MiR-532-3p suppresses cell viability, migration and invasion of clear cell renal cell carcinoma through targeting TROAP

Abstract

Clear cell renal cell carcinoma (ccRCC) is a subtype of renal cell cancer with the highest mortality, infiltration, and metastasis rate, threatening human health. Despite oncogenic role of TROAP in various cancers, its function in ccRCC remains to be unraveled. The differentially expressed mRNAs (DEmRNAs) and miRNAs (DEmiRNAs) were obtained by analyzing the related data sets of ccRCC in TCGA. The expression levels of mRNAs and miRNAs in the cell were detected by qRT-PCR, while the protein levels were characterized by western blot. The viability, migratory and invasive abilities of ccRCC cells were determined by MTT, wound healing and cell invasion assays. The combination of miRNA target site prediction and dual-luciferase reporter gene assay verified the binding relationship between miR-532-3p and TROAP. Research on ccRCC displayed that TROAP expression was upregulated, while miR-532-3p was down-regulated. Besides, upregulation of TROAP could accelerate viability, migratory and invasive potentials of ccRCC cells. On the contrary, miR-532-3p could downregulate TROAP level, but TROAP upregulation reversed the viability, migration, and invasion of ccRCC cells. MiR-532-3p could attenuate the viability, migration and invasion of ccRCC cells by targeting TROAP. This may generate novel insights into molecular therapeutic targets for ccRCC.

Keywords: Clear cell renal cell carcinoma; TROAP; cell viability; invasion; miR-532-3p; migration.

Figure 1.

The expression level of TROAP is up-regulated in ccRCC (a): Volcano map of DEmRNAs in ccRCC, the red dots represent up-regulated mRNAs, and the green dots represent down-regulated mRNAs; (b): The expression of TROAP in ccRCC; (c): Survival curves of TROAP expression on the prognosis of patients; (d-h): Box plots of TROAP expression in different G, stage, T, M, and N stages of ccRCC, respectively; (i): The results of qRT-PCR detection of mRNA expression of TROAP in human renal tubular epithelial cell line HKC and three ccRCC cell lines including A498; (j): The results of western blot of protein expression of TROAP in human renal tubular epithelial cell line HKC and three ccRCC cell lines including A498; * p<0.05

Figure 2.

TROAP promotes cell viability, migration and invasion of ccRCC (a): The results of qRT-PCR detection of TROAP mRNA expression in cells after transfected with oe-TROAP; (b): The effect of overexpressed TROAP on cell viability was detected by MTT assay; (c): The effect of overexpression of TROAP on cell migration was assessed through wound healing assay (40×); (d): The effect of overexpressed TROAP on cell invasion was measured by cell invasion assay (100×); * p<0.05

Figure 3.

MiR-532-3p can target and suppress the expression of TROAP (a): Volcano map of DEmiRNAs in ccRCC dataset, the red dots represent up-regulated miRNAs, and the green dots represent down-regulated miRNAs; (b): Venn diagram of the predicted upstream target miRNAs of TROAP and down-regulated DEmiRNAs in TCGA database; (c): Pearson correlation analysis of miRNAs (miR-532-3p; miR-138-5p) and TROAP expression in ccRCC; (d): Expression of miR-532-3p in ccRCC; (e): The results of qRT-PCR of miR-532-3p expression in normal tissue cell line and cancer cell lines such as A498; (f): The efficiency of overexpression of miR-532-3p in cancer cells was measured through qRT-PCR; (g): The results of qRT-PCR of TROAP mRNA expression level with overexpressed miR-532-3p in cancer cells; (h): The results of western blot of TROAP protein level with overexpressed miR-532-3p in cancer cells; (i): The binding sites of miR-532-3p and TROAP, and verification results of dual-luciferase reporter gene assay of targeted binding relationship; * p < 0.05

Figure 4.

MiR-532-3p inhibits cell viability, migration and invasion of ccRCC by targeting TROAP (a): The results of qRT-PCR of TROAP mRNA expression in cells after various treatments; (b): The results of western blot of TROAP protein expression in cells after various treatments; (c): Changes in cell proliferative ability after various treatments were measured by MTT assay; (d): Cell migratory ability after various treatments was detected via wound healing assay (×40); (e): Cell invasion ability after various treatments was assessed through cell invasion assay (×100); * p < 0.05