TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro

Abstract

Purpose: To evaluate the expression of tripartite motif-containing 33 (TRIM33) in ccRCC tissues and explore the biological effect of TRIM33 on the progress of ccRCC.

Method: The Cancer Genome Atlas (TCGA) database was used to examine the mRNA expression levels of TRIM33 in ccRCC tissues and its clinical relevance. Immunohistochemistry (IHC) was performed to evaluate its expression in ccRCC tissues obtained from our hospital. The correlation between TRIM33 expression and clinicopathological features of the patients was also investigated. The effects of TRIM33 on the proliferation of ccRCC cells were examined using the CCK-8 and colony formation assays. The effects of TRIM33 on the migration and invasion of ccRCC cells were explored through wound healing and transwell assays, along with the use of Wnt signaling pathway agonists in rescue experiments. Western blotting was used to explore the potential mechanism of TRIM33 in renal cancer cells. A xenograft model was used to explore the effect of TRIM33 on tumor growth.

Result: Bioinformatics analysis showed that TRIM33 mRNA expression in ccRCC tissues was downregulated, and low TRIM33 expression was related to poor prognosis in ccRCC patients. In agreement with this, low TRIM33 expression was detected in human ccRCC tissues. TRIM33 expression levels were correlated with clinical characteristics, including tumor size and Furman's grade. Furthermore, TRIM33 overexpression inhibited proliferation, migration, and invasion of 786-O and ACHN cell lines. The rescue experiment showed that the originally inhibited migration and invasion capabilities were restored. TRIM33 overexpression reduced the expression levels of β-catenin, cyclin D1, and c-myc, and inhibited tumor growth in ccRCC cells in vivo.

Conclusion: TRIM33 exhibits an abnormally low expression in human ccRCC tissues. TRIM33 may serve as a potential therapeutic target and prognostic marker for ccRCC.

Figure 1

TRIM33 is downregulated in various tumors and is related to the progress of ccRCC. (a and b) TRIM33 expression in pan-cancer; red represents tumor tissue and blue represents normal tissue. (c) Analysis of differences in TRIM33 mRNA levels between ccRCC tissues and normal tissues through the UALCAN website based on TCGA. (d–i) The difference in expression of TRIM33 according to individual cancer stages, patient's gender, patient's age, tumor grade, KIRC subtypes, and nodal metastasis status. (j) Relationship between TRIM33 expression and survival of renal cell carcinoma patients. (k) KM plot depicting the association of TRIM33 expression levels and tumor grade with patient survival. (l) KM plot depicting the association of TRIM33 expression levels and gender with patient survival. (m and n) The relationship between the expression of TRIM33 in ccRCC and the survival of patients through the HPA website. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 2

The expression of TRIM33 in kidney cancer tissues and normal tissues, and its relationship with the prognosis. (a) The expression of TRIM33 in kidney cancer tissues and normal tissues through the ccRCC data of the TCGA database; blue represents normal tissue and red represents tumor tissue, P = 7.708e‐19. (b and c) The relationship between OS, DFS, and TRIM33 expression in renal cell carcinoma patients. The cutoff value is 75%. (d and e) Immunohistochemical pictures of three typical kidney cancer tissues and normal tissues, and their H score. ^∗P < 0.05. (f) Analysis of immunohistochemistry results of 80 cases of kidney cancer tissues and normal tissues. P < 0.0001. (g) The survival curve is drawn according to TRIM33 expression. P = 0.0233.

Figure 3

The expression of TRIM33 in HK-2, 786-O, A498, and ACHN cell lines and verification of the overexpression effect. (a) Quantitative real-time PCR was used to detect the expression of TRIM33 in HK-2, 786-O, A498, and ACHN cell lines. (b and c) Western blot analysis of the expression of TRIM33 in HK-2, 786-O, A498, and ACHN cell lines and data analysis. (d) Quantitative real-time PCR was used to verify TRIM33 overexpression in two kidney cancer cell lines. (e and f) Western blot analysis confirming TRIM33 overexpression in two kidney cancer cell lines and data analysis. ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 4

Cell proliferation experiment in vitro. (a and b) CCK-8 assay. The OD values of the OV-TRIM33 group and the OV-NC group in 786-O cells and ACHN cells were measured by the CCK-8 assay, and the relative growth rate of each group was evaluated. (c and d) Colony formation assay. Image-Pro Plus software was used to calculate the number of colonies formed. ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 5

TRIM33 overexpression can inhibit migration, invasion, and EMT in ccRCC; here, the potential molecular mechanisms are explored. (a and b) Wound healing assay. (c–e) Transwell assay, including migration and invasion experiments. (f–h) Western blot assay for TRIM33, E-cadherin, N-cadherin, vimentin, β-catenin, cyclin D1, c-myc, and β-actin. (i–k) Western blot assay after incubation with a Wnt signaling pathway agonist for β-catenin, cyclin D1, c-myc, and β-actin. ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001; ns represents no statistical significance.

Figure 6

TRIM33 overexpression can inhibit tumor growth in vivo. (a) Pictures of nude mice and tumors. (b) Scatter plot of tumor weight between the OV-TRIM33 group and the OV-NC group. P < 0.0001. (c) Tumor volume measured every 3 days after the second week. (d, e) Immunohistochemistry staining for TRIM33, E-cadherin, N-cadherin, and vimentin. A histogram is used to display semiquantitative analysis results. ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.

Figure 7

Schematic diagram of TRIM33 affecting renal cancer cell proliferation and EMT through the Wnt/β-catenin pathway (requires further experimental proof).