| Objective:Lung cancer has become the malignant tumor with the highest morbidity and mortality worldwide.Lung cancer is histologically divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for 85%of all lung cancers.Lung adenocarcinoma(LUAD)is the most common pathological type of NSCLC.Although comprehensive treatment such as surgery,radiotherapy,chemotherapy,targeted therapy and immunotherapy has made great progress,However,the treatment results of LUAD are still unsatisfactory,mainly because the exact pathogenesis of LUAD is unknown.Microtubules are cytoskeleton components that play important roles in cell motility,polarization,mitosis,and transport.Microtubule-associated proteins play an important role in microtubule homeostasis regulation.Recent research has discovered that some microtubuleassociated proteins are overexpressed in tumor cells.These proteins disrupt the microtubule dynamic system and play an important role in the occurrence and progression of tumors.MAP7 is a protein that belongs to the microtubule-associated protein family and is involved in cell proliferation and differentiation.MAP7 is overexpressed in cervical cancer,colon cancer,and acute myeloid leukemia and is linked to a poor prognosis.MAP7 expression is increased in LUAD,according to databases such as The Cancer Genome Atlas(TCGA),but its mechanism in the development of LUAD remains unknown.The purpose of this study is to investigate the effects of MAP7 on the proliferation,invasion,and migration of LUAD,and its possible mechanism.Methods:1.Download the LUAD transcriptome data from TCGA and use R to examine the mRNA level of MAP7 expression.The Gene Expression Profiling Interactive Analysis(GEPIA)database was used to confirm expression.The online database CPTAC was used to perform a differential analysis of MAP7 protein levels.The online database HPA was used to analyze MAP7 immunohistochemical staining.2.To investigate whether MAP7 is related to the prognosis of LUAD patients,use the online database K-M plotter for survival analysis.3.Real-time quantitative PCR(RT-qPCR)and Western Blot experiments were used to detect MAP7 expression in lung epithelial cells(Beas-2B)and three LUAD(A549,H1299,and PC9)cell lines.And,based on the level of MAP7 expression in the LUAD cell line,choose the appropriate cell line for further functional experiment research.4.Small interfering RNA(siRNA)was transfected into A549 and PC9 cell lines,and MAP7 overexpression plasmid was transfected into PC9 and H1299 cell lines.After confirming the efficiency of the knockdown or overexpression,CCK-8,EDU proliferation experiments,scratch experiments,Transwell invasion and migration experiments were carried out.5.To investigate the effect of MAP7 on LUAD proliferation in vivo,a PC9 stable transfection strain overexpressing and knocking down MAP7 was created using lentivirus,and subcutaneous tumor formation experiments were performed in nude mice after successfully construction.6.Gene set enrichment analysis(GSEA)was used to perform pathway enrichment analysis on the MAP7 high expression group in order to investigate the mechanism of action of MAP7 on LUAD,and the enrichment analysis results were validated in LUAD cell lines using western blot experiments.CIBERSORT was used to investigate the relationship between MAP7 expression and immune cell infiltration as well as immune checkpoint genes in LUAD.Results:1.MAP7 expression is increased in LUAD and is associated with a poor prognosis in LUAD patients.The differential analysis of mRNA levels of MAP7 in TCGA showed that the expression of MAP7 was higher in tumor tissues of LUAD patients than in normal tissues.The pairwise difference of MAP7 expression in cancer tissues and paracancerous tissues of LUAD patients in TCGA showed that MAP7 was higher in cancer tissues than in paracancerous tissues.The GEPIA database was used to verify the expression of MAP7,and the results also showed that the expression of MAP7 was higher in LAUD tumor tissues.The results of CPTAC database also showed that the expression level of MAP7 protein in LAUD tumor tissue was higher than that in normal tissue.Immunohistochemical staining of HPA database lung tissue showed high expression of MAP7 in LAUD cancer tissue.Survival analysis results of K-M plotter database showed that the survival time of patients with low expression of MAP7 was longer than that of high expression of MAP7.2.MAP7 promotes the proliferation,invasion and migration of LUAD cells in vitro.Results from RT-qPCR and western blotting experiments revealed that MAP7 expression levels in A549,PC9,and H1299 cell lines were higher than those in bronchial epithelial cells,with A549 having the highest and H1299 having the lowest levels.Following functional experiments,MAP7 was knocked down in the A549 and PC9 cell lines.It was discovered that the MAP7 knockdown group’s cell proliferation,invasion,and migration abilities were lower than those of the control group.Subsequent functional experiments of overexpression of MAP7 in PC9 cell line and H1299 cell line showed that the cell proliferation,invasion and migration abilities of the overexpression group were stronger than those of the control group.3.MAP7 promotes the proliferation of LUAD cells in vivo.The knockdown and overexpression of MAP7 stably transfected strains in the PC9 cell line were created using lentivirus to conduct subcutaneous tumor formation experiments in nude mice.The results showed that the tumor volume of the MAP7 knockdown group was smaller than that of the control group,and the growth curve was also lower.Ki-67 immunohistochemical staining of embedded sections of subcutaneous tumors revealed that the knockdown group had lower Ki-67 expression than the control group.The tumor volume of the MAP7 overexpression group was larger than that of the control group,the growth curve was steeper than that of the control group,and the Ki-67 immunohistochemical staining was stronger than that of the control group.4.MAP7 regulates wnt/β-catenin signaling pathway in LUAD.The results of GSEA analysis of LUAD transcriptome data in TCGA revealed that the MAP7 high expression group was primarily enriched in the wnt/β-catenin signaling pathway,non-small cell lung cancer,and so on.The enrichment results were confirmed at the cellular level by western blot experiments,which revealed that after knocking down MAP7,the expression levels of β-catenin,cyclinD1,and c-myc decreased in A549 and PC9 cell lines.MAP7 overexpression increased the expression of-catenin,cyclinD1,and c-myc in PC9 and H1299 cell lines.Further investigation of the protein interaction network using the STRING database revealed an interaction relationship between MAP7 and MAPRE3,and MAPRE3 can influence APC2.APC2 is a homologous gene of the wnt/β-catenin signaling pathway suppressor gene APC.In LUAD cell lines,MAP7 increased the expression of MAPRE3 while decreasing the expression of APC2.MAPRE3 also represses the expression of APC2.5.In LUAD,MAP7 expression levels are related to immune cell infiltration and immune checkpoints.Using LUAD transcriptome data from the TCGA to investigate the relationship between MAP7 expression and immune cell infiltration and immune checkpoints,the researchers discovered that MAP7 expression was positively correlated with naive B cells and M0 macrophages,but negatively correlated with resting dendritic cells.MAP7 expression was also found to be negatively related to immune checkpoint genes.TICA data was used to analyze the response of LUAD patients to immune checkpoint inhibitors,and it was discovered that PD-1 inhibitors alone and in combination with PD-1 inhibitors and CTLA4 inhibitors benefited more patients in the MAP7 low expression group.Conclusion:1.In LUAD,MAP7 expression is increased and is linked to a poor prognosis for LUAD patients.2.MAP7 promotes proliferation,invasion and migration of LUAD cells.3.MAP7 up-regulates MAPRE3,inhibits the expression of APC2 and activates the wnt/βcatenin signaling pathway.4.Immune checkpoint genes have a negative correlation with MAP7 expression. |
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