Aspirin targets P4HA2 through inhibiting NF-κB and LMCD1-AS1/let-7g to inhibit tumour growth and collagen deposition in hepatocellular carcinoma

Abstract

Background: Abnormal construction of the extracellular matrix (ECM) is intimately linked with carcinogenesis and the development of solid tumours, especially hepatocellular carcinoma (HCC). As the major component of the ECM, collagen plays a pivotal role in carcinogenesis. P4HA2, the essential enzyme during collagen formation, becomes an important target in HCC treatment. Here, we tried to decipher whether aspirin (ASA), a classic anti-inflammatory drug, could improve the prognosis of HCC through targeting P4HA2.

Methods: Western blotting, qRT-PCR assay, immunofluorescence staining, luciferase reporter gene assay, and ChIP assay were applied to demonstrate the molecular mechanism of the regulation of P4HA2 expression by aspirin. A mouse xenograft model, cell viability assay, colony formation assay, and immunohistochemistry analysis were used to evaluate the anti-fibrosis effect of aspirin through targeting the NF-κB/P4HA2 axis and LMCD1-AS1/let-7g/P4HA2 axis in vitro and in vivo. The TCGA database was used to evaluate the correlation among P4HA2, let-7g, LMCD1-AS1 and overall survival of HCC patients.

Findings: In xenograft mice, aspirin was capable of targeting P4HA2 to decrease collagen deposition, resulting in the inhibition of liver tumour growth. TCGA database analysis revealed the close association between a higher P4HA2 concentration in HCC patients and shorter overall survival or a higher cancer stage and the pathological grade. Mechanistically, NF-κB can bind to the promoter of P4HA2 to activate its transcription. Moreover, lncRNA LMCD1-AS1 functions as a molecular sponge of let-7g to post-transcriptionally induce the target gene of let-7g, namely, P4HA2.

Interpretation: Our findings disclose the novel role and regulatory mechanism of aspirin in the suppression of HCC by disrupting abnormal collagen deposition.

Funds: 973 Program, National Natural Scientific Foundation of China, Fundamental Research Funds for the Central Universities, Project of Prevention and Control of Key Chronic Non-Infectious Diseases.

Keywords: Aspirin; Collagen deposition; LMCD1-AS1; Let-7g; NF-κB; P4HA2.

Fig. 1

Aspirin reduces collagen deposition-associated growth of liver tumours in mice. HepG2 cells were implanted into BALB/c nude mice. (A) Tumours of the saline- and aspirin-treated groups were dissected and weighted 28 days after HCC cell implantation (n = 5). Scale bar, 1 cm. (B and C) The tumour growth curve (B) and the body weight (C) of mice under saline as the control or aspirin treatment are shown. (D) Tumour sections were stained with Ki 67 antibody. The bar graph is the quantification of Ki 67 staining. Scale bar, 20 μm. (E) Masson's trichrome staining of tumour sections (blue, collagen fibres; dark blue, nuclei; red, cytoplasm). Scale bar, 50 μm. (F) The levels of collagen type I and collagen type IV were detected by western blotting; the bar graph shows the quantification of western blotting results. (G) IHC staining of collagen I and IV in saline- and aspirin-treated mice tumours is shown. Scale bar, 20 μm. The data are representative of three independent experiments (means ± SD). (H) The protein level of CTGF in xenograft tumours were detected by IHC assay. Scale bar, 20 μm. ***P < 0.001; **P < 0.01; *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Aspirin targets P4HA2 to hamper collagen synthesis in liver tumour growth. (A) Kaplan−Meier analysis of the correlation between P4HA2 and the relapse free survival of 228 cases of HCC patients from GEO database. (B) RT-PCR analysis of P4HA2 in HepG2 cells treated with aspirin at elevated concentrations (0, 2, 4 mM) is shown. The band graph shows the quantification of RT-PCR results. (C) The levels of P4HA2 and collagen type I were tested using western blotting. The band graph shows the quantification of western blotting. (D) Immunofluorescence analysis of P4HA2 and collagen type I in HepG2 cells under aspirin treatment. (E) Tumours from the indicated treatment groups were collected and weighted 32 days after HCC cell implantation (n = 5). Scale bar, 1 cm. (F) Growth curve of the tumours from the indicated groups. (G) Masson's trichrome staining of the tumour sections (blue, collagen fibres; dark blue, nuclei; red, cytoplasm). (H) The levels of collagen type I and P4HA2 were detected by western blotting in mouse tumours from the indicated groups. (I) IHC staining of P4HA2 and collagen type I in mouse tumours from the indicated groups is shown. The data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

NF-κB/p65 is responsible for activating the promoter of P4HA2 in aspirin-regulated hepatoma. (A) Luciferase reporter gene assay of HepG2 cells treated with aspirin and/or pGL3-P0 transfection. (B) Relative luciferase activity of the full-length promoter and the other four truncated promoter regions of P4HA2. (C) RT-PCR and western blotting assays of HepG2 cells treated with an elevated dose of aspirin or PDTC for 24 h; the bar graph shows the quantification of the western blotting results. (D) RT-PCR and western blotting of HepG2 cells treated with TNF-α alone or in combination with aspirin; the bar graph shows the quantification of the western blotting results. (E) Immunofluorescence assay of P4HA2 under PDTC (30 μM) or TNF-α (20 ng/mL) treatment. (F) Luciferase reporter gene analysis of P4HA2 promoter activity in HepG2 cells transfected with p65 siRNAs including sip65#1 or sip65#2. (G and H) P4HA2 expression at the mRNA and protein levels was evaluated by RT-PCR, western blotting (G) and immunofluorescence (H) assays in HepG2 cells transfected with sip65#2. (I and J) ChIP assay of the binding of NF-κB/p65 with the P4HA2 promoter region after aspirin treatment (I) or TNF-α stimulation (J). (K) Luciferase reporter gene assay of P4HA2 promoter activity after the NF-κB/p65 binding sites in the P4HA2 promoter were mutated. The data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01; *P < 0.05; N, not significant (Student's t-test).

Fig. 4

Let-7g acts as a mediator of aspirin-reduced P4HA2 in liver tumour. (A) Kaplan−Meier analysis of the correlation between let-7g and the overall survival of 371 HCC patients from the TCGA database. (B) TCGA analysis of let-7g expression in 50 paired tumour and para-tumour tissues. (C) Luciferase reporter gene assay of P4HA2 3'UTR after miRNA treatment. (D and E) HepG2 cells were transfected with let-7g (50 nM) for 36 h. Then, RT-PCR, western blotting (D) and immunofluorescence (E) were performed to evaluate the change of P4HA2 at mRNA and protein levels. (F) RT-PCR and western blotting assays of HepG2 cells treated with elevated concentration of let-7g inhibitor (50, 100 nM) or aspirin (4 mM) in combination with anti-let-7g (50 nM). (G) Luciferase reporter gene assay of HepG2 cells transfected with let-7g and pGL3-P4-UTR-wt or pGL3-P4-UTR-mut. (H) Luciferase reporter gene assay of HepG2 cells transfected with anti-let-7g and pGL3-P4-UTR-wt or pGL3-P4-UTR-mut. (I) Luciferase reporter gene assay of pGL3-P4-UTR-wt activity in HepG2 cells treated with aspirin with or without the presence of anti-let-7g is shown. All experiments were repeated at least three times independently. The data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01; *P < 0.05; N, not significant (Student's t-test).

Fig. 5

The lncRNA LMCD1-AS1/let-7g axis participates in aspirin-decreased P4HA2 expression in liver tumour. (A) Kaplan−Meier analyses of the correlation between LMCD1-AS1 and overall survival of 371 patients with HCC from the TCGA database. (B) Expression of lncRNAs including CFAP53, LMCD1-AS1 and GGT3P in the control and aspirin-treated (4 mM) groups was measured by RT-PCR. (C) The binding sequence of let-7g with LMCD1-AS1. (D and E) Quantitative real-time PCR and RT-PCR analysis of let-7g and LMCD1-AS1 in HepG2 cells transfected with LMCD1-AS1 siRNAs (si-LMCD1-AS1 #1 and si-LMCD1-AS1 #2) (D) or pcDNA-LMCD1-AS1 (E). (F and G) Luciferase activity analysis of P4HA2 3'UTR reporter plasmid in HepG2 cells co-transfected with LMCD1-AS1 siRNAs (F) or pcDNA-LMCD1-AS1 (G). (H and I) RT-PCR and western blotting assays were performed to detect the expression of P4HA2 after transfecting with si-LMCD1-AS1 #2 (H) and pcDNA-LMCD1-AS1 (I). (J and K) Luciferase reporter gene assay was performed to detect the activity of P4HA2 3'UTR reporter plasmid after transfecting with pcDNA-LMCD1-AS1 (or pcDNA-LMCD1-AS1 combined with let-7g) (J) and si-LMCD1-AS1 #2 (or si-LMCD1-AS1 #2 combined with anti-let-7g) (K). The data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 6

Aspirin bates the axis of NF-κB/P4HA2 and LMCD1-AS1/let-7g/P4HA2 to block the growth of liver tumour in vitro and in vivo. (A and B) MTT assay (A) and colony formation assay (B) of HepG2 and Huh-7 cell lines under treatment with the TNF-α, pcDNA-LMCD1-AS1, TNF-α + pcDNA-LMCD1-AS1, or TNF-α + pcDNA-LMCD1-AS1 + aspirin. (C) Quantitative real-time PCR, RT-PCR and western blotting assays were performed to detect the expression of let-7g, p65 and P4HA2 in HepG2 and Huh-7 cell lines after indicated treatment. (D and E) IHC (D) and western blotting (E) detection of the level of NF-κB/p65 in mice tumour tissues from saline- and ASA-treated groups. (F to H) Quantitative real-time PCR detection of NF-κB/p65 (F), let-7g (G), or LMCD1-AS1 (H) expression in mice tumour tissues from saline- and ASA-treated groups. (I) Graphic model of aspirin (ASA)-ameliorated liver tumour development. As a classic downstream factor of aspirin, NF-κB/p65 is able to activate the transcription of P4HA2. Meanwhile, lncRNA LMCD1-AS1 acts as a sponge to decrease the level of tumour suppressor miRNA let-7g and subsequently enhances the target gene of let-7g, i.e., P4HA2. Aspirin suppresses collagen deposition and further liver tumour growth through targeting the axis NF-κB/P4HA2 and LMCD1-AS1/let-7g/P4HA2. The data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01; *P < 0.05.

Supplementary Fig. S1

Aspirin targets P4HA2 to hamper collagen synthesis in liver tumour growth. (A) Kaplan−Meier analysis of the correlation between P4HA family members (P4HA1, P4HA2 and P4HA3) and overall survival of 371 patients with HCC. P values were estimated by Graphpad Prism software. (B) The mRNA levels of P4HA family members in 50 paired para-tumour vs liver tumour tissues from TCGA database. (C and D) Expression of P4HA2 at different clinical stage (C) and pathology grade (D) of HCC patients from TCGA database. (E) The protein level of P4HA2 in xenograft tumours were detected by western blotting. (F) Tumour sections from saline-treated or aspirin-treated mice were stained with P4HA2 antibody. (G to I) The same experiments were exerted in Huh-7 cells as those in HepG2 cells in Fig. 2B–D. (J) Statistic analysis of tumour weight in Fig. 2E. (K) Tumour sections were stained with Ki 67 antibody; bar graph shows the quantification of Ki 67 staining. (L) Primary HCC cell line was transfected by P4HA2 overexpression plasmid and/or treated with aspirin, and the proliferation of cells was detected by MTT assay. Data are representative of three independent experiments (means ± SD). ***P < 0.001; N, not significant.

Supplementary Fig. S2

NF-κB/p65 is responsible for activating the promoter of P4HA2 in aspirin-regulated hepatoma. (A) Huh-7 cells were treated with elevated dose of aspirin or PDTC for 24 h. Cells were collected for analysis of P4HA2 by RT-PCR and western blotting. The band graph shows the quantification of western blotting results. (B) Huh-7 cells were treated with elevated dose of TNF-α alone or in combination with aspirin for 24 h. Cells were collected for analysis of P4HA2 by RT-PCR and western blotting. The band graph shows the quantification of western blotting results. (C) The efficiency of two p65 siRNAs including sip65#1 and sip65#2 were demonstrated by RT-PCR and western blotting assays. (D) Correlation between mRNA levels of P4HA2 and NF-κB/p65. We analysed the expression of P4HA2 and NF-κB/p65 of 371 HCC patients in TCGA database. Data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01.

Supplementary Fig. S3

Let-7g acts as a mediator of aspirin-reduced P4HA2 in liver tumour. (A) Prediction of potential miRNAs which might target P4HA2 in three websites. (B) Fold changes of several candidate miRNAs after aspirin treatment were detected by qRT-PCR. (C) Kaplan−Meier analyses of correlation between the predicted miRNAs and overall survival of 371 HCC patients from TCGA database. (D) Expression of miR-30e, miR-494 and miR-495 in 50 paired tumours and their adjacent nontumor parts of HCC patients in TCGA database. (E and F) Analysis of P4HA2 expression in Huh-7 cells treated with let-7g (E), anti-let-7g, ASA (4 mM) or ASA combined with anti-let-7g (50 nM) (F). (G) The binding of let-7g with 3'UTR of P4HA2 mRNAs. Sequences of P4HA2 3'UTR containing wild-type and mutated let-7g binding site (pGL3-P4-UTR-wt and pGL3-P4-UTR-mut). (H to J) Luciferase reporter gene assay of pGL3-P4-UTR-wt and pGL3-P4-UTR-mut in Huh-7 cells treated with let-7g (H), anti-let-7g (I), ASA, ASA combined with anti-let-7g (J). Data are representative of three independent experiments (means ± SD). **P < 0.01; *P < 0.05; N, not significant.

Supplementary Fig. S4

LncRNA LMCD1-AS1/let-7g axis participates in aspirin-decreased P4HA2 expression in liver tumour. (A) Kaplan−Meier analyses of the correlation between 15 lncRNAs and overall survival of 371 patients with HCC. P values are estimated by Graphpad prism software. (B) The analysis of LMCD1-AS1 expression in 50 paired tumour and para-tumour tissues from TCGA database. (C and D) The relationship between LMCD1-AS1 with tumour staging (D) and grading (E) of HCC patients basing on TCGA database were analysed. Data are representative of three independent experiments (means ± SD). ***P < 0.001; **P < 0.01; *P < 0.05.