CPSF6 links alternative polyadenylation to metabolism adaption in hepatocellular carcinoma progression

Abstract

Background: Alternative polyadenylation (APA) is an important mechanism of gene expression regulation through generation of RNA isoforms with distinct 3' termini. Increasing evidence has revealed that APA is actively involved in development and disease, including hepatocellular carcinoma (HCC). However, how APA functions in tumor formation and progression remains elusive. In this study, we investigated the role of cleavage factor I (CFIm) subunit CPSF6 in human hepatocellular carcinoma (HCC).

Methods: Expression levels of CPSF6 in clinical tissues and cell lines were determined by qRT-PCR and western blot. Functional assays, including the cell number, MTT, colony formation and transwell, were used to determine the oncogenic role of CPSF6 in HCC. Animal experiments were used to determine the role of CPSF6 in HCC tumorigenicity in vivo. Deep sequencing-based 3 T-seq was used to profile the transcriptome-wide APA sites in both HCC cells and CPSF6 knockdown HCC cells. The function of CPSF6-affected target NQO1 with distinct 3'UTRs was characterized by metabolism assays.

Results: We observed CPSF6 was upregulated in HCC and the high expression of CPSF6 was associated with poor prognosis in patients. Overexpression of CPSF6 promoted proliferation, migration and invasion of HCC cells in vitro and in vivo. Transcriptome-wide APA profiling analysis indicated that high expression of CPSF6 promoted the favorable usage of the proximal poly(A) site in the 3'UTR of NQO1. We demonstrated CPSF6-induced tumorigenic activities were mediated by the NQO1 isoform with short 3'UTR. Furthermore, we found that CPSF6 induced metabolic alterations in liver cells through NQO1.

Conclusion: CPSF6 plays a critical role in HCC progression by upregulating NQO1 expression through APA. These findings provide evidence to demonstrate that APA of NQO1 contributes to HCC progression and may have implications for developing new therapeutic strategy against this disease.

Keywords: Alternative polyadenylation; CPSF6; Hepatocellular carcinoma; Metabolism; NQO1.

Fig. 1

CPSF6 is upregulated and predicts poor prognosis in HCC. a IHC staining of CPSF6 in surrounding non-tumor and HCC tissues. Percentage of CPSF6 IHC was shown in bar graph. Scale bar, 50 μm. b Western blot assay for detecting CPSF6 expression in 16 paired surrounding non-tumor (N) and HCC tumor (T) tissues. β-actin was used as internal control. c qRT-PCR analysis of CPSF6 mRNA in 36 paired surrounding non-tumor (N) and HCC tumor (T) tissues. GAPDH was used as internal control. d, e Kaplan–Meier analysis of the correlation of CPSF6 protein expression with OS (d) and DFS (e). The expression level of CPSF6 protein was detected by immunohistochemistry. f qRT-PCR analysis of CPSF6 mRNA in TCGA liver cancer samples. g,h Kaplan–Meier analysis of OS (g) and DFS (h) data from TCGA liver cancer data containing 364 patients. The data of g and f can be obtained through online website (http://kmplot.com/analysis/).**p < 0.001, Student’s t-test

Fig. 2

CPSF6 accelerates the growth of HCC cells in vitro and in vivo. a The protein level of CPSF6 in different cell lines was assessed by western blotting. β-actin was used as internal control. b The protein level of CPSF6 in the CPSF6-overexpressing (CPSF6-OE) HL-7702 was assessed by western blotting. β-actin was used as internal control. c CPSF6 knockdown efficiency in Huh-7 with two CPSF6 specific shRNAs (shCPSF6–1 and shCPSF6–2) was examined by western blotting. β-actin was used as internal control. d Proliferation curves of the CPSF6-overexpressing HL-7702 cells and the control (Ctrl) were shown. e Proliferation curves of the CPSF6 knockdown Huh-7 cells and the control (shCtrl) were shown. f The cell viability of HL-7702 cells with CPSF6 overexpression and the control was examined by MTT assay. g The cell viability of CPSF6 knockdown Huh-7 cells and Huh-7 cells (the control) was examined by MTT assay. h,i Colony formation analysis of the indicated cell lines. j HL-7702 xenograft tumor growth with or without overexpression of CPSF6. k The weight of HL-7702 tumors at the end point was shown. l Huh-7 xenograft tumor growth with or without CPSF6 knockdown. m The weight of Huh-7 tumors at the end point was shown. n, o IHC analysis of CPSF6 and Ki67 expression in tumors. Scale bars, 100 μm. **p < 0.001, Student’s t test

Fig. 3

CPSF6 promotes migration, invasion and metastasis of HCC cells. a Effect of CPSF6 overexpression on HL-7702 cell migration. b Effect of CPSF6 knockdown on Huh-7 cell migration. c Effect of CPSF6 overexpression on HL-7702 cell invasion. d Effect of CPSF6 knockdown on Huh-7 cell invasion. Representative pictures of H&E staining of lungs and incidence of lung metastasis from mice inoculated with (e) HL-7702 and (f) Huh-7 cells. Red arrows indicated the lung metastases. Results represented mean ± SD using bar graph. Scale bars, 100 μm. **p < 0.001, Student’s t test

Fig. 4

Characteristics of 3 T-seq data. a, b Genomic locations of qualified 3 T-seq reads mapped to the reference genome. c, d Genomic distribution of the poly(A) sites. e, f The statistics of genes with various number of detected poly(A) sites. g, h Scatterplot of CULI for measuring the 3′UTR alteration in CPSF6-overexpressing or CPSF6 knockdown cells when compared with the corresponding control cells (false discovery rate (FDR) = 0.05). i CPSF6-modulated 7 candidate genes. These genes had the shortened 3′UTR in CPSF6-overexpressing HL-7702 cells and the lengthened 3′UTR in CPSF6 knockdown Huh-7 cells. j CPSF6-induced APA shift of NQO1. Left, Integrative Genomics Viewer (IGV) genome browser exhibited the poly(A) site usage of NQO1 3′UTR. Right, histogram showed the relative expression of the isoform with distal polyadenylation site (dPAS) relative to the one with proximal PAS (pPAS). k, l The protein levels of NQO1 in the CPSF6-overexpressing HL-7702 cells (k) and the CPSF6 knockdown Huh-7 cells (l) were assessed by western blotting. β-actin was used as internal control

Fig. 5

The NQO1 short 3’UTR isoform has oncogenic function and increases aggressiveness in liver cells. a Schematic illustration of the NQO1 isoform with long or short 3’UTR. Positions of the binding sites of miRNA were indicated by yellow horizontal lines. The activity of short 3’UTR (3’UTR-S) and long 3’UTR (3’UTR-L) of NQO1 after enforced expression of the specific miRNAs was examined using luciferase reporter assay in HL-7702 (b) and Huh-7 (c) cells. d Luciferase expression from a reporter containing the short 3’UTR of NQO1, as compared to that from the reporter containing the long 3’UTR of NQO1 in 4 liver cell lines. e The protein levels of NQO1 in HL-7702 cells stably transfected with NQO1-overexpressing plasmids were assessed by western blotting. β-actin was used as internal control. f Proliferation curves of HL-7702 cells stably transfected with NQO1-overexpressing plasmids or control (Ctrl) were shown. g The cell viability of HL-7702-NQO1-OE and HL-7702-Ctrl cells was examined by MTT assay. h Colony formation analysis of HL-7702-NQO1-OE and HL-7702-Ctrl cells. i Transwell analysis of the migration ability of HL-7702 cells. j Transwell analysis of the invasion ability of HL-7702 cells. k HL-7702 xenograft tumor growth with or without overexpression of different NQO1 isoforms. l The weight of HL-7702 tumors at the end point was shown. m IHC analysis of NQO1 and Ki67 expression in tumors. Scale bars, 100 μm. n Representative pictures of H&E staining of lungs and incidence of lung metastasis from mice inoculated with HL-7702 cells. Black arrows indicate the lung metastases. **p < 0.001, Student’s t test

Fig. 6

Function of CPSF6 is NQO1-dependent. a Western blot analysis of the efficiency of NQO1 knockdown and overexpression of CPSF6 in HL-7702 cells. b Proliferation curves of HL-7702 cells were shown. c The cell viability of HL-7702 cells was examined by MTT assay. d Colony formation analysis of HL-7702 cells. e Transwell analysis of cell migration of HL-7702. f Transwell analysis of the cell invasion of HL-7702. g Western blot analysis of the efficiency of NQO1 overexpression (NQO1-OE) and CPSF6 knockdown in Huh-7. h Proliferation curves of Huh-7 cells were shown. i The cell viability of Huh-7 cells was examined by MTT assay. j Colony formation analysis of Huh-7 cells. k Transwell analysis of the cell migration of Huh-7. l Transwell analysis of the cell invasion of Huh-7. **p < 0.001, Student’s t test

Fig. 7

CPSF6 upregulates glycolysis and promotes aerobic glycolysis through NQO1. a Glucose uptake in control and NQO1-overexpressing HL-7702 cells. b Cellular lactate levels in control and NQO1-overexpressing HL-7702 cells. OCR (c) and ECAR (d) of control and NQO1- overexpressing HL-7702 cells measured by the Seahorse Bioscience XF96 analyzer. e Glucose uptake in control and CPSF6-overexpressing HL-7702 cells with or without NQO1 knockdown. f Cellular lactate levels in control and CPSF6-overexpressing HL-7702 cells with or without NQO1 knockdown. OCR (g) and ECAR (h) of control and CPSF6-overexpressing HL-7702 cells with or without NQO1 knockdown measured by the Seahorse Bioscience XF96 analyzer. i Glucose uptake in control and CPSF6 knockdown Huh-7 cells with or without overexpression of NQO1. j Cellular lactate levels in control and CPSF6 knockdown Huh-7 cells with or without overexpression of NQO1. OCR (k) and ECAR (l) of control and CPSF6 knockdown Huh-7 cells with or without overexpression of NQO1 measured by the Seahorse Bioscience XF96 analyzer. **p < 0.001, Student’s t test

Fig. 8

Expression of NQO1 in human HCC tissues. a IHC staining of NQO1 in surrounding non-tumor and HCC tissues. Scale bar, 50 μm. b Western blot analysis of NQO1 expression in 16 paired surrounding non-tumor (N) and HCC tumor (T) tissues. β-actin was used as internal control. c The correlation analysis between NQO1 expression and CPSF6 level in 124 HCC tissues (TNM, III/IV) with linear regression and pearson’s correlation significance (P < 0.0001, ANOVA test). d The proposed mechanism of CPSF6 in regulation of HCC progression