CD146 Promotes Migration And Invasion Of Non-small Cell Lung Cancer By Regulating EMT

BackgroundLung cancer is the leading cause of cancer death globally,with highest morbidity and mortality rate rank first among malignant tumors in China.According to its pathological type,lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC is the most common pathological type,accounting for about 85%of the total number of lung cancers.With the continuous development,precision therapy strategies including targeted and anti-angiogenesis therapy improve prognosis and quality of life of patients with lung cancer to a certain extent yet this disease still cannot completely be cured.The overall survival period of lung cancer patients is still relatively short.The annual survival rate is still low.Therefore,it is urgent to understand the underlying mechanisms of pathogenesis and development of lung cancer from a molecular perspective,thus gain new insight for lung cancer treatment that may improve patients’ survival and quality of life.CD 146 is a Ca2+ independent adhesion molecule,a member of the immunoglobulin superfamily,whose function is involved in cell transmembrane signaling.This molecule was first identified in melanoma and is closely associated with tumor metastasis and poor prognosis.With the deepening of the research on CD 146,it has been found that it has more extensive and complex biological functions,and participates in a variety of physiological processes,such as angiogenesis embryo and neural tube development.In addition,studies have shown that CD146 also plays an important role in tumor,and is associated with the progression,metastasis and poor prognosis of many different types of tumor.However,the role of CD146 in NON-small cell lung cancer is still unclear.Therefore,this study focuses on the role of CD146 in metastasis of non-small cell lung cancer,in order to enrich the theory of lung cancer metastasis and provide new ideas for the diagnosis and treatment of lung cancer metastasis.Part One:The expression of CD146 and its clinical valuePurpose:Taking CD 146 as the research object,we analyzed the expression level and clinical value of CD 146 in tumors in the public database,and explored the molecular pathway of CD 146 regulating non-small cell lung cancer.Method:1.Calculate the expression level of CD 146 in normal tissues or organs from the public database GTEx.2.Use the public database TCGA to calculate the expression level of CD146 in 32 kinds of tumors and their corresponding normal tissues.3.Use analysis of variance to evaluate the expression of CD146 in different tumor stages.4.Use the Cox proportional hazard model to model the expression level of CD 146 and the overall survival,use the R language to draw forest mapse,and valuate the impact of CD 146 on the survival rate.5.Use the CCLE public data platform to map the expression level of CD 146 in human lung cancer cell lines and its methylation table level.6.GSVA algorithm was used to analyze lung adenocarcinoma and lung squamous cell carcinoma in TCGA database,and the enrichment pathways of genes with different expression with CD 146 were analyzed.7.Use the GSEA algorithm to analyze the regulatory relationship between lung adenocarcinoma,squamous cell lung carcinoma,CD 146,and EMT pathway in the TCGA database.8.In the lung adenocarcinoma and squamous cell lung carcinoma’s expression profile data,the 2000 genes with the most significant correlation with the target gene expression were concentratedly screened,and the hallmark data set was used for KEGG analysis.9.CD146 was divided into high and low expression groups by median,and heat maps of lung adenocarcinoma lung squamous cell carcinoma tissue samples and EMT-related genes were displayed.Result:1.The expression level of CD146 is higher in large blood vessels and lower in normal tissues.2.The expression of CD146 in tumor tissues was higher than that in adjacent tissues in 5 tumors,and the expression of CD146 in 6 tumors was lower than that in adjacent tissues(P<0.01).3.The expression of CD146 in the overall tumor is significantly related to the tumor stage.A separate analysis of each tumor type found that the expression of CD 146 in 6 tumors was significantly associated with the tumor stage.However,the expression of CD 146 did not follow the tumor stage.It didn’t increase as the increase of tumor stage(P<0.05).4.The expression of CD 146 in 5 tumors was significantly correlated with the overall survival of patients(P<0.05).5.The expression of CD146 is the highest in melanoma cell lines,while the expression of acute myeloid leukemia tumors in the blood system is the lowest.The RRBS data of these cell lines showed that the expression of CD 146 was highly correlated with promoter DNA methylation(P<0.05).6.GSVA analysis showed that in lung adenocarcinoma,28 pathways have a positive regulatory effect on CD 146,and 14 pathways have a negative regulatory effect;in squamous cell lung carcinoma,29 pathways have a positive regulatory effect on CD146,and 18 pathways have a negative regulation(P<0.05).7.GSEA analysis shows that lung adenocarcinoma and squamous cell lung carcinoma show that CD146 has a positive regulatory relationship with EMT(P<0.05).8.KEGG pathway analysis showed that both lung adenocarcinoma and squamous cell lung carcinoma showed a significant correlation between CD 146 and EMT activation(P<0.05).9.The results of single gene heatmap analysis showed that CD146 is correlated with EMT pathway genes(P<0.05).Part Two:Correlation analysis between CD146 expression and NSCLC clinical dataPurpose:To analyze the correlation between the expression of CD 146 protein and the clinical data and prognosis of NSCLC.Method:1.Collect cancer tissue specimens of NSCLC patients according to the inclusion and exclusion criteria of this subject,use immunohistochemistry to detect the expression level of CD 146 in cancer tissues,and analyze the correlation between the expression level of CD146 and the clinical characteristics of NSCLC patients.2.Assess the correlation between the expression level of CD146 and the prognosis of patients.Result:1.The expression level of CD146 is correlated with lymph node metastasis,distant metastasis,and tumor TNM staging in NSCLC patients(P<0.05),but does not correlate with patient gender,age,tumor pathological type,and tumor size(P>0.05).2.CD146 is an independent risk factor for the poor prognosis of NSCLC patients(P<0.05).Part Three:The effect of CD146 on the biological behavior of non-small cell lung cancer and its mechanismPurpose:To explore the effect and mechanism of regulating the expression of CD146 on the migration and invasion of NSCLC in vitro.Method:1.Use western blot and flow cytometry to screen NSCLC cell lines with high and low expression of CD146.2.Use immunofluorescence staining technology to locate the expression position of CD146.3.H460 and A549 was transfected with lentivirus to construct stable cell lines and verify the transfection efficiency.4.The effect of CD 146 on cell migration was detected by scratch assay and Transwell assay.6.Transwell(matrigel)invasion assay was used to detect the effect of CD146 on cell invasion ability7.Screen the differential genes related to CD146 by mRNA sequencing.8.The differential genes were analyzed by GO analysis,KEGG pathway enrichment and GSEA analysis to find the possible molecular pathway.9.Use the Western blot method to detect changes in EMT-related proteins,transcription factors,and PI3K/Akt pathway proteins.10.Use the Western blot to detect changes in the knockdown or overexpression group on EMT-related proteins and PI3K/Akt signaling pathways by using PI3K activators or inhibitors.Result:1.Screen 8 NSCLC cell lines;the CD146 high-expressing cell line is H460,and the low-expressing cell line is A549.2.Immunofluorescence experiments show that CD146 is located on the cell membrane.3.Successfully used lentiviral transfection technology to construct a stable knockdown CD146 cell line of H460 cells and a stably transfected cell line of A549 overexpressing CD 146(P<0.05).4.The results of the scratch experiment showed that in H460 cells,compared with the control group,the wound healing ability of the shRNA knockdown CD 146 group was weakened;in the A549 cells,compared with the control group,the wound healing ability of the CD146 overexpression group was enhanced(P<0.05).Transwell experiment results showed that in H460 cells,compared with the control group,the number of transmembrane cells in the shRNA knockdown CD146 group decreased;in A549 cells,the number of transmembrane cells in the CD146 overexpression group increased compared with the control group(P<0.05).5.The results of the Transwell(Matrigel)invasion experiment showed that in H460 cells,compared with the control group,the number of transmembrane cells in the shRNA knockdown CD146 group was reduced;in A549 cells,compared with the control group,the number of transmembrane cells increased in the group in the CD146 overexpression group(P<0.05).6.By sequencing with a 2-fold difference as the standard,a total of 324 differential genes were screened,174 genes were up-regulated,and 150 genes were down-regulated(P<0.05).7.Through the different gene set enrichment methods of GO,KEGG,GSEA,the meaningful enrichment pathways are all related to cell migration and invasion,such as cell adhesion,cell junction,focal adhesion,and cell migration(P<0.05).8.EMT-related indicators and PI3K/Akt pathway protein detection results showed that in H460 cells,the expression of N-cadherin,Vimentin,Snail,Twist,p-PI3K and P-Akt decreased in the knockdown group.In A549 cells,the expression of E-cadherin decreased in overexpression group,and the expression of N-cadherin,Vimentin,Snail,Twist,P-PI3K and P-Akt increased(P<0.05).9.The results of Western blot showed that in H460 cells,the expression of N-cadherin,vimentin,snail,twist,p-PI3K and p-Akt increased in PI3K activator group compared with knockdown group;In A549 cells,compared with the overexpression group,the expression of E-cadherin increased,and the expression of N-cadherin,vimentin,snail,twist,p-PI3K and p-Akt decreased in the PI3K inhibitor group(P<0.05).Part Four:The effect of regulating the expression of CD146 in vivo on the metastasis of NSCLC in nude micePurpose:To explore the effect of regulating the expression of CD 146 on the formation of metastasis in NSCLC nude mice.Method:1.The H460-shCtrl and H460-shRNA#1 knockdown CD146 group containing GFP tags and the A549-pcDNA3.1,A549-pcDNA3.1-CD 146 overexpression CD 146 group were injected into nude mice through the tail vein,respectively.A small animal live imaging device was used to observe the fluorescent signal of metastasis formed in nude mice every week.2.After the nude mice were sacrificed,a gross specimen of lung tissue was removed,and the number of metastatic tumors formed was observed.3.HE staining and immunohistochemical techniques to detect the formation of metastases and the expression level of EMT-related proteins.Result:1.Successfully established a nude mouse metastasis model.The experimental results showed that in H460 cells,compared with the control group,the fluorescence signal of the shRNA knockdown CD 146 group was significantly weakened;in the A549 cells,compared with the control group,the CD146 overexpression group The fluorescence signal was significantly enhanced(P<0.05).2.The statistical results of the number of metastatic tumors in nude mouse lung specimens showed that in H460 cells,compared with the control group,the H460-shRNA#1-luc group had fewer metastatic tumors;in A549 cells,compared with the control group,A549-pcDNA3.1-CD 146-luc group’s metastatic tumors increased(P<0.05).3.The results of HE staining showed that in H460 cells,compared with the control group,the number of cancer nests in the pathological tissue of the H460-shRNA-luc group was less,and the area was small;in the A549 cells,compared with the control group,A549-pcDNA3.1-CD146-luc group had more cancer nests in pathological tissues and the area was larger(P<0.05).Conclusion1.The expression of CD 146 in non-small cell lung cancer may be related to the positive activation of the EMT pathway.2.The expression of CD 146 in non-small cell lung cancer tissues is related to tumor lymph node metastasis,distant metastasis,and TNM staging and is an independent risk factor for poor prognosis of patients.3.The expression of CD146 promotes the migration and invasion of NSCLC.4.CD146 mediates the EMT of non-small cell lung cancer by activating the PI3K/Akt signaling pathway and promotes the migration and invasion of non-small cell lung cancer.