MicroRNA-142-5p promotes cell growth and migration in renal cell carcinoma by targeting BTG3

Abstract

Purpose: Some microRNA (miRNA) levels have been found to be dysregulated in cancer patients, suggesting the potential usefulness of miRNAs in cancer therapies. The purpose of this study was to investigate the expression of miR-142-5p in human renal cell carcinoma (RCC) and its potential role in tumor growth and metastasis.

Methods: The expression level of miR-142-5p in human RCC tissue and cell lines was determined by quantitative reverse transcription polymerase chain reaction analysis. MTT, colony formation, Transwell, and cell cycle assays were performed to explore the potential functions of miR-142-5p in human RCC cells. The potential target gene of miR-142-5p was identified and confirmed via luciferase reporter assays.

Results: miR-142-5p expression was elevated in RCC tissues and cell lines. Overexpression of miR-142-5p significantly promoted cell proliferation and colony formation and could prevent G1 phase arrest among RCC 786-O cells. Meanwhile, the migration potential of 786-O cells was greater than that of control cells. BTG3 was identified as a direct target of miR-142-5p, and re-expression of BTG3 reversed the miR-142-5p-induced cell proliferation.

Conclusion: miR-142-5p promoted the proliferation and migration of RCC cells by targeting BTG3. With this potential onco-miRNA role in the progression of RCC, miR-142-5p may be a therapeutic target for the treatment of RCC.

Keywords: BTG3; miR-142-5p; proliferation; renal cell carcinoma.

Figure 1

miR-142-5p was commonly upregulated in RCC specimens and cell lines. A. Relative expression of miR-142-5p in 30 paired clinical RCC specimens and adjacent normal tissues as measured by qRT-PCR. B. Expression level of miR-142-5p in 3 RCC cell lines (786-O, A498, and Caki-1) and one normal renal cell line (HK-2) as analyzed by qRT-PCR. U6 served as an internal reference. All data are expressed as mean ± SD for three independent experiments. *P<0.05.

Figure 2

miR-142-5p promotes proliferation of RCC cells. A. miR-142-5p expression by qRT-PCR after transfecting of 786-O cells with miR-142-5p or negative control (NC). B. Cell proliferation of 786-O cells by MTT assay after transfection with different concentrations of miR-142-5p or NC for 72 h. C. Growth curves for 786-O cells after 48 h of transfection with miR-142-5p or NC. D. Numbers of colony in miR-142-5p- and NC-transfected groups. E. Cell cycle distribution based on flow cytometry 48 h after transfection of 786-O cells with miR-142-5p or NC. All data are expressed as mean ± SD for three independent experiments. *P<0.05.

Figure 3

miR-142-5p enhances the migratory ability of RCC cells. A. Representative images (at 100× magnification) of crystal violet-stained migrating 786-O cells after transfection with miR-142-5p or NC. B. Quantification of 786-O cell migration. The data represent mean ± SD. *P<0.05.

Figure 4

miR-142-5p inhibits BTG3 expression in 786-O cells by directly targeting its 3’-UTR. A. Binding sites for miR-142-5p in the 3’-UTR of BTG3 mRNA. B. Luciferase reporter assay showing reduced luciferase reporter activity in 786-O cells containing the BTG3 WT 3’-UTR fragment. C. qRT-PCR analysis of BTG3 mRNA expression in 786-O cells transfected with miR-142-5p or NC. D. Western blot analysis of BTG3 protein expression in 786-O cells transfected with miR-142-5p or NC. The data represent mean ± SD. *P<0.05.

Figure 5

BTG3 reverses miR-142-5p-induced cell proliferation. A. BTG3 protein expression by Western blot analysis. B. Effect of BTG3 on 786-O cell proliferation. C. Growth curve showing that re-expression of BTG3 reversed the miR-142-5p-induced cell proliferation. The data represent mean ± SD. *P<0.05.