RNA-seq reveals novel mechanistic targets of Livin in bladder cancer

Abstract

Background: Bladder cancer is a very common malignancy with a high recurrence rate. The survival of patients with muscle-invasive bladder cancer is poor, and new therapies are needed. Livin has been reported to be upregulated in bladder cancer and influence the proliferation of cancer cells.

Materials and methods: The Livin gene in human bladder cancer cell line T24 was knocked out, and the differentially expressed genes were identified by RNA-seq and qPCR.

Results: Livin knockdown affects gene expression and has strong negative effects on some cancer-promoting pathways. Furthermore, combined with bladder cancer clinical sample data downloaded from TCGA and GEO, 2 co-up-regulated genes and 58 co-down-regulated genes were identified and validated, which were associated with cancer proliferation and invasion.

Conclusion: All these results suggest that Livin plays an important role in bladder cancer and could be a potential anticancer target in clinical therapy.

Keywords: Bladder cancer; Livin; RNA-seq; T24 cell.

Fig. 1

Effect of Livin knockdown in T24 cells. A The knockdown efficiency of Livin was detected in T24 cells. B Migration and invasion analysis 72 h after Livin knockdown, crystalline violet staining was performed on migrated and invading cells. C Efficiency of cell migration and invasion after 72 h of Livin knockdown. D Western blotting assays were performed to detect protein levels of E-cadherin and MMP-9 in NC group and si-Livin group, normalized by GAPDH. *P < 0.05, **P < 0.01. versus NC group

Fig. 2

Effect of Livin knockdown in T24 cells on cell proliferation. A Cell apoptosis was detected by flow cytometry in NC group and si-Livin group. B Western blotting assays were performed to detect protein levels of VEGFA, EGFR, and HIF-1α in NC group and si-Livin group, normalized by GAPDH. C The protein levels of Bcl-2 in NC group and si-Livin group, normalized by GAPDH. *P < 0.05, **P < 0.01. versus NC group

Fig. 3

RNA-Seq revealed differences in gene expression between T24 normal cells and Livin knockdown cells. A The heatmap of correlation coefficients within and between sample groups. B Volcano diagram displayed genes upregulated and downregulated in two groups. C Heatmaps depicting differentially expressed genes in two groups

Fig. 4

Functional analyses of the differentially expressed genes between T24 normal cells and Livin knockdown cells. A GO enrichment analysis for expression of upregulated genes. BP: biological process, CC: Cellular component, MF: molecular function. B GO enrichment analysis for expression of downregulated genes. C Bubble diagram of KEGG enrichment analysis results of differential genes

Fig. 5

GSEA functional enrichment analysis. A GO enrichment analysis. B KEGG enrichment analysis

Fig. 6

Identification of co-differentially expressed genes from T24 cell data combined with TCGA and GEO clinical data. A Venn diagrams of the three datasets of upregulated genes. B Venn diagrams of the three datasets of downregulated genes. C RT-qPCR validation of common genes on T24 cells. *P < 0.05, **P < 0.01, ***P < 0.001. versus NC group