MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies.

Methods: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness.

Results: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs.

Conclusions: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

Keywords: Cancer biomarkers; Cell cycle arrest; Intrahepatic cholangiocarcinoma; MFAP5; NOTCH.

Fig. 1

MFAP5 expression was upregulated in ICC patients and correlated with poor prognosis. a Heatmap showed the expression of genes in CCA tissues and non-cancerous tissue from the dataset GSE76297. b Venn diagram represented 7 genes that were differently expressed in the two GEO datasets. c Relative expression level of MFAP5 was detected by RT-QPCR. d Protein expression level of MFAP5 in liver tissues were detected by IHC. e, f Prognostic results based on MFAP5 expression in ICC tissues. *P<0.05, **P<0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

MFAP5 serum level was elevated in ICC patients. a MFAP5 expression level in CCA and HCC tissues from dataset GSE76297. b MFAP5 serum level (ELISA) in healthy volunteers, ICC patients and HCC patients (samples = 8,32,13 respectively). c, d AUC of ICC patients,healthy volunteers and HCC patients based on MFAP5 serum level. e MFAP5 serum level (ELISA) of pre-operation and 7 days after operation in 8 ICC patients. f, g, h IHC results and box plots of MFAP5 protein level in ICC tissues grouped by ICC TNM stages f, lymph node metastasis g and five-year overall survival h. Scale bar = 50 μm *P<0.05, **P<0.01, ***P < 0.001

Fig. 3

MFAP5 promoted proliferation of ICC cells in vitro and in vivo. a, b Cell viability results showed the different proliferation rate after co-cultured with recMFAP5 and after transfected MFAP5 shRNAs. c, d Colony formation assay results showed the different colony numbers in co-cultured experiments of recMFAP5 and transfected MFAP5 shRNAs cells. e Tumor growth curves after injected ICC cells. f Xenograft tumors from respective groups were shown. g Tumor weight of different xenograft tumors groups. h Boxplot showed Ki-67 level in sh-MFAP5 and sh-Control derived xenograft tumors which evaluated by IHC scores. *P < 0.05,**P < 0.01,***P<0.001

Fig. 4

MFAP5 facilitated ICC cell cycle transition. a, c Cell cycle flow cytometry analysis showed the different G0/G1 percentage after co-cultured with recMFAP5 and after transfected sh-MFAP5. b, d Three replicate experiments on cell cycle analysis. e Western blot assay showed the expression level of CCND1, CDK4, CDK6 CDC25A, P21 in ICC cells line after co-cultured with recMFAP5 and after transfected sh-MFAP5. g, h RT-PCR results showed the relative expression level of cell cycle genes in sh-MFAP5 ICC cell lines. *P < 0.05,**P < 0.01,***P<0.001

Fig. 5

MFAP5 positively regulated Notch1 pathway transcription. a Heat map showed results of RNA-seq of SSP-25 and RBE transformed sh-control, sh-MFAP5–1# and sh-MFAP5–2#. b, c GSEA results showed down-regulation of Notch pathway in cells transfected sh-MFAP5 compared with sh-Control. d, e RT-PCR results showed the mRNA level of genes in NOTCH1 pathway after transfected sh-MFAP5 in ICC cell lines. f, g Western blot analysis of Notch1 pathway target genes in sh-MFAP5 and sh-Control cells (f) and co-culture with recMFAP5 and control cells (G, each “+” represented 50μg/ml recMFAP5). h, i Co-IP results showed that MFAP5 could bind directly with NOTCH1. *P < 0.05,**P < 0.01,***P<0.001

Fig. 6

Chromatin Accessibility by ATAC-seq Analysis Reveals a Role of MFAP5 for NOTCH1 Activation. a, b CCK-8 assay showed the proliferation differences of ICC cells co-cultured with recMFAP5 and combination (recMFAP5 + FLI-06). c Western blot results of cell cycle genes in ICC cell lines after co-cultured with recMFAP5 and FLI-06. d ATAC-seq heatmap results showed the appearing peaks status in different groups. e GSEA analysis revealed genes in the G1/S phase following recMFAP5 co-culture. f The gene peaks results revealed changes in different groups in RBE cell line. g Proposed schematic models illustrated MFAP5 facilited the aggressiveness of ICC via modulating Notch pathway/G1S signaling axis