HIF2α-induced upregulation of RNASET2 promotes triglyceride synthesis and enhances cell migration in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC), the most common malignant subtype of renal cell carcinoma, is characterized by the accumulation of lipid droplets in the cytoplasm. RNASET2 is a protein coding gene with a low expression level in ovarian cancers, but it is overexpressed in poorly differentiated neuroendocrine carcinomas. There is a correlation between RNASET2 upregulation and triglyceride expression levels in human serum but is unknown whether such an association is a factor contributing to lipid accumulation in ccRCC. Herein, we show that RNASET2 expression levels in ccRCC tissues and cell lines are significantly higher than those in both normal adjacent tissues and renal tubular epithelial cells. Furthermore, its upregulation is associated with increases in ccRCC malignancy and declines in patient survival. We also show that an association exists between increases in both cytoplasmic lipid accumulation and HIF-2α transcription factor upregulation, and increases in both RNASET2 and triglyceride expression levels in ccRCC tissues. In addition, DGAT1 and DGAT2, two key enzymes involved in triglyceride synthesis, are highly expressed in ccRCC tissues. By contrast, RNASET2 knockdown inhibited their expression levels and lowered lipid droplet accumulation, as well as suppressing in vitro cell proliferation, cell invasion, and migration. In conclusion, our data suggest HIF2α upregulates RNASET2 transcription in ccRCC cells, which promotes both the synthesis of triglycerides and ccRCC migration. As such, RNASET2 may have the potential as a biomarker or target for the diagnosis and treatment of ccRCC.

Keywords: HIF2α; RNASET2; cancer stages; clear cell renal cell carcinoma (ccRCC); triglycerides synthesis.

Fig. 1

RNASET2 expression is higher in clear cell renal cell carcinoma. (A) CCLE database shows that RNASET2 mRNA is overexpressed in kidney cancer cell lines; (B) RNASET2 expression is significantly higher in ccRCC tissues compared with that in normal kidney tissues; (C) RNASET2 expression in different stages of ccRCC; (D) Overall survival of ccRCC patients with different expression levels of RNASET2 (normalized by ACTB), red line: high expression, blue line: low expression; (E) disease‐free survival (DFS) of RNASET2 expression in patients using TCGA datasets (normalized by ACTB).

Fig. 2

RNASET2 expression in ccRCC exceeds that in ANT. (A) The relative mRNA expression level of RNASET2, n = 28; (B) The relative protein expression level of RNASET2 (upper, the results of western blot, N1 ~ N6: adjacent normal tissues, T1 ~ T6: ccRCC tissues; lower, analyses of western blot by gray value), n = 28; (C) Hematoxylin–eosin (H&E) staining and immunohistochemistry (IHC) of RNASET2 in ccRCC and ANT, scale bars: 50 μm; (D) IHC chip of RNASET2 in ccRCC and ANT, scale bars: 50 μm; (E) The relative intensity in the IHC chip of RNASET2 expression level in different ccRCC grades, Number of different grades of cases: n _{Grade 1} = 5, n _{Grade 2} = 37, n _{Grade 3} = 25, n _{Grade 4} = 7; (F) The relative intensity in the IHC chip of RNASET2 expression level in different ccRCC T stages, Number of different stages of cases: n _{stage 1} = 35, n _{stage 2} = 25, n _{stage 3} = 14. Values in bar graphs are the mean with SD. Statistical analysis was performed using the Student's t‐test (A and B) and one‐way ANOVA (E and F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Fig. 3

High expressions of DGAT1 and DGAT2 and low expression of RIP140 in ccRCC accompanied lipid droplet accumulation. (A) Oil Red O staining monitored cytoplasmic lipid droplet accumulation in the ccRCC, scale bars, 50 μm; (B) TEM evaluation of cytoplasmic lipid droplets that accumulated in the ccRCC, M: mitochondria; N: nucleus; LD: lipid droplet; scale bars (from left to right): 5 μm, 1 μm, and 500 nm; (C) IHC analysis compared DGAT1, DGAT2, and RIP140 expression levels in ccRCC with their corresponding values in ANT, scale bars: 50 μm; (D) Analysis of the overall survival curves of DGAT1, DGAT2, and RIP140 expressions base on the TCGA database (normalized by endoplasmic reticulum marker CANX for both DGAT1 and DGAT2, ACTB for RIP140), red line: high expression; blue line: low expression.

Fig. 4

RNASET2 expression influenced the expressions of lipids‐associated genes, cell proliferation, and cell migration. (A) mRNA expression level of RNASET2 in HK‐2, 769‐P, and 786‐O cells, respectively, n = 3; (B, C) RNASET2 was knocked down in ccRCC cells by RNASET2 shRNA, n = 3; (D) RNASET2 knockdown in ccRCC cells decreased both DGAT1 and DGAT2 expressions, n = 6; (E) Oil Red O staining, scale bars: 50 μm; (F) Assessment of lipi‐red staining, scale bars: 100 μm; (G) Real‐time cell analysis (RTCA) of cell proliferation showed that it was inhibited after RNASET2 knockdown in ccRCC cells; (H) Transwell assay to evaluate cell invasion after RNASET2 knockdown in ccRCC cells, scale bars: 100 μm, n = 6; (I) The scratch wound healing test showed that RNASET2 deficiency inhibited the migration of ccRCC cells, scale bars: 100 μm, n = 6. Values in bar graphs are the mean with SD. Statistical analysis was performed using the Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

HIF2α directly interacted with RNASET2 in ccRCC cells. (A) TCGA database analysis shows that there is a negative correlation between RNASET2 and VHL expression levels, P = 9.9e‐05, R = −0.16; (B) Following exposure to CoCl₂ for 24 h, both HIF2α and RNASET2 proteins were highly upregulated in 786‐O and 769‐P cells with HIF1α deficiency; (C) The RNASET2 mRNA expression level increased significantly in 786‐O and 769‐P cells after exposure to 150 μm CoCl₂ for 24 h (reference gene: ACTB), n = 6; (D) Sequence of ChIP‐PCR amplicon corresponding to RNASET2 gene structure (green fragments: exons); (E, F) Fragments pulldown with HIF2α antibody by chromatin immunoprecipitation (ChIP) was analyzed by regular PCR and quantitative real‐time PCR, n = 3; (G) RNASET2 transcription in 786‐O and 769‐P cells after exposure to different doses of PT2385 for 24 h (control: DMSO, reference gene: ACTB), n = 6; (H) VEGF transcription in 786‐O and 769‐P cells after exposure to different doses of PT2385 for 24 h (control: DMSO, reference gene: ACTB), n = 6; (I) A schematic representation showing the regulatory mechanism of RNASET2 transcription in ccRCC. Values in bar graphs are the mean with SD. Statistical analysis was performed using the Student's t‐test (C and F) and one‐way ANOVA (G and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.