miR-1202 regulates BPH-1 cell proliferation, apoptosis, and epithelial-to-mesenchymal transition through targeting HMGCL

Abstract

Benign prostatic hyperplasia (BPH) is the expansion of the prostate gland that results in urinary symptoms. Both the epithelial-to-mesenchymal transition (EMT) and the Wnt signaling pathway are associated with BPH pathology. In this study, we find that miR-1202 is increased in BPH samples. Overexpression of miR-1202 in TGF-β-treated BPH-1 cells enhances cell survival and DNA synthesis and inhibits cell apoptosis, whereas miR-1202 inhibition partially abolishes the effects of TGF-β on BPH-1 cells. miR-1202 overexpression reduces E-cadherin level but elevates vimentin, N-cadherin, and snail levels, whereas miR-1202 inhibition partially attenuates the effects of TGF-β on EMT markers. Regarding the Wnt/β-catenin pathway, miR-1202 overexpression significantly enhances, whereas miR-1202 inhibition partially decreases, the promotive effects of TGF-β on Wnt1, c-Myc, and cyclin D1 proteins. 3-Hydroxy-3-methylglutaryl-CoA lyase (HMGCL) is a direct downstream target of miR-1202, and miR-1202 inhibits HMGCL expression through binding to its 3'UTR. Overexpression of HMGCL significantly reduces the effect of miR-1202 overexpression on the phenotypes of BPH-1 cells by inhibiting cell survival and promoting apoptosis. Similarly, HMGCL overexpression has the opposite effects on EMT markers and the Wnt/β-catenin signaling, and markedly alleviates the effects of miR-1202 overexpression. Finally, in the BPH rat model, Ki67 and vimentin levels are elevated, but E-cadherin and HMGCL levels are reduced. In conclusion, miR-1202 is upregulated in benign prostatic hyperplasia; miR-1202 enhances epithelial cell proliferation, suppresses cell apoptosis, and promotes EMT by targeting HMGCL. The Wnt/β-catenin pathway may participate in the miR-1202/HMGCL axis-mediated regulation of BPH-1 cell phenotypes.

Keywords: 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL); Wnt/β-catenin pathway; benign prostate hyperplasia (BPH); epithelial-to-mesenchymal transition (EMT); miR-1202.

Figure 1

miR-1202 is upregulated in benign prostatic hyperplasia (BPH) (A,B) Differentially expressed miRNAs between BPH and normal samples were analyzed using the threshold of |log fold change|>0.3, P<0.05 based on GSE61741 (including 35 BPH samples and 94 normal samples) and GSE113234 (including 51 BPH samples and 27 normal samples). (C) Overlapping miRNAs were compared, and miR-1202 was identified. (D–F) miR-1202 expression in BPH and normal tissue samples according to the GSE61741, GSE113234 and GSE34933 cohorts. (G–I) BPH and normal samples were collected, and the expression levels of miR-1202 and TGF-β were examined via qRT-PCR or western blot analysis. (J) RWPE-1 and BPH-1 cells were treated with or without 10 ng/mL TGF-β for 24 h, after which the miR-1202 expression level was subsequently examined via qRT-PCR.

Figure 2

Effects of miR-1202 overexpression and inhibition on TGF-β-stimulated BPH-1 cells BPH-1 cells were transduced with miR-1202 mimics/NC mimics or miR-1202 inhibitor/NC inhibitor or nontransduced, stimulated or nonstimulated with TGF-β, and examined for miR-1202 expression for transduction efficiency by qRT-PCR (A), cell viability by MTT (B), DNA synthesis using EdU (C,D), cell apoptosis by flow cytometry (E), and the protein levels of BAX, BCL2, cleaved caspase 3, and caspase 3 by western blot analysis (F). **P<0.01, *** P<0.001, compared to the control group; ##P<0.01, compared to the mimics NC group; and &&P<0.01, compared to the inhibitor NC group.

Figure 3

Effects of miR-1202 overexpression and inhibition on the Wnt/β-catenin signaling and EMT BPH-1 cells were transduced with miR-1202 mimics/NC mimics or miR-1202 inhibitor/NC inhibitor or nontransduced, stimulated or nonstimulated with TGF-β, and then examined for fibroblast-like morphology with a phase contrast microscope (A), the levels of E-cadherin and vimentin by immunofluorescence staining (B,C), the protein levels of E-cadherin, N-cadherin, vimentin, and snail by western blot analysis (D), as well as the protein levels of Wnt1, c-Myc, and cyclin D1 by western blot analysis (E). **P<0.01, compared to the control group; ##P<0.01, compared to the mimics NC group; and &&P<0.01, compared to the inhibitor NC group.

Figure 4

HMGCL is a direct downstream target of miR-1202 (A) miRDB and RNAInter were used to predict the mRNAs that might be targeted by miR-1202; overlapping candidates were compared and identified. (B,C) Candidate genes were subjected to Gene Ontology (GO) functional enrichment annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment annotation. (D) The expression levels of candidates related to epithelial cell migration were analyzed based on the GSE205378 dataset, and genes with significantly different expression levels were identified. (E) The expressions of MAP4K3, PFN2, ARID5B, HMGCL, DYNLL2, and LTBP2 were examined in BPH and normal tissue samples by qRT-PCR. (F) The protein levels of HMGCL were examined in BPH and normal tissue samples by western blot analysis. (G) RWPE-1 and BPH-1 cells were treated with or without 10 ng/mL TGF-β for 24 h, after which the MAP4K3, HMGCL and DYNLL2 mRNA expression levels were examined by qRT-PCR. (H) The protein levels of HMGCL were examined in normal RWPE-1 and BPH-1 cells or in TGF-β-stimulated RWPE-1 and BPH-1 cells by western blot analysis. (I,J) Dual-luciferase reporter assays were performed to validate the predicted binding between miR-1202 and HMGCL. (K–L) BPH-1 cells were transduced with miR-1202 mimics/NC mimics or miR-1202 inhibitor/NC inhibitor, and the mRNA and protein expression levels were examined via qRT-PCR and western blot analysis, respectively. *P<0.05, **P<0.01, *** P<0.001, compared to normal, RWPE-1, BPH-1 or mimic NC cells; ##P<0.01, compared to the inhibitor NC group.

Figure 5

miR-1202 exerts its effects on TGF-β-stimulated BPH-1 cells Target cells were cotransduced with miR-1202 mimics/NC mimics and HMGCL, stimulated with TGF-β, and examined for HMGCL expression for transduction efficiency by qRT-PCR (A), cell viability by MTT (B), DNA synthesis using EdU (C), cell apoptosis by flow cytometry (D), and protein levels of BAX, BCL2, cleaved caspase 3, and caspase 3 by western blot analysis (E). *P<0.05, **P<0.01, compared to the vector+mimics NC group.

Figure 6

Dynamic effects of the miR-1202/HMGCL axis on the Wnt/β-catenin signaling and EMT Target cells were cotransduced with miR-1202 mimics/NC mimics and HMGCL and stimulated with TGF-β, after which the levels of E-cadherin and vimentin were examined by IF staining (A), the protein levels of E-cadherin, N-cadherin, vimentin, and snail were determined by western blot analysis (B), and the protein levels of Wnt1, c-Myc, and cyclin D1 were determined by western blot analysis (C). **P<0.01, ***P<0.001, compared to the vector+mimics NC group.

Figure 7

The levels of EMT proteins and HMGCL in BPH model rats The BPH model was induced in rats by subcutaneous injection of testosterone, and the histopathological characteristics of the rats were examined via H&E staining (A), the protein levels of Ki-67, vimentin, E-cadherin, and HMGCL were measured via IHC staining (B–E), and HMGCL mRNA and protein expressions were measured via qRT-PCR and western blot analysis, respectively (F,G). *P<0.01, ** P<0.01, ***P<0.001, compared to the control group.

Figure 8

Schematic diagram of the mechanism by which miR-1202 regulates BPH-1 cell proliferation, apoptosis and EMT miR-1202 promotes epithelial cell proliferation, inhibits cell apoptosis, and promotes the EMT process through targeting HMGCL and regulating the Wnt/β-catenin pathway.