MicroRNA-186 suppresses cell proliferation and metastasis in bladder cancer

Abstract

Purpose: Bladder cancer (BCa) is a common malignancy in the urinary system. This study aims to explore the role of miR-186 in BCa tumorigenesis.

Methods: The expression of miR-186 and ADAMTS12 in clinical BCa tissues and cell lines was detected. BCa cell lines T24, 5637 and EJ were used to transfect miR-186 mimics or inhibitors. Luciferase reporter gene detection confirmed the correlation between miR-186 and ADAMTS12. MTT method and flow cytometry were used to detect cell viability and apoptosis. Cell migration and invasion ability was detected by transwell assay. The protein level of ADAMTS12, β-catenin, GSK-3β and p-GSK-3β was determined using western blot analysis.

Results: MiR-186 was negatively correlated with the expression of ADAMTS12 in BCa tissues. Further research confirmed that ADAMTS12 is the direct target of miR-186. In addition, overexpression of miR-186 down-regulated the expression of ADAMTS12, inhibiting cell viability and apoptosis, while knockout of miR-186 led to the opposite result. miR-186 also inhibits the phosphorylation of GSK-3 β and β-catenin without changing the total GSK-3β level. Our study shows that miR-186 has a negative regulatory effect on the expression of ADAMTS12 in clinical specimens and in vitro. miR-186 can inhibit the proliferation and invasion of BCa cells.

Conclusions: miR-186 has the potential to be used as a biomarker in the early detection of BCa.

Keywords: ADAMTS12; bladder cancer; metastasis biomarker; miR-186; proliferation.

Figure 1

The correlation between miR-186 and ADAMTS12 in BCa samples. (A) Starbase 2.0 data showed that ADAMTS12 was expressed in BCa tissues and paired normal tissues (B) Kaplan-Meier analysis of ADAMTS12 expression and BCa patient total Correlation of survival rate (C) Data from starbase2.0 shows that miR-186 is expressed in BCa tissues and paired normal tissues (D) Data from Starbase 2.0 shows the expression of miR-186 in a two-tailed Pearson correlation analysis It is negatively correlated with the expression of ADAMTS12 (E) Immunohistochemical staining of ADAMTS12 in normal bladder tissue, NMIBC tissue and MIBC tissue (F) Block diagram shows the relative expression of ADAMTS12 in BCa tissue and paired normal tissues (G) Block diagram shows the relative expression of miR-186 in BCa tissues and matched normal tissues.

Figure 2

MiR-186 directly regulate ADAMTS12 expression in vitro. (A) Expression of miR-186 in bladder cancer cell lines (T24, 5637 and EJ) and human bladder epithelial cells (SV-HUC-1) (*P<0.05) vs. SV-HUC-1 group) (B) Detection of miR-186 in T24 cells transfected with miR-186 inhibitor and 5637 cells transfected with miR-186 mimics by qRT-PCR (*P<0.05 compared with the corresponding NC group) (C) Sequence alignment of miR-186 binding site predicted in ADAMTS12 3′UTR and its mutation sequence in luciferase reporter gene analysis ( D, E) Luciferase reporter analysis was performed in T24 and 5637 cells co-transfected with miR-186 mimic and a reporter vector containing ADAMTS12 3′UTR or mutated ADAMTS12 3′UTR. The relative activity (F, G) of luciferase was introduced in the Western blot and qRT PCR analysis of ADAMTS12 expression of miR-186 inhibitor transfected in T24 cells and ADAMTS12 expression of miR-186 mimic transfected in 5637 cells (*Compared with the corresponding NC group, P<0.05) (*Compared with the corresponding NC simulation group, P<0.05).

Figure 3

miR-186 regulates malignant phenotypes of BCa in vitro. (A, B) CCK-8 assay was performed to determine the cell viability in BCa cells after transfection. Detect absorbance values at 24, 48, 72, and 96 hours after transfection (*Compared with the corresponding NC group, P<0.05) (C, D) Flow cytometry to detect miR-186 inhibitor-transfected T24 cells and transfected cells Apoptosis of 5637 cells infected with miR-186 mimics (*P<0.05 compared with the corresponding NC group) (E, F) After transfection of T24 and 5637 cells, cross-well migration and invasion experiments were performed to detect cell Migration and invasion capabilities. The number of cells in 5 random areas was counted in units of 200 × magnification (*P<0.05 compared with the corresponding NC group).

Figure 4

miR-186 involves in the regulation of Wnt/β-catenin signalling pathway. (A, B) Protein level β-catenin, p-GSK-3β, GSK-3β western blot detects T24 cells transfected with miR-186 inhibitor and 5637 cells transfected with miR-186 analog. GAPDH was used as an internal control (*P<0.05 compared with the corresponding NC group).