Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, MEM medium, Trypsin-EDTA, and phosphate-buffered saline (PBS) buffer were purchased from M&C Gene Technology (Beijing) Ltd. TRIzol was obtained from Invitrogen (Carlsbad, CA). A reverse transcription kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Sohlh2 and Klotho antibodies were purchased from Abcam Inc. (Cambridge, MA). Ki67, DNMT3a, E-cadherin, ZO-1, N-cadherin, GAPDH, and Actin antibodies were bought from Affinity Biosciences Ltd.

The human RCC cell lines (ACHN, A498, 786-O) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The tissue microarray of RCC was purchased from Shanghai Xinchao Biotechnology Co., Ltd.

RCC cells were cultured in a 6-well plate (1 × 10³ cells/well) for 2 weeks. Culture medium change is not necessary for the first 3 days to prevent cells’ detachment from the culture plate. After that, the medium was changed every 3 days. Colonies (≥50 cells/colony) were visualized and counted after they were fixed with methanol for 30 min and stained with crystal violet for 20 min.

Cells measuring 2 × 10³ per well in 100 μl of culture medium were incubated in 96-well plates with PBS buffer in the neighboring wells to keep the humidity. Cell Counting Kit-8 (CCK-8) solution measuring 10 μl was added to each well at an appropriate length of time (24, 48, 72, 96, and 120 h), and the absorbance was measured at 450 nm using a microplate reader.

Cells were seeded in a 6-well plate at a density of 6 × 10⁴ cells/well. When the cells reached ~95% confluence, a 100-μl pipette tip was used to make a straight scratch, simulating a wound, and the cell debris were washed away by PBS buffer two times. Pictures were taken at 0, 24, or 48 h after the scratch, and the horizontal wound widths were measured using ImageJ software.

Cell migration was performed by using a chamber with a pore size of 8 mm. In a 24-well plate, 500 μl of culture medium containing 20% bovine serum albumin was added, and the chamber was placed on top of the medium. In a chamber, 200 μl of serum-free cell suspension (5 × 10⁴ cells/well) was added, and it was cultured in a 37°C cell incubator for 6–8 h. The number of migrating cells was counted according to the cells stained with crystal violet under the microscope. For the invasion assay, a layer of matrigel (BD Biosciences) was laid at the bottom of the chamber and dried for 30 min in a 37°C incubator. The other steps are the same as the migration assay.

Six-week-old male BALB/c nude mice were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Every nude mouse was injected subcutaneously with 2 × 10⁶ stable Sohlh2 overexpression or Sohlh2 knockdown ACHN cells. After 8 weeks (the tumor volume ≤1,000 mm³), mice were euthanized, and the subcutaneous tumors were removed, photographed, and weighed.

Six-week-old male BALB/c nude mice were randomly divided into two groups. Cells measuring 5 × 10⁵ were injected via tail veins. Four weeks later, mice were euthanized, the livers and lungs were taken out, and the number of metastatic nodules was counted.

The tissues were fixed with 4% paraformaldehyde for 2 days and embedded with paraffin. The thickness of the tissue section was 4 μm. Sections were immunostained with an appropriate proportion of anti-Sohlh2 or anti-Klotho, etc. The images were captured under the microscope (upper, middle, lower, left, and right field). Image pro-plus and SPSS analysis software were used for statistical analysis.

The procedure before staining is the same as that of immunohistochemistry. The nucleus was stained with hematoxylin for 3 min. The cytoplasm was stained with eosin for 2 min. The acquisition of images was performed according to the Olympus computerized image system.

Total RNA was isolated from the RCC cells or tumor tissues using TRIzol reagent (Invitrogen). The cDNA was generated with a reverse transcription kit (Thermo Fisher Scientific Inc.). Real-time PCR was performed in 20 μl of SYBR Green PCR Master Mix. The specific primers are listed at the end of this article ( Table 1 ).

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beijing Solarbio Science & Technology Co., Ltd). The target protein was isolated from 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel and transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore Corp, Billerica, MA). The membrane was detected with a specific antibody at 4°C overnight. The next morning, the PVDF membrane was incubated with anti-rabbit or anti-mouse IgG antibodies for 1.5 h at 20°C–30°C. The expression of the target protein was detected by a chemiluminescence kit (Thermo Fisher Scientific Inc.). GAPDH or Actin was used as internal reference proteins.

All data analyses were processed with GraphPad Prism 5, ImageJ, Image pro-plus, SPSS, Photoshop, and other related software. The difference between different treatment groups was tested by a small-sample t-test. It is considered statistically significant when p < 0.05.

Sohlh2 expression was detected in 85 cases of RCC samples, including 5 cases of adjacent non-cancerous tissues, 6 cases of grade 1 RCC tissues, 66 cases of grade 2 RCC tissues, and 8 cases of grade 3 RCC tissues. The results of the immunohistochemical staining showed that Sohlh2 expression was negatively correlated with the grading of RCC (p < 0.0001; Figures 1A, B ). We continued to analyze the relationship between Sohlh2 and the clinical characteristics of RCC patients. The results indicated there was no significant difference between Sohlh2 expression and age or sex (p < 0.01; Figure 1C ).

In order to detect the function of Sohlh2 in the proliferation of RCC cells in vitro, colony formation assay and CCK-8 assay were performed. First, we detected the expression of Sohlh2 in 3 RCC cell lines by Western blotting. The results showed that the Sohlh2 expression level in ACHN and 786-O cell lines was lower than that in the A498 cell line. Then, ACHN and 786-O were used to establish Sohlh2 overexpression cell lines, and A498 was used to establish the Sohlh2 knockdown cell line ( Figure 2A ). The result of colony formation showed that Sohlh2 overexpression inhibited the proliferation of ACHN and 786-O cells (p < 0.0001; Figure 2B ). Conversely, Sohlh2 knockdown promoted the proliferation of A498 cells (p < 0.0001; Figure 2C ). The above results were further confirmed by the CCK-8 assay (p < 0.05; Figure 2D ).

To detect the regulation of Sohlh2 on epithelial–mesenchymal transition (EMT) of RCC cells, qPCR and Western blotting were performed. The result of qPCR showed that Sohlh2 overexpression upregulated the expression of epithelial cell markers E-cadherin, ZO-1, and Claudin and downregulated the expression of mesenchymal cell markers ZEB1, Fibronectin, and Vimentin. Sohlh2 knockdown had the opposite effects (p < 0.05; Figure 3A ). The results of Western blotting were consistent with the qPCR results (p < 0.05; Figure 3B ). We then detected the effect of Sohlh2 on the migration and invasion of RCC cells by wound healing assay, transwell migration assay, and matrigel invasion assay. The results of the wounding healing assay showed that Sohlh2 overexpression slowed down the closure of the wound compared with the control group, and Sohlh2 knockdown had the opposite effects (p < 0.05; Figures 3C, D ). We obtained similar results in the transwell migration assay and matrigel invasion assay. Sohlh2 overexpression decreased the passing through a number of RCC cells, while Sohlh2 knockdown had the opposite effects (p < 0.01; Figures 3E, F ).

After exploring the effect of Sohlh2 on RCC cells in vitro, we continued to detect the role of Sohlh2 in RCC through subcutaneous implantation and tail vein injection of RCC cells in nude mice to assess whether Sohlh2 affects tumor growth and metastasis in vivo. The results showed that Sohlh2 overexpression inhibited RCC tumor growth, while Sohlh2 knockdown had the opposite effects (p < 0.05; Figures 4A, B ). The expression of EMT and proliferation-related markers were also detected by qPCR and Western blotting. The results showed that Sohlh2 overexpression inhibited EMT and proliferation (p < 0.05; Figures 4C, D ). The results of immunohistochemical staining were consistent with the qPCR and Western blotting results (p < 0.05; Figures 4E, F ). The results of the metastasis assay showed that Sohlh2 overexpression decreased the number of metastases in the livers and lungs compared with the control group. H&E staining results also confirmed the above results (p < 0.001; Figures 4G, H ). Finally, we detected if Sohlh2 regulated RCC through DNMT3a/Klotho by qPCR, Western blotting, and immunohistochemical staining. The results showed that Sohlh2 overexpression promoted Klotho expression but inhibited DNMT3a expression (p < 0.05; Figures 4I–K ), suggesting that Sohlh2 may attenuate RCC malignancy through DNMT3a/Klotho.

To detect whether Sohlh2 suppressed RCC via DNMT3a/Klotho, we first used knockdown and overexpression of Sohlh2 RCC cell lines to detect the expression of Klotho and DNMT3a by qPCR and Western blotting. The results showed that Sohlh2 overexpression upregulated the expression of Klotho and downregulated the expression of DNMT3a in RCC cells, while Sohlh2 knockdown had the opposite effects (p < 0.01; Figures 5A, B ). Thereafter, the effect of DNMT3a overexpression on the expression of Sohlh2 and Klotho was detected, and the results showed that DNMT3a did not affect the Sohlh2 expression but downregulated the expression of Klotho. We then detected the effects of DNMT3a on the expression of Sohlh2 and Klotho by overexpression of DNMT3a in Sohlh2 overexpression RCC cell lines and by adding an inhibitor of DNMT3a in Sohlh2 knockdown RCC cell lines. The results showed that DNMT3a overexpression or DNMT3a inhibitor could not block the effect of Sohlh2 on Klotho expression (p < 0.05; Figures 5C–E ). We also detected if Klotho could affect the effects of Sohlh2 on the proliferation of RCC cell lines at 0 and 48 h by CCK-8. The results showed that Klotho could partially affect the effects of Sohlh2 on the proliferation of RCC cell lines (p < 0.001; Figure 5F ). The results of transwell migration and invasion assay showed that the number of traversed cells in Klotho knockdown plus Sohlh2 overexpression group is more than that of the Sohlh2 overexpression only group, and the number of traversed cells was less in the Klotho overexpression plus Sohlh2 knockdown RCC cell lines than that in the Sohlh2 knockdown only group, indicating that Klotho could mediate Sohlh2 inhibiting the migration and invasion of RCC cells (p < 0.05; Figure 5G ). In order to investigate if Klotho regulates Sohlh2 expression, we also detected the effects of Klotho overexpression and Klotho knockdown on the expression level of Sohlh2 by qPCR and Western blotting. The results showed that Klotho overexpression or knockdown did not regulate the expression of Sohlh2 ( Figure 5H ).

We finally investigated the correlation between Sohlh2, Klotho, and DNMT3a by immunohistochemical staining. The results showed that the expression of Sohlh2 correlated with Klotho positively and with DNMT3a positively in human RCC tissues ( Figures 6A, B , p < 0.001).