Sohlh2 Inhibits the Malignant Progression of Renal Cell Carcinoma by Upregulating Klotho via DNMT3a

Abstract

Background: Renal cell carcinoma is the most common malignant tumor of the kidney. The 5-year survival of renal cell carcinoma with distant metastasis is very low. Sohlh2 is a newly discovered tumor suppressor gene playing inhibitory roles in a variety of tumors, but its role in renal cell carcinoma has not been reported.

Methods: To clarify the role of Sohlh2 in the occurrence and development of renal cell carcinoma, we constructed stably transfected human renal cell carcinoma cell lines with Sohlh2 overexpression and Sohlh2 knockdown, separately. First, we studied the effects of Sohlh2 on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of renal cell carcinoma cells in vitro and in vivo. Then, we detected whether Sohlh2 functions through DNMT3a/Klotho using Western blotting, qPCR, and Cell Counting Kit-8 (CCK-8) assay. Finally, we collected 40 resected renal cell carcinoma samples to study the relevance between Sohlh2, DNMT3a, and Klotho by immunohistochemistry.

Results: Our results showed that Sohlh2 was downregulated in renal cell carcinoma, and its expression level was negatively correlated with tumor staging. Both in vitro and in vivo experiments confirmed that Sohlh2 overexpression inhibited the proliferation, migration, invasion, metastasis, and EMT of renal cell carcinoma. Sohlh2 functions through demethylation of Klotho by downregulating the expression of DNA methyltransferase of DNMT3a. In renal cell carcinoma, Sohlh2 was positively correlated with Klotho and negatively correlated with DNMT3a.

Conclusion: Sohlh2 functions as a tumor suppressor gene in renal cell carcinoma by demethylation of Klotho via DNMT3a. Sohlh2 correlated with Klotho positively and with DNMT3a negatively in renal cell carcinoma. Our study suggests that Sohlh2 and DNMT3a/Klotho can be used as potential targets for the clinical treatment of renal cell carcinoma.

Keywords: DNMT3a; Klotho (KL); Sohlh2; malignant progression; renal cell carcinoma (RCC).

Figure 1

Sohlh2 is downregulated in RCC. (A) The results of immunohistochemical staining of Sohlh2 in adjacent non-cancerous tissue and grade 1, grade 2, and grade 3 RCC tissues labeled as Con, I, II, and III, respectively. (B) The statistical analysis of immunohistochemical staining of Sohlh2. (C) The correlation between Sohlh2 and the clinical characteristics of RCC patients. RCC, renal cell carcinoma. It is statistically significant (***P < 0.0001).

Figure 2

Sohlh2 repressed proliferation of RCC cells in vitro. (A) The Western blotting results of Sohlh2 expression in 3 kinds of RCC cell lines (left) and the establishment of Sohlh2 overexpression and knockdown cell lines. (B) The result of the Sohlh2 overexpression group by colony formation. (C) The results of Sohlh2 knockdown groups. (D) CCK-8 assays were performed to analyze the proliferative difference between Sohlh2 overexpression and Sohlh2 knockdown groups. Statistical analysis was performed by GraphPad Prism 5. RCC, renal cell carcinoma; CCK-8, Cell Counting Kit-8. (*P < 0.05; **P < 0.001; ***P < 0.0001).

Figure 3

Sohlh2 represses capacities of the EMT, migration, and invasion in RCC cells. (A, B) The results of expression of epithelial and mesenchymal cell markers by qPCR and Western blotting. (C, D) The results of the wounding healing assay in the Sohlh2 knockdown group and Sohlh2 overexpression group. (E) The results of the transwell migration assay and matrigel invasion assay in the Sohlh2 overexpression group. (F) The passing through a number of RCC cells in the Sohlh2 knockdown group. EMT, epithelial–mesenchymal transition; RCC, renal cell carcinoma. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 4

Sohlh2 inhibited RCC growth and metastasis in vivo. (A) Pictures of mice and tumors from the control and Sohlh2 overexpression ACHN groups. n = 6. (B) The quantitative analysis of tumor volume (above) and weight (below) in the control and Sohlh2 overexpression ACHN xenografts. (C) The Western blotting results of the effects of Sohlh2 on RCC EMT and proliferation. (D) The qPCR results of the effects of Sohlh2 on RCC EMT. (E, F) The representative immunohistochemical staining results of the effects of Sohlh2 on RCC proliferation and EMT. (G) The pictures of mice, lungs, and livers from the control and Sohlh2 overexpression ACHN groups (left) and statistical analysis of metastases in the lung and liver (right). n = 5. (H) The H&E staining results of tumor metastases in the lungs (above) and livers (below). (I) The immunohistochemical staining results of the effects of Sohlh2 on Klotho and DNMT3a expression levels. (J, K) The Western blotting and qPCR results of the effects of Sohlh2 on Klotho and DNMT3a expression levels. RCC, renal cell carcinoma; EMT, epithelial–mesenchymal transition. (*P < 0.05; **P < 0.001; ***P < 0.0001).

Figure 5

Sohlh2 suppressed RCC through DNMT3a and Klotho. (A, B) The qPCR and Western blotting results of the effect of Sohlh2 on Klotho and DNMT3a expression level in RCC cell lines. (C–E) The qPCR and Western blotting results of the effects of DNMT3a on Sohlh2 and Klotho expression level in RCC cell lines by overexpression of DNMT3a and adding DNMT3a inhibitor 5-Aza. (F) The CCK-8 results of Klotho on the effects of Sohlh2 on the proliferation of RCC cell lines. (G) The role of Klotho on the effects of Sohlh2 on the migration and invasion of RCC cell lines by transwell migration and matrigel invasion assay. (H) The qPCR and Western blotting results of the role of Klotho on the Sohlh2 expression level. RCC, renal cell carcinoma; CCK-8, Cell Counting Kit-8. NS, no significance. (*P < 0.05; **P < 0.001; ***P < 0.0001).

Figure 6

Correlation analysis of Sohlh2, Klotho, and DNMT3a in human RCC tissues. (A) Immunohistochemical staining results of Sohlh2, DNMT3a, and Klotho in human RCC samples. (B) Statistical analysis results of panel (A) RCC, renal cell carcinoma. (***P < 0.0001, which is statistically significant).

Figure 7

A possible signaling pathway for Sohlh2 to function as a tumor suppressor in renal cancer cells. At present, targeted therapeutic molecules have been developed for certain signaling pathways such as PT 2385 and PT 2399. PT 2385 and PT 2399 are small molecule inhibitors of HIF-2, which can selectively interfere with the heterodimerization of HIF-2α and HIF-1β. Clinical data show that these small molecules inhibitors could effectively block the proliferation and tumor angiogenesis of RCC cells (49). The signaling pathways are very perplexing in the tumor microenvironment; lncRNA, miRNA, and many other signaling pathways are also involved in the occurrence and development of RCC cells (–53). We hope that our findings could provide new therapeutic targets and new view sights for the therapy of RCC. RCC, renal cell carcinoma.