LncRNA MBNL1-AS1 represses cell proliferation and enhances cell apoptosis via targeting miR-135a-5p/PHLPP2/FOXO1 axis in bladder cancer

Abstract

LncRNAs have been shown to play essential roles in bladder cancer (BC) progress. Our microarrays of clinical samples firstly screened that lncRNA muscleblind-like 1 antisense RNA 1 (MBNL1-AS1) was poorly expressed in BC tissues. However, its biological function in BC remains not well understood. Here we examined the clinical correlations with MBNL1-AS1 in BC patients. Then, 5673 and T24 cell lines were employed to investigate the role of MBNL1-AS1 in the proliferation and apoptosis of BC cells in vitro and in vivo. Furthermore, miR-135a-5p (miR-135a)/PHLPP2/FOXO1 axis was focused to explore its regulatory mechanism in BC. The results showed that MBNL1-AS1 was significantly downregulated in bladder tumor tissues, and associated with BC progression. In vitro, MBNL1-AS1 knockdown increased the number of viable cells and bromodeoxyuridine-positive cells, accelerated cell cycle, and dysregulated proliferative regulators (Ki67, p21, p27, and Cyclin D1) in BC cells. The apoptotic cells and the cleavages of caspase-3/9 were reduced in MBNL1-AS1-silenced BC cells. Overexpression of MBNL1-AS1 had opposite effects on BC cell proliferation and apoptosis. Moreover miR-135a was demonstrated to interact with MBNL1-AS1, and inhibiting miR-135a reversed the effects of shMBNL1-AS1 on BC cells. The downstream effectors (PHLPP2 and FOXO1) were positively regulated by MBNL1-AS1, but negatively regulated by miR-135a. Similar results were also observed in xenograft tumors. In conclusion, this study firstly suggests that MBNL1-AS1 acts as a tumor suppressor of BC by targeting miR-135a/PHLPP2/FOXO1 axis, providing a novel insight for BC diagnosis and treatment.

Keywords: FOXO1; MBNL1-AS1; PHLPP2; bladder cancer; miR-135a-5p.

Figure 1

MBNL1‐AS1 was downregulated in BC patients. A, Differently expressed lncRNAs were shown with Hierarchical Clustering between tumor tissues and adjacent nontumor tissues. B, qRT‐PCR validated that lncRNA MBNL1‐AS1 was downregulated in bladder tumor tissues. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; N, nontumor tissues; qRT‐PCR, quantitative real‐time PCR; T, tumor tissues. ***P < .001

Figure 2

Knockdown of MBNL1‐AS1 enhanced the proliferation of BC cells. A, Relative expression of MBNL1‐AS1 in 5673 and T24 cells was detected by qRT‐PCR. B, MTT assay was applied to examine cell viability in 5673 and T24 cells. C, Cell cycle progression of 5673 and T24 cells was analyzed using flow cytometry. D, Brdu incorporation assay was used to detect the proliferation of 5637 and T24 cells. *P < .05, **P < .01, ***P < .001. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; qRT‐PCR, quantitative real‐time PCR

Figure 3

Knockdown of MBNL1‐AS1 suppressed the apoptosis of BC cells. A, Flow cytometry assay was employed to evaluate the apoptotic ratio in 5673 and T24 cells. B, The apoptotic cells were stained with TUNEL staining in 5673 and T24 cells. ***P < .001. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling

Figure 4

Overexpression of MBNL1‐AS1 inhibited the proliferation and induced the apoptosis of BC cells. A, Relative expression of MBNL1‐AS1 in 5637 and T24 cells was assessed by qRT‐PCR. B, The viable cells were evaluated using MTT assay in 5637 and T24 cells. C, Flow cytometry analysis was performed to determine the cell cycle distribution of 5637 and T24 cells. D, The percentage of apoptotic cells was detected by flow cytomentry in 5637 and T24 cells. *P < .05, **P < .01, ***P < .001. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; qRT‐PCR, quantitative real‐time PCR

Figure 5

MBNL1‐AS1 was directly targeted by miR‐135a and positively regulated PHLPP2/FOXO1 expression. A, The sequences of MBNL1‐AS1 binding with miR‐135a. B, Luciferase reporter assay was carried out to evaluate the binding activity between MBNL1‐AS1 and miR‐135a. C, Relative expression of miR‐135a was determined using qRT‐PCR. D, The protein levels of PHLPP2, FOXO1, p‐AKT, and AKT were detected with western blot. *P < .05, **P < .01, ***P < .001. MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; qRT‐PCR, quantitative real‐time PCR

Figure 6

MBNL1‐AS1 regulated the proliferation and apoptosis of BC cells via miR‐135a/PHLPP2/FOXO1 axis. A, Cell viability of 5673 and T24 cells was assessed by MTT. B, The proliferative cells were detected using Brdu staining. C, Flow cytometry analysis was carried out to examine the apoptotic cells. D, Western blot analysis of cell proliferative regulators (Ki67, p21, p27, and cyclin D1) and apoptotic regulators (cleaved caspase‐3 and cleaved caspase‐9) in 5673 and T24 cells. E, Western blot analysis of PHLPP2/FOXO1 signals (PHLPP2, FOXO1, p‐AKT, and AKT) in 5673 and T24 cells. *P < .05, **P < .01, ***P < .001. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1

Figure 7

Inhibition of MBNL1‐AS1 promoted the tumorigenesis of BC cells through the regulation of miR‐135a/PHLPP2/FOXO1 in vivo. The nude mice were injected with 5673 and T24 cells stably transfected with shMBNL1‐AS1, respectively. After 19 days, mice were sacrificed. A, The tumor xenografts were collected. B, Tumor size was measured every 3 days. C, The isolated tumor was weighed at day 19. D and E, qRT‐PCR analysis of relative expression of MBNL1‐AS1 and miR‐135a. F, The immunopositive materials of Ki67 were detected using immunohistochemistry staining. G, The protein levels of cell proliferative regulators (p21, p27, and cyclin D1) and apoptotic regulators (cleaved caspase‐3 and cleaved caspase‐9) were tested by western blot. H, The protein levels of PHLPP2, FOXO1, p‐AKT, and AKT were detected using western blot. **P < .01, ***P < .001. BC, bladder cancer; MBNL1‐AS1, muscleblind‐like 1 antisense RNA 1; qRT‐PCR, quantitative real‐time PCR