Hypoxia Related Gene TGFBI Promotes The Migration,invasion And Tumor Related Angiogenesis Of Renal Clear Cell Carcinoma

Objective: renal cancer is a common tumor in Department of Urology,and its incidence rate in urological tumors is only inferior to that of prostate cancer and bladder cancer.Among the histological types of renal cell carcinoma,renal clear cell carcinoma is the most common,accounting for about 7-8% of all renal cell carcinoma.Locally advanced renal cell carcinoma and metastatic renal cell carcinoma are the difficulties of clinical treatment of renal cell carcinoma,so various new adjuvant treatment schemes have been developed one after another.However,due to the complex tumor characteristics of renal clear cell carcinoma,the response rate of patients to the same treatment is uneven.Therefore,distinguishing the characteristics of patients with renal clear cell carcinoma and then precise treatment has become a new direction of renal cancer treatment.Hypoxia is an inevitable obstacle in the growth of solid tumors.The hypoxic environment not only affects tumor metabolism,but also changes a series of tumor characteristics,including tumor microenvironment.These changes not only make the tumor have their own metabolic and immune characteristics,but also further affect the tumor response to drug therapy.Renal cell carcinoma is a typical solid tumor,and hypoxia related pathways are generally over activated;Therefore,patients were differentiated according to the hypoxic phenotype of renal clear cell carcinoma,so as to evaluate the prognostic risk and possible response to related treatment.TGFBI is a member of hypoxia related genes.It was first found in lung adenocarcinoma cell line A549.It mainly exists in extracellular matrix.As a member of cytokines,TGFBI participates in the composition of tumor microenvironment and immune related regulation.The role of TGFBI in tumors has attracted much research attention.Many studies have pointed out that TGFBI plays a role in promoting cancer in some tumors,promoting cancer in some tumors,and inhibiting cancer in some tumors.Even at different stages of development of the same tumor,the role of TGFBI may be completely different.TGFBI can be expressed by a variety of cells in tumor tissues,and its main expression sources in different tissues are also different.In addition,the specific mechanism of TGFBI promoting and inhibiting cancer is not clear.Although many studies have proposed a variety of possible mechanisms,there is no unified view to describe the specific function of TGFBI.Therefore,in-depth understanding of the role and mechanism of TGFBI in renal clear cell carcinoma has high innovative value and pioneering.The ability of tumor migration and invasion is the key for tumor to break through the limitation of normal tissue,cross the vascular barrier into blood circulation and metastasis of distal organs.As an extracellular matrix protein,some studies have pointed out that TGFBI is involved in regulating cell adhesion function,and many studies have pointed out that TGFBI can promote tumor migration and invasion.Therefore,studying the effect of TGFBI on the invasion and migration ability of renal clear cell carcinoma will help to clarify the cancer promoting effect of TGFBI on renal clear cell carcinoma.Renal clear cell carcinoma has obvious angiogenesis characteristics.At the same time,it is also due to the formation of a large number of tumor blood vessels,which solves the problem of hypoxia in the process of volume increase of renal clear cell carcinoma.Antiangiogenic therapy for renal clear cell carcinoma is also the first-line adjuvant therapy for renal clear cell carcinoma.Since a large number of previous studies have pointed out that TGFBI is related to tumor angiogenesis,it is a feasible research direction to study the relationship between TGFBI and angiogenesis of renal clear cell carcinoma.Methods: Firstly,we used bioinformatics technology to conduct GSEA enrichment analysis based on the gene transcription data of renal clear cell carcinoma samples and adjacent samples in TCGA database and geo database,and screened the valuable tumor biological characteristics.We focused on the hypoxia gene set,The enrichment degree of hypoxia phenotype and the difference of PCA characteristic score in TCGA solid tumors were compared by ss GSEA and PCA scoring methods.Next,we distinguished the characteristics of KIRC samples of TCGA and e-mtab-1980 samples by NMF clustering,and explored the prognostic differences of patients with different types of hypoxia characteristics.The prognosis model of different hypoxia related genes was constructed based on the difference of hypoxia related genes of Las Sox.By analyzing the prognosis and expression characteristics of the genes constituting the model,we selected the TGFBI with in-depth research value for intensive study.Through the correlation analysis of immune characteristics based on transcriptional data,the potential association between TGFBI and immune cells was analyzed,and the differences of immune characteristics between patients with different types of hypoxia were evaluated.Based on the existing single-cell sequencing data set,we explored the main expression source cells of the genes constituting the model,and further explored the expression source of TGFBI.In addition,we evaluated the correlation between hypoxia risk model and its genes through TCGA methylation data and SNVs data.Based on the above analysis and the research evidence obtained by consulting a large number of literatures,we first collected renal clear cell carcinoma and its adjacent samples for immunohistochemistry to verify the high expression of TGFBI in tumor tissues,and the high expression of TGFBI is related to the poor prognosis of patients.Then we studied the difference of TGFBI expression level in different renal clear cell carcinoma cell lines based on CCLE database and self-collected cell lines,and selected769-P and 786-O for follow-up research and experimental design.In vitro,we first constructed environmental hypoxia and chemical hypoxia through hypoxia incubator and Co Cl2,and demonstrated the relationship between TGFBI expression level of renal cell carcinoma cell line and macrophage and hypoxia stimulation.And exogenous TGF was added-β1 and TGF-β1 antibody neutralization test to observe the effect of TGFBI on TGF-β1 response to stimulation.SiRNA knockdown of TGFBI and plasmid overexpression of TGFBI were used to observe the effects of TGFBI on the proliferation,migration and invasion of renal cell carcinoma and macrophage line,so as to explore the potential function of TGFBI in renal cell carcinoma.In addition,we stimulated HUVEC by exogenous addition of TGFBI,and further observed the changes of cell proliferation,migration,invasion,cell budding and tube forming ability.Results: Based on the analysis of database data,we found that the hypoxic phenotype in renal clear cell carcinoma was generally highly enriched compared with normal renal tissue,and the hypoxic phenotype of renal carcinoma was specific at the pan cancer level.Cluster analysis showed that patients with renal clear cell carcinoma with high hypoxia characteristics had a poor prognosis compared with patients with low hypoxia characteristics.The prognosis model based on hypoxia related genes had a certain ability to predict the prognosis of patients,and it was an independent risk factor for the prognosis of patients.The constituent gene TGFBI of the prognosis model is highly expressed in tumors and is associated with poor prognosis,and its expression abundance is negatively correlated with the purity of tumors.In addition,the immune score of high hypoxia characteristic samples is higher than that of low hypoxia characteristic samples,and macrophages and tumor cells may be involved in the expression of TGFBI;The hypoxia type of patients is related to the SNVs of the sample,and the expression of TGFBI is affected by its gene methylation level;Through the histochemistry of the collected samples and sorting out the clinical information,we found that TGFBI was highly expressed in tumor tissues,and the high expression of TGFBI was associated with poor prognosis.Macrophages of TGFBI in renal cancer tissues may be involved in the expression of TGFBI,and TGFBI may mainly come from renal cancer cells.In an in vitro study based on renal cell carcinoma cell lines,we found that the expression of TGFBI in some renal clear cell carcinoma cell lines(769-P)was responsive to hypoxia stimulation,but the TGFBI of renal cell carcinoma cell lines in this study was sensitive to TGF-β1 was corresponding.However,the expression of TGFBI in different types of macrophages had corresponding effects on hypoxia stimulation in vitro.Cell experiments showed that TGFBI could promote the migration and invasion of renal clear cell carcinoma and macrophages,but had no significant effect on their proliferation;Exogenous TGFBI can promote the migration,invasion,tube formation and budding of HUVEC cells.Conclusion: The risk scoring model based on hypoxia related genes has a certain ability to evaluate the prognosis of patients,which is helpful to distinguish the characteristics of hypoxia and then treat accurately.TGFBI is highly expressed in tumors,and its high expression is associated with poor prognosis.TGFBI is mainly expressed by tumor cells,and macrophages are also involved in the expression of TGFBI;The expression of TGFBI in renal cell carcinoma cells is regulated by TGF-β1,TGFBI expression in macrophages has a corresponding effect on hypoxia stimulation.TGFBI can promote the migration and invasion of renal clear cell carcinoma,macrophages and endothelial cells,and promote the budding and tubulation of endothelial cells.