COL6A6 Inhibits The Proliferation And Metastasis Of Non-small Cell Lung Cancer Through The JAK Signaling Pathway

Background:By far,the most common malignancy worldwide has been lung cancer,which is a significant contributor to tumor mortality.Non-small cell lung cancer(NSCLC)the most prominent type of lung cancer is divided into three major groups: lung squamous cell carcinoma(LUSC),lung adenocarcinoma(LUAD)and large cell carcinoma.Surgery is the most frequent treatment for those with early-stage NSCLC,whereas combination therapy is favored for those with mid-to late-stage NSCLC,and conventional radiotherapy is still the primary treatment for NSCLC.Most patients with late-stage NSCLC have a mean 5-year survivability rate of merely 19%,notwithstanding the recent developments in tumor molecular biology,genomics,and targeted therapies.Notably,tumorigenesis may be significantly associated with changes in essential genes.The rise of targeted drugs has alleviated the overall survival rate of NSCLC patients,but the development of highly effective and less toxic targeted drugs for rare and refractory targets in patients who are resistant to some sensitive gene-targeted drugs has been an urgent clinical challenge.Hence,exploring the mechanism of NSCLC and seeking the latest diagnostic targets for the early treatment of NSCLC to improve the normal amount of life of patients remains imperative.Objectives:Type Ⅵ collagen alpha 6 chain(COL6A6)belongs to the type Ⅵ collagen(COL6)family and is encoded by a 6789-bp coding region.COL6A6,as an extracellular matrix protein,significantly expressed in a various human organizations including the lungs,kidneys,liver,spleen,spleen and thymus,as well as heart and skeletal muscle,playing an essential role in the maintenance of cellular integrity and adjustment of cell function.It established that COL6A6 is involved in the development of a variety of tumors: COL6A6 inhibits the growth and metastasis of pituitary adenomas,meanwhile a significant correlation between high COL6A6 expression and early pathological stages of breast cancer has also been demonstrated.Furthermore,numerous other COL6 family member have been shown to act as essential actors in tumor inception and progress.While JAK-STAT plays an important role in the development of colorectal,gastric,bladder and other tumors,it remains unclear whether COL6A6 is involved in NSCLC.Non-small cell lung cancers are over-expressed of JAK-STAT,promoting cell proliferation.Investigations revealed that JAK-STAT accelerated the invasion and metastasis of non-small cell lung cancer,which was associated with poor patient prognosis.Nonetheless,the relationship between COL6A6 and JAK-STAT signaling pathway has not been explored.Methods:Part I: Bioinformatics study of COL6A6 gene based on public databases and clinical validation.1.Detect the m RNA levels of COL6A6 gene in various human tumor tissues and paraneoplastic tissues using public databases(TCGA,GTEx,GEPIA2,TIMER).2.The TCGA-LUAD and TCGA-LUSC datasets from the TCGA database were used to analyze the divergence in COL6A6 gene expression between paired and unpaired NSCLC samples.3.Collecting 17 NSCLC samples and the corresponding precancerous tissue specimens.4.By wb assay,the expression of COL6A6 protein was detected..5.Downloading immunohistochemical images of the COL6A6 gene in The Human Protein Atlas(HPA)database.6.Analyzing the differences between clinical variable groupings of the target gene COL6A6 based on TCGA-LUAD dataset and TCGA-LUSC dataset,analyzing the relationship between COL6A6 and clinical information such as pathological stage,TNM stage,gender,and age of patients,and elucidating the prognostic and diagnostic significance of COL6A6 gene in NSCLC.Part II: COL6A6 regulates the proliferation,migration and invasion ability of lung cancer cells.1.Cell lines(A549,H1299,H520,H1975)from adult lung cancers and normal lung epithelial cells(Beas-2B)were cultured in vitro.2.The expression of COL6A6 protein in each group of cell lines was detected by Western blot.3.H1299 cells were selected to construct a knockdown COL6A6 cell line(COL6A6-si),while a negative control group(NC-si)was constructed.4.By employing the cell proliferation assay(CCK8),the effect of COL6A6’s knockdown on NSCLC cells’ proliferation capacity can be determined.5.By scratch assay,the migration capacity of NSCLC was impacted by the knockdown of COL6A6.6.To determine the effect of COL6A6 knockdown on NSCLC cell invasion,a Transwell invasion assay was utilized.Part III: Molecular mechanism of COL6A6 regulation of NSCLC cell growth1.Batch correlation analysis was performed between COL6A6,and all genes based on TCGA-LUAD and TCGA-LUSC datasets to find the correlated genes of COL6A6 separately and form a gene set.2.Perform GO/KEGG enrichment analysis on the gene set.3.GSEA Gene Set Enrichment Analysis(GSEA)was performed for COL6A6 gene.4.WB examined the protein JAK2,p-JAK2,STAT3 and p-STAT3 expression ability in the JAK signaling pathway knocked down by COL6A6.Results:Part I: Bioinformatics study of COL6A6 gene based on public databases and clinical validation.1.The m RNA expression level of COL6A6 gene in various human cancer cells as well as paraneoplastic cells was significantly different,with COL6A6 gene showing lower expression in LUAD and LUSC tissues than in normal lung tissues.2.In both paired and unpaired samples of NSCLC,the expression of COL6A6 gene was significantly lower in the tumor than in the collateral cancer samples.3.In contrast to the NSCLC paired tissues,the expression of the COL6A6 gene protein in collateral tissues was remarkably low.4.The immunohistochemistry results revealed the COL6A6 protein was mainly targeted to the cytoplasm and membrane of NSCLC;5.COL6A6 gene was closely correlated with the clinical data of pathological stage,TNM stage,gender and age of NSCLC patients,and high expression of COL6A6 gene had good prognostic and diagnostic significance for NSCLC.6.Univariate COX regression analysis showed that COL6A6 gene expression level and clinicopathological stage were strongly correlated with the overall survival among NSCLC patients,and high COL6A6 gene expression was a well-prognostic factor for NSCLC patients.Results of multifactorial cox regression study showed the expression of COL6A6 gene,which is a favorable independent prognostic factor in NSCLC.Part II: COL6A6 regulates the proliferation,migration and invasion ability of NSCLC cells1.Human NSCLC cell lines(A549,H1299,H520,H1975)expressed significantly less COL6A6 protein than normal epithelial cell lines(BEAS-2B),and the H1299 cell line showed a significantly higher level of COL6A6 protein.2.H1299 cells were selected to construct a knockdown COL6A6 cell line(COL6A6-si),and a negative control group(NC-si)was also constructed.The PCR and WB of H1299 cell lines revealed a significant decrease in both m RNA and protein expression levels of COL6A6 following its knockdown,when compared to the control group.3.The knockdown of COL6A6 was demonstrated to significantly augment the proliferation capacity of NSCLC cells in comparison to the control group,as CCK8 revealed.4.Scratch assay showed that the migration ability of NSCLC cells was significantly accelerated after knockdown of COL6A6.5.The knockdown of COL6A6 caused a marked augmentation in the invasion capacity of NSCLC cells,as demonstrated by the transwell invasion assay.Part III: Molecular mechanism of COL6A6 regulation of NSCLC cell growth1.The JAK-STAT signaling pathway was revealed to be enriched by GO/KEGG in genes related to COL6A6.2.GSEA gene enrichment analysis of COL6A6 gene showed that COL6A6 gene was highly associated with JAK-STAT signaling pathway.3.The WB assay revealed a marked rise in the expression of related proteins in the JAK signaling pathway when COL6A6 was knocked out,compared to the control group.Conclusions:1.COL6A6 protein expression in NSCLC tissue was less than that of neighboring normal lung tissue.2.The degree of COL6A6 protein expression was significantly correlated with the clinicopathological stage,TNM stage,gender and age of NSCLC patients.3.The prognosis of NSCLC patients could be predicted independently by the COL6A6,which has a strong diagnostic value and is linked to the overall survival of those with the condition.4.NSCLC cells exhibited a diminished expression of the COL6A6 gene compared to normal lung epithelial cells.5.The expression of COL6A6 being low could potentially foster the growth,movement,and infiltration of NSCLC cells.6.COL6A6 can be involved in the regulation of NSCLC by affecting the JAK signaling pathway.