The Function And Mechanism Of SIRT7 Promoting The Research Progress Of Renal Clear Cell Carcinoma

Objective: Due to lack of early sensitive tumor biomarkers,15% patients of aclear cell renal cell carcinoma(cc RCC)are diagnosed metastasia at first check.And patients with cc RCC usually cannot fully benefit from targeted therapy and immune checkpoint inhibitor therapy due to the abundant vascularity and high heterogeneity of the tumor microenvironment.SIRT7 can be involved in tumorigenesis and development as an important molecule in remodeling tumor microenvironment in various solid tumors,and plays an important role in inhibiting immune cell infiltration to assist tumors to develop immune tolerance and immune escape.The aim of this study is to explore the role and related regulatory mechanisms of SIRT7 in the occurrence and development of cc RCC and in tumor microenvironment remodeling,and to provide s new potential screening and therapeutic target for clinical diagnosis and treatment.Materials and Methods: 1.Western Blot and immunohistochemical staining were used respectively to detect the expression of SIRT7 in 10 pairs of cc RCC and adjacent normal tissue samples and the previously constructed tissue microarray(containing 144 cc RCC tissue samples and 106 adjacent normal tissue samples).Then combined with clinical and follow-up information,the correlation between SIRT7 and clinicopathological characteristics was analyzed to explore the prognostic value.2.Proliferation assay,wound healing assay and Trans-well assay were performed to detect the effects of SIRT7 knockdown on cell proliferation,migration and invasion in 786-O and SN-12 cell lines.High-throughput RNAsequencing with bioinformatics were used to comprehensively identify the the downstream target molecule LOXL2 regulated by SIRT7.CCK-8 detection,Ed U staining,scratching,Trans-well experiment,TUNEL staining,and flow cytometry apoptosis detection assay were performed to explore the role of LOXL2 in the malignant progression of cc RCC.3.Mice orthotopic allograft and xenograft model were established in vivo to verify the effect of SIRT7 knockdown on tumor cell proliferation and metastasis.The regulatory function of SIRT7 on the immune microenvironment was tested by tumor-infiltrating lymphocyte clustering in flow cytometry and immunohistochemical staining was used to perform the infiltration of CD4+ and CD8+T lymphocytes and macrophages in tumor microenvironment after SIRT7 knockdown.Results: 1.Western Blot and immunohistochemistry staining for tissues samples showed an overall SIRT7 upregulation in cc RCC tissue samples compared with adjacent normal tissue samples.The expression of SIRT7 was significantly correlated with advanced nuclear grade,lymph nodes and distant metastases.Kaplan–Meier survival curve analysis showed that high SIRT7 expression level often indicates poor postoperative prognosis.2.Proliferation test,wound healing and Transwell assay indicated that stable knockdown of SIRT7 significantly inhibits the ability of proliferation,migration and invasion of cc RCC cells.3.The results of high-throughput RNA-sequencing suggested that LOXL2 is regulated by SIRT7 and participates in the remodeling of the tumor microenvironment in cc RCC.4.CCK8 detection and Ed U staining suggest that SIRT7 regulates LOXL2 to promote the proliferation of cc RCC cells.Wound healing and Trans-well assay suggest that SIRT7 regulates LOXL2 to promote the migration and invasion of cc RCC cells.TUNEL staining and flow cytometry apoptosis detection assay suggest that SIRT7 promotes genome stability through LOXL2 and inhibits cell apoptosis.5.Overexpression of LOXL2 rescue experiments confirmed that SIRT7 regulates LOXL2 to remodel the tumor microenvironment and promote the malignant progression of cc RCC.6.Mice orthotopic allograft and xenograft models showed that the size and weight of orthotopic renal tumor and the number of pulmonary metastases within group of SIRT7 knockdown were significantly smaller and lesses than those in the control group.Tumor infiltrating lymphoid subpopulation by flow cytometry detection confirmed that knockdown of SIRT7 significantly increased infiltration extend of CD4+ T lymphocytes,B lymphocytes and macrophages within tumor but decreased NKT cells.The immunohistochemical staining performed that significant infiltration of CD4+ and CD68+ macrophages around and inside the tumor in the group of SIRT7 knockdown,but there was no significant difference in infiltration CD8+ lymphocytes.Western Blot experiments further verified that after knockdown of SIRT7,the expression level of PD-L1 was significantly up-regulated,independent of IFNG-γ.Conclusion: This study is the first to explore the biological functions of SIRT7 in the development of cc RCC and the tumor microenvironment.Upregulation of SIRT7 in cc RCC tissues is significantly correlated with advanced tumor grades,distant metastases and worse prognosis.SIRT7 promotes proliferation,migration,invasion and maintenance of genomic stability in renal clear cell carcinoma by regulating LOXL2 and inhibits apoptosis.Most importantly,SIRT7 could also participate in the construction and remodling of tumor microenvironment by regulating the infiltration of CD4+ and CD8+T lymphocytes,which did not depend on the expression of PD-L1.SIRT7 also participates in the remodeling of the tumor immune microenvironment by regulating the infiltration of CD4+ T lymphocytes,B lymphocytes and macrophages,and upregulates the expression of PD-L1 independently of IFNG-γ.