The Study Of Correlation Between HSP60 And Malignant Phenotype And Prognosis Of Non-small Cell Lung Cancer

Background:Lung cancer is not only one of the most common malignant tumors,but also the main cause of cancer death in the world.Lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer.Lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)are the two most common histological subtypes of NSCLC.Patients with early diagnosis of NSCLC can get a good prognosis after surgical resection.However,most patients are in the middle and advanced stage of lung cancer at the time of diagnosis.Although,with the in-depth study of the occurrence and development mechanism of lung cancer,great progress has been made in the treatment of patients with advanced lung cancer(including molecular targeted therapy)in recent years,there are still many problems such as tumor drug resistance,recurrence and metastasis,which seriously restrict the improvement of the five-year survival rate of patients.Therefore,identifying new biomarkers is of great significance for the early diagnosis,prognosis and targeted therapy of NSCLC.Heat shock protein 60(HSP60)is an important molecular chaperone protein.Most HSP60 are distributed in mitochondria and usually play biological functions with the help of heat shock protein 10(HSP10)to correct the folding of newborn proteins,restore the structure of misfolded proteins,and maintain the stable state of mitochondrial proteins.In the environment of malignant stress,tumor cells will hijack HSP60 and HSP10 proteins to protect key proteins from degradation,so as to maintain their survival and development.As a molecular chaperone,HSP60 plays an important role in mediating the occurrence and development of tumors by acting on other substrate proteins.For example,HSP60 interacts with tumor suppressor protein p53 to down regulate the expression of p21 and Bax to promote the survival of cancer cells.HSP60 can interact with survivin,an inhibitor of apoptotic protein,to maintain its structural stability.Knockdown of HSP60 promotes survivin instability and protein degradation,thus inducing apoptosis.HSP60 can also interact with cyclophilin D(cypD),a pro-apoptotic component of mitochondrial permeability transition pore,and inhibit its pro apoptotic function.In addition,HSP60 can interact with β-catenin to promote cancer cell metastasis.However,the expression and clinical significance of HSP60 and HSP10 in NSCLC are unclear and need to be further discussed.Based on the above research background,this study analyzed the expression of HSP60 and HSP10 at mRNA level and the correlation between them,as well as the relationship with clinicopathological features and prognosis of patients by bioinformatics technology.At the same time,the expression levels of HSP60 and HSP10 protein in 354 cases of NSCLC and 53 cases of non-cancerous lung tissues(Non-CLT)were detected by immunohistochemical(IHC)staining,and the correlation between their protein expression and clinicopathological features and prognosis was analyzed,in order to evaluate whether HSP60 and HSP10 can be used as new independent prognostic markers for NSCLC patients.Further,we chose HSP60 as the main research object to explore the effect of HSP60 on the biological behavior of NSCLC cells at the cellular level.At the same time,we explored the enrichment gene set related to the high expression of HSP60 mRNA by GSEA enrichment analysis,so as to further explore the biological function and possible internal mechanism of HSP60 on NSCLC.Research methods:1.LUAD and LUSC TCGA data sets were downloaded from UCSC Xena website to analyze the expression of HSP60 and HSP10 mRNA in NSCLC and their relationship with clinicopathological features and prognosis of NSCLC patients.2.All patients had a definite diagnosis and did not receive radiotherapy,chemotherapy and targeted therapy before operation.The expression of HSP60 and HSP10 protein in NSCLC and Non-CLT was detected by tissue microarray(TMA)and IHC.The correlation between HSP60 and HSP10 protein and clinicopathological features and prognosis was statistically analyzed.3.The effect of HSP60 on the biological behavior of NSCLC was detected by molecular biology experiment.(1)The expression of HSP60 in NSCLC cell lines and human bronchial epithelial cell line was detected by WB.(2)RT-qPCR and WB assay were used to detect the expression of HSP60 in NSCLC after transient transfection.(3)CCK-8 proliferation test and plate clone formation test were used to detect the effects of HSP60 knockdown and KHS101 drug treatment on the proliferation of NSCLC cell line.(4)Ann exin V/PI apoptosis assay was used to detect the effects of HSP60 knockdown and KHS101 drug treatment on the apoptosis of NSCLC cell line.(5)WB assay was used to detect the changes of apoptosis-related protein level of NSCLC cell lines after HSP60 knockdown.4.The high expression of HSP60 was analyzed from the LUSC mRNA and HSP60 signal set of patients.Results:1.In both LUAD and LUSC,the mRNA levels of HSP60 and HSP10 were higher than those in normal lung tissues and paired adjacent tissues.HSP60 mRNA level was positively correlated with HSP10 mRNA level.2.High expressed HSP60 mRNA,high level of HSP10 mRNA and the co-high expression of HSP60 and HSP10 mRNA were correlated with gender,histological type,clinical stage,size and site of primary tumor and lymph node metastasis(LNM)in patients with NSCLC.3.LUAD patients with high expression of HSP60 mRNA had lower overall survival time than patients with low expression.4.HSP60 and HSP10 protein levels were higher than those in normal lung tissue.HSP60 and HSP10 protein levels were positively correlated.5.The high expression of HSP60 protein was associated with histological type,clinical stage and LNM in patients with NSCLC.The high expression of HSP10 protein and the high combination of HSP60 and HSP10 protein expression were related to the clinical stage of patients.6.Both individual and combined HSP60 and HSP10 protein expression were significantly correlated with the poor prognosis of patients.7.Cox multivariate regression analysis showed that the high expression of HSP60 and HSP10 protein could be used as independent predictors for poor prognosis of NSCLC independent of clinical stage,LNM and pathological grade.8.HSP60 inhibitor KHS101 can significantly inhibit the proliferation of NSCLC cell line and induce apoptosis.9.HSP60 knockdown can significantly inhibit the proliferation of NSCLC cell lines and activate mitochondrial apoptotic pathway.10.GSEA gene enrichment analysis showed that the high expression of HSP60 was positively enriched in the gene set related to proliferation and mTORCl pathway in LUAD and LUSC patients,and the high expression of HSP60 was negatively enriched in the gene set related to apoptosis and p53 pathway in LUSC.Conclusions:1.HSP60 knockdown can inhibit NSCLC cell proliferation and induce cell apoptosis through mitochondrial apoptotic pathway.2.The high expression of HSP60 is positively correlated with the gene set related to proliferation and mTORCl pathway,and negatively correlated with the gene set related to apoptosis and p53 pathway.3.HSP60 and HSP10,which are highly expressed in NSCLC and are associated with poor prognosis,can be used as independent predictors of poor prognosis of NSCLC independent of clinical stage,LNM and pathological grade,respectively.