LncRNA ERVH48-1 Contributes to the Drug Resistance of Prostate Cancer and Proliferation through Sponging of miR-4784 to the Activation of the Wnt/β-Catenin Pathway

Abstract

Long noncoding RNAs (LncRNAs) are very important in the way that docetaxel resistance (DR) happens in prostate cancer (PCa) patients. ImmuneScore and StromalScore were calculated using PCa-related expression data from TCGA and the ESTIMATE algorithm. We finally found the DEGs that were related to the immune system and the stroma of the patients by making profiles of the DEGs in ImmuneScore and StromalScore. The CancerSubtypes algorithm identified prognosis-related PCa subtypes, and the GSVA assessed their pathway activity. A UniCox regression analysis was used to identify a prognosis-related differential gene set. We then used intersection analysis to identify immunological and prognostic (IP)-related genes (IPGs). The coexpression of long noncoding RNAs (lncRNAs) and IPGs was used to identify IP-related lncRNAs (IPLs). Three methods (SVM-RFE, random forest, and LASSO) were used to find genes that overlap in the GEO database. A gene signature was then validated by building an ROC curve. CIBERSORT technology was used to look at the possibility of a link between the gene signature and immune cells. LncRNA-miRNA pairs and miRNA-mRNA pairs from the miRDB and TargetScan databases were used to construct the ERVH48-1-miR-4784-WNT2B ceRNA regulation network. The concentration of docetaxel elevated the expression of ERVH48-1. Overexpression of ERVH48-1 increased PCa-DR cell proliferation, invasion, and migration while inhibiting apoptosis. ERVH48-1 increased the tumorigenicity of PCa-DR cells in nude mice. ERVH48-1, acting as a ceRNA, targeted miR-4784 to increase WNT2B expression. ICG001 therapy increased Wnt/-catenin signaling activity in PCa-DR cells by inhibiting ERVH48-1. Finally, ERVH48-1 increased docetaxel resistance in a WNT2B-dependent manner via the miR-4784/Wnt/-catenin pathway.

Keywords: ERVH48-1; WNT2B; docetaxel resistance; miR-4784; prostate cancer.

Figure 1

Correlation between three categories of scores with the prognosis and clinical characteristics of individuals with prostate cancer. (A) Kaplan–Meier survival analysis for high- and low-score PCa patients, grouped by ImmuneScore median; (B) StromalScore survival analysis; (C) ESTIMATEScore survival analysis. (D–F) Association between PCa patients’ age, N classification, and T classification and ImmuneScore. (G–I) Association between PCa patients’ age, N classification, and T classification and StromalScore. (J–L) Association between PCa patients’ age, N classification, and T classification and ESTIMATEScore. (M,N) the DEGs were visualized in heatmaps.

Figure 2

PCa subgroups identified to have clinical relevance. (A) GSVA computed the enrichment score (ES) heat map for 4922 gene sets; (B) the optimal number of clusters (K) was determined using the factoextra package; (C) cluster results were visualized using the factoextra package; and (D) PCa samples were clustered using the nonnegative matrix factorization (NMF) method. The CancerSubtypes tool was used to generate silhouette width plots. (E) The CancerSubtypes tool was used to generate silhouette width plots. (F) Survival patterns were analyzed using the CancerSubtypes program; (G) a link was revealed between their expression and other clinical variables; and (H) the differential enrichment score of gene sets was computed between each grouping and intersection. (I) The association between 17 common gene sets and PCa clinical characteristics; (J) heat map of 619 differentially expressed IPGs; (K) the coexpression network of PCa-associated IPGs and lncRNAs.

Figure 3

Construction of IP-related lncRNA–miRNA–mRNA regulatory network. (A) Heat map of DEGs from GSE69223, GSE103512, and GSE116918 datasets; (B) volcano plot of DEGs; (C) LASSO algorithm; (D) heat map of 50 DEGs; (E) SVM-RFE algorithm; (F) heat map of 31 DEGs; (G,H) RFS-FS algorithm; (I) heat map of 65 DEGs; (J) heat map of DEMs; (K) volcano plot of DEMs; (L) Venn plot of overlapping miRNAs; (M) Venn plot of candidate characteristics; (N) WNT2B expression in the tumor group; (O) ROC curve for WNT2B; (P,Q) the CIBERSORT tool showed that WNT2B had a potential correlation with T cells; (R) an ERVH48-1-miR-4784-WNT2B regulatory network was constructed.

Figure 4

High ERVH48-1 expression was detected in PCa tumor tissue and Dox-resistant PCa cells and was connected with poor survival. RT-PCR analysis of ERVH48-1 expression (A); miR-4784 expression (B); and WNT2B expression (C). Correlation analysis of ERVH48-1 expression and miR-4784 expression (D); WNT2B expression and miR-4784 expression (E), ERVH48-1 expression and WNT2B expression (F). Survival analysis of high ERVH48-1 expression and low ERVH48-1 expression (G), ERVH48-1_High and ERVH48-1_High+miRNA_Low+WNT2B_High (H), ERVH48-1_High and WNT2B_High (I), ERVH48-1_High and ERVH48-1_High+WNT2B_High (J), ERVH48-1_High+miRNA_Low+WNT2B_High and ERVH48-1_Low+miRNA_High+WNT2B_Low (K). (L) CCK8 analysis of cell viability for PCa cell lines. (M) RT-PCR analysis of ERVH48-1 expression in PC3-DR and DU145-DR cells. (N) RT-PCR analysis of ERVH48-1 expression in the different groups. ** p < 0.01. All experiments were repeated three times.

Figure 5

ERVH48-1 promotes cell growth, invasion, and migration in PC3-DR and DU145-DR cells while inhibiting apoptosis. In ERVH48-1-overexpressed PC3-DR and DU145-DR cells, clone formation, transwell, scratch-wound, flow cytometry, and Western blot assays were used to detect cell proliferation (A), invasion (B), migration (C,D), and apoptosis (E,F). In ERVH48-1-inhibition PC3-DR and DU145-DR cells, clone formation, transwell, scratch-wound, flow cytometry, and Western blot assays were used to detect cell proliferation (G), invasion (H), migration (I,J), and apoptosis (K,L). ** p < 0.01. All experiments were repeated three times.

Figure 6

ERVH48-1 regulates related protein expression. In ERVH48-1-overexpressed PC3-DR and DU145-DR cells, Western blot assays were used to detect the expression of Ki67, Bax, Bcl-2, cleaved caspase-3, E-cadherin, and MMP2 (A). In ERVH48-1-inhibited PC3-DR and DU145-DR cells, Western blot assays weres used to detect the expression of Ki67, Bax, Bcl-2, cleaved caspase-3, E-cadherin, and MMP2 (B). ** p < 0.01. All experiments were repeated three times.

Figure 7

ERVH48-1 has an impact on the oncogenicity of Dox-resistant PCa cells in vivo. (A) Subcutaneous injections of PC3-DR cells transfected with siRNA against ERVH48-1 were given to null mice. (B,C,E,F) Tumor weights and volumes. (D) Nude mice were subcutaneously injected with DU145-DR cells that had been transfected with vectors expressing ERVH48-1. (G) IHC analysis of Ki67 expression, cleaved caspase-3 expression, and E-cadherin expression. ** p < 0.01. All experiments were repeated three times.

Figure 8

ERVH48-1 increased the expression of WNT2B by suppressing miR-4784. (A) The location of ERVH48-1 was discovered in PC3-DR and DU145-DR cells. (B) RT-PCR quantification of ERVH48-1 in cells treated with ERVH48-1-targeting siRNA. (C,D) RT-PCR analysis of miR-4784 expression in cells treated with miR-4784 mimics. (E) Potential binding sites of ERVH48-1 for miR-4784 were predicted. (F) The wild-type 3′-UTR reporter for ERVH48-1 was less active in cells containing miR-4784 mimics based on a luciferase reporter test. (G) Anti-IgG or anti-Ago2 antibodies were added to PC3-DR and DU145-DR cell lysates, and RT-PCR was used to determine ERVH48-1 and miR-4784 levels. (H) Potential binding sites of WNT2B for miR-4784 were predicted. (I) The WNT2B-WT reporter’s luciferase activity was tested after transfection with particular plasmids. (J) PC3-DR and DU145-DR cell lysates were mixed with anti-IgG or anti-Ago2 antibodies before RT-PCR analysis of the captured WNT2B and miR-4784. (K,L) RT-PCR analysis of WNT2B expression after transfection with specific plasmids. (M,N) RT-PCR analysis of WNT2B expression after transfection with miR-4784 mimics. * p < 0.05; # p < 0.05. All experiments were repeated three times.

Figure 9

ERVH48-1 activates the Wnt/β-catenin signaling pathway via the miR-4784/WNT2B axis to regulate cell viability and apoptosis. (A,B) RT-PCR analysis of WNT2B expression. (C) CCK8 assays were used to detect cell viability. (D) Detection of caspase-3 activity. RT-PCR (E–G) and Western blot (H) assays were used to detect the expression of MMP2, β-catenin, and Ki67. Western blot (I) and RT-PCR (J) assays were used to detect the expression of WNT2B and β-catenin in ICG001-treated cells. (K,N) CCK8 assays were used to detect cell viability. (L,O) Detection of caspase-3 activity. (M,P) Detection of TOP/FOP transcriptional activity. * p < 0.05, # p < 0.05, and ^ p < 0.05. All experiments were repeated three times.