Expression And Clinical Significance Of TRIM13 In Renal Clear Cell Carcinoma And Molecular Mechanism Of Inhibiting Migration And Invasion Of 786-O Cells

Background and objectives:Kidney cancer is common in China and the world.Among the vast majority of adults,the most common type is renal cell carcinoma(RCC),which is the main cause of all types of urinary tumors.In all RCC types,the clear cell human renal carcinoma(ccRCC)is clinically usually public.At present,for early patients with clear cell renal cell carcinoma,surgery is generally selected for complete resection,but for metastatic clear cell renal cell carcinoma,whether or not surgery will change its poor prognosis,the 5-year survival rate of patients is extremely low.The main reason may be unfamiliar with the the progress of ccRCC and its pathogenesis.TRIM protein is a general term for a class of structurally conserved proteins containing triplet motifs.They play important functions in many aspects,such as malignant tumor,cell death,and antiviral immunity.TRIM 13 studied in this subject is a member of the TRIM family.Abnormal changes in its expression level will affect the occurrence and development of tumors,but little research has been done in renal cancer.This paper we focus on exploring TRIM 13 protein expression in human kidney cancer tissue and its possible relationship with clinicopathological factors and prognosis,and to study TRIM13 involved in the development of human ccRCC,thus hoped that early diagnosis,prognostic analysis and targeted therapy of renal cancer can bring new ideas and strategies.Methods:1.The levels of TRIM 13 protein in differentiated tissues of ccRCC were detected by immunohistochemical staining.The relationship between the TRIM 13 protein expression of human ccRCC and the corresponding pathological clinical indicators was analized.Western blot analysis was done to measure TRIM 13 protein level(human ccRCC and non-ccRCC tissues).The relationship between TRIM13 protein expression level and ccRCC prognosis through survival analysis(Kaplan Meier method).2.Real-time PCR analysis was used in TRIM13 mRNA expression in renal cancer cell lines.The ccRCC 786-O cell line overexpressing TRIM 13 was used to verify TRIM 13 protein expression and related signaling pathways in the control group and overexpression group after transfection,while comparing cell migration and invasion levels.CCK8 was to examine ratio of cell proliferation following by TRIM13 overexpressing 786-O cells,and flow cytometry was used to detect cell cycle simultaneously.3.The tissue sections of transplanted tumor in nude mice were analyzed by conventional HE staining and Ki67 immunohistochemical staining.Results:1.By immunohistochemical staining analysis of TRIM 13 expression in renal cancer tissues with different degrees of differentiation,we found that TRIM 13 protein existed in the ccRCC cell’s cytoplasm,and the expression in positive ratio was lower in ccRCC tissues that differentiated as poorly significantly.In medium and high-differentiated kidney cancer tissues,the difference is statistically different(P<0.05).Correlation analysis of TRIM 13 expression in the tissues of 87 patients with human ccRCC combined with corresponding pathological indicators,we found TRIM 13 expression was associated with ccRCC tissues grade(P=0.011)and metastases(P=0.001)significantly,but at the same time,it has no correlation with the other pathological indicators,such as the age of the patients,male or female,and ccRCC tumor size.Western blot analysis was done to measure TRIM13 protein level in ccRCC and the adjacent non-ccRCC tissues.The experiment results suggested that TRIM 13 protein was under expressed in human ccRCC tissues significantly.Kaplan Meier survival analysis of 87 patients with ccRCC was to investigate whether differential TRIM 13 expression was correlated with the ccRCC patients’ prognosis.After statistical analysis,we found that the prognosis of ccRCC patients in group of the low TRIM 13 protein expression was worse than those in group of the high TRIM 13 protein expression significantly(P<0.01).2.The TRIM13 mRNA level in ccRCC cell lines was by real-time quantitative PCR.The results showed TRIM 13 mRNA expression level was generally under expressed in ccRCC cell lines cell lines.When TRIM13 overexpressing in 786-O,the protein expressions of TRIM13 and related signaling pathways in the control group and overexpression group were detected after Western blot transfection,and the levels of cell migration and invasion were compared.The results showed that after over-expression of TRIM13 in 786-O cells,the protein expression levels of AKT/NF-kB signaling pathway-related proteins p-AKT,NFkB and MMP-9 were significantly reduced(P<0.05).Compared to the ctrl group,the 786O cells migration and invasiveness were all suppressed significantly in the TRIM 13 overexpression group(P<0.05).We tested cell viability by the CCK8 method,and the results pointed group of TRIM 13 overexpression was suppressed significantly,compared to the ctrl group(P<0.05).Simultaneous cell cycle analysis showed G1 phase capture 786-O cells occurred in the over-expressing TRIM 13 group,compared to the ctrl(the number of cells increased significantly during this phase).While S phase,that is cell synthesis phase,cell counts were reduced in the phase significantly(P<0.05).3.The experiment vivo,the HE conventional staining and Ki67 immunohistochemical staining were used to analyze the transplanted tumor tissue sections.HE staining showed that the nuclei of the two groups of renal cancer cells were large and deeply stained.Compared with the empty control group,the positive rate of Ki67 in TRIM 13 overexpressed group was significantly reduced.Conclusions:1.The TRIM 13 protein expression in ccRCC is significantly lower,which is significantly associated with ccRCC grade and metastasis.2.The TRIM13 over expression inhibited migration and invasion of the 786-O cell line significantly.TRIM 13 may participated in course of ccRCC metastasis or invasion.3.TRIM13 can reduce the expression of p-AKT/NF-kB/MMP-9.The AKT/NFkB/MMP-9 pathway may participate in TRIM 13-mediated cancer cell migration and/or invasion.4.Further research on the TRIM13’s tumor suppressive function and mechanism will allow us to deeply understand the occurrence and development of RCC and provide a new strategy for the diagnosis or treatment of RCC.