The Anti-tumor Mechanism Of RUNX3 In Renal Clear Cell Carcinoma

Background: Among the tumors of the urinary system in our country,renal cell carcinoma(RCC)is the most common malignant tumor that seriously threatens the life of patients.Worldwide,renal cell carcinoma accounts for 2.4% of all cancers diagnosed,and approximately 1.7% of cancer patients die from it.According to the pathology,it can be divided into clear cell renal cell carcinoma(CCRCC),chromophobe renal cell carcinoma(CRCC),papillary renal cell carcinoma(PRCC),mucinous tubular,and spindle cell carcinoma(MTSCC),etc.Among them,CCRCC is the most common type during clinical diagnosis.In recent years,more and more researchers have paid attention to it due to its increasing morbidity and mortality.The treatment of early-stage CCRCC is usually based on surgery.However,due to the inconspicuous clinical symptoms and signs,a large number of patients are diagnosed with advanced CCRCC at the initial diagnosis and miss the best time for surgery.Advanced CCRCC is not sensitive to conventional radiotherapy and chemotherapy,resulting in poor long-term survival and quality for life of patients.In addition,patients with advanced CCRCC have higher recurrence and metastasis rates,which not only affect the long-term quality of life and survival expectations of patients,but also increase the burden of medical insurance.Therefore,it is essential to explore the mechanism of CCRCC tumorigenesis and metastasis.The Runt-associated transcription factor family(RUNX)consists of RUNX1,RUNX2 and RUNX3 in mammals.As one of the transcription factor members,the heterodimer of RUNX3 and the β subunit form a complex,which generates a new protein by combining with the core DNA sequence in the corresponding promoter and enhancer to further activate or inhibit transcription.RUNX3 is generally considered to be a tumor-inhibiting gene in the human body.RUNX3 methylation occurs in a variety of cancers which are known to humans,and this process can result in the silencing or loss of this gene.Loss of RUNX3 can enhance the ability of tumor cells to proliferate,invade and migrate,thereby affecting tumor progression.Studies have shown that it has a non-negligible effect on various malignant tumors such as lung cancer,colorectal cancer,gastric cancer and bladder cancer.However,the mechanism of action of RUNX3 on CCRCC is still unclear,and it is necessary to explore it fully.High invasiveness and high metastases are essential factors that threaten the life and quality of patients with malignant tumors.Epithelial-mesenchymal transition(EMT)can promote the transformation of early-stage tumors to aggressive malignant tumors.It is an important link in the malignant transformation of tumor cells and is very important in the process of tumor metastasis and invasion.EMT is a complex and dynamic process in organisms,accompanied by the decline of multiple epithelial cell markers(including β-catenin and E-cadherin)and the increase of mesenchymal markers(including vimentin and N-cadherin),which have been confirmed contributes to the metastasis and invasion of various malignant tumors.Therefore,elucidating the molecular mechanism of EMT in the process of malignant tumors and studying EMT-related pathways is one of the effective means to prevent tumor metastasis.Objective: By detecting the expression levels of RUNX3 factor and EMT-related proteins,including epithelial cadherin(E-cadherin)and Zeste homolog enhancer 2(EZH2)in CCRCC tissue and adjacent noncancerous tissue samples,the expression of RUNX3 and E-cadherin in CCRCC was preliminarily determined.The role of this gene in CCRCC and its effect on EMT were verified by constructing RUNX3-overexpressing human renal clear cell carcinoma cell lines(786-O and ACHN).To explore the possibility of RUNX3 as a prognostic marker for CCRCC and a therapeutic target for this tumor drug.Methods: The tumor tissue and corresponding adjacent non-cancerous tissue specimens of30 patients diagnosed with CCRCC in Qingdao University Affiliated Hospital were collected,and RUNX3,E-cadherin and EZH2 were detected by immunohistochemistry,qRT-PCR and other experimental methods.Human CCRCC cell lines(ACHN and 786-O)were transfected with a lentiviral vector encoding RUNX3 and an empty control vector,and RUNX3-overexpressing and RUNX3-low-expressing cell lines were constructed to validate the previous hypothesis.The proliferation rate and ability of different RUNX3 expression cell lines were evaluated by cell plate clone and MTT;the invasion and migration ability of tumor cells in the experimental group and the control group in the ACHN and 786-O cell lines were detected by Transwell and cell scratching;the number of apoptotic cells was detected by flow cytometry and the cell cycle was analyzed;the expression of the target protein RUNX3 and E-cadherin which is important in EMT were determined by qRT-PCR and western blot.Finally,the tumorigenic ability of RUNX3 in vivo was verified by a subcutaneous tumorigenic experiment in nude mice.Results: Compared with normal kidney tissue,the expression of RUNX3 in CCRCC tissue was lower,and the expression of E-cadherin was also down,while the expression of EZH2 was higher than that in normal kidney tissue.In vitro experiments of ACHN and 786-O cell lines overexpressing RUNX3 inhibited the tumor proliferation rate and invasion and migration ability;at the same time,when the cell cycle and the number of apoptotic cells were counted,it was found that the cell lines overexpressed RUNX3 had prolonged cell cycle and apoptosis.The number of apoptotic cells increased;in addition,the expression of E-cadherin and RUNX3 were detected synergistically,and the expression trend of Ecadherin also increased when RUNX3 was overexpressed in the OE group,and vice versa.In addition,in nude mice,compared with the control group,the tumor volume and weight of nude mice injected with RUNX3 gene-overexpressing tumor cells were smaller,and the expression of E-cadherin was increased.Conclusion: The expression of RUNX3 and E-cadherin was low in CCRCC tissues.In vitro and in vivo experiments confirmed that the high expression of RUNX3 can slow down the proliferation rate of CCRCC and hinder the invasion and migration ability of the tumor,which has a significant impact on the prognosis;the expression of RUNX3 is synergistic with E-cadherin,we believe that the inhibition of CCRCC cell proliferation by RUNX3 gene may not be sufficient.The mechanism of action of invasion and migration is realized by EMT.RUNX3 can be used as a biomarker for prognostic monitoring of CCRCC and may become a new therapeutic target for this cancer.