistry and qRT-PCR showed that COL10A1 was highly expressed in patients with lung adenocarcinoma,positively correlated with lymph node metastasis,and was an independent prognostic factor for lung adenocarcinoma.In vitro and in vivo experiments confirmed that COL10A1 could promote the invasion and metastasis of lung adenocarcinoma cells.Mechanism studies confirmed that C0L10A1 interacts with DDR2 receptor-dependent activation of FAK signaling pathway to regulate invasion and metastasis of lung adenocarcinoma.Through this study,the molecular mechanism of COL10A1 in promoting the invasion and metastasis of lung adenocarcinoma can be elucidated theoretically,which will provide new ideas for the early warning and treatment of lung adenocarcinoma patients.