MicroRNA-497 suppresses renal cell carcinoma by targeting VEGFR-2 in ACHN cells

Abstract

Abnormal expression of miRNAs contributed to cancers through regulation of proliferation, apoptosis and drug resistance of cancer cells. The present study was designed to investigate the effect of miR-497 on renal cell carcinoma (RCC) and its possible mechanism. Forty paired clear cell RCC (ccRCC) tissues and adjacent normal kidney tissues were obtained from patients, who were not treated by chemotherapy or radiotherapy. RT-PCR was performed to detect expression of miR-497 in the ccRCC tissues. Effects of miR-497 on cell viability, apoptosis, migration and invasion were detected in ACHN cells. Western blotting (WB) was employed to detect the downstream targets of miR-497 We found that miR-497 in ccRCC tissues was decreased. We treated ACHN cells with miR-497 mimics and inhibitors in vitro and found that miR-497 inhibited viability, migration and invasion of ACHN cells. miR-497 promoted ACHN cells' apoptosis. VEGFR-2 was predicted as a possible target of miR-497 Luciferase reporter assay proved that miR-497 suppressed VEGFR-2 directly by binding to its 3'-UTR. Further studies showed that miR-497 influenced the MEK/ERK and p38 MAPK signalling pathways. Our findings demonstrated that miR-497 could suppress RCC by targeting VEGFR-2.

Keywords: MEK/ERK; VEGFR-2; ccRCC; miR-497; p38 MAPK.

Figure 1. Expression of miR-497 in vivo

Level of miR-497 that was detected in ccRCC tissues was significantly lower than the level of miR-497 detected in the corresponding adjacent normal kidney tissues (**, P<0.01).

Figure 2. miR-497 inhibits viability in ACHN cells

Cell viability was determined by CCK-8 assay after transfection. Data represent mean ± S.E.M. from three independent experiments (**, P<0.01).

Figure 3. (A,C) Exogenous expression of miR-497 reduces the migration of ACHN cells. Photographs represented the cells travelled through the membrane by Transwell assay (×200 magnification). (B,D) Photographs represented the cells passing through the matrigel by Matrigel invasion assay (×200 magnification).

Data represent mean ± S.E.M. from three independent experiments (**, P<0.01).

Figure 4. The appearance of apoptosis characteristics in each treated group of ACHN cells

(A,B) Detection of apoptotic ACHN cells by TUNEL and DAPI staining assay. Nuclear TUNEL staining (green) is superimposed on the phase contrast image of the cells to show the contour of the cells. The mean percentage of TUNEL-positive cells (n≥3 batches of cells for each column) were counted from five fields for each group. Original magnification: ×200. Data represent mean ± S.E.M. (**, P<0.01). (C,D,E) Expression levels of Bax, Bcl-2 and caspase-3 proteins in transfected cells. Cell lysates were prepared and used for Western blot of Bax, Bcl-2 and cleaved caspase-3 in different groups. Data are expressed as mean ± S.E.M. from three independent experiments (**, P<0.01, ***, P<0.001).

Figure 5. miR-497 modulates VEGFR-2 directly

(A) The expression of VEGFR-2 was significantly decreased in protein level after restoration of miR-497 by miR-497 mimics. (B) Schematic diagram showed the predicted region where miR-497 is expected to bind VEGFR-2 3′-UTR and the mutated version lacking the binding site for miR-497. Wild-type and mutated miR-497 target sites in the 3′-UTR of VEGFR-2 were cloned into luciferase reporter vectors. (C) Wild-type and mutated miR-497 target sites in the 3′-UTR of VEGFR-2 were cloned into luciferase reporter vectors. Dual-luciferase reporter assay was performed in ACHN cells. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. The luciferase experiments were repeated three times.

Figure 6. miR-497 inhibited MEK/ERK and activated p38 MAPK signalling.

(A) The p38 (B) p-p38 (C) MEK (D) p-MEK (E) ERK and (F) p-ERK were measured by Western blot. Data are expressed as mean ± S.E.M. from three independent experiments (**, P<0.01).