CircMEMO1 modulates the promoter methylation and expression of TCF21 to regulate hepatocellular carcinoma progression and sorafenib treatment sensitivity

Abstract

Background: Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples.

Methods: The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity.

Results: We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment.

Conclusion: Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.

Keywords: Circ RNA; HCC; TCF21; TET; miR-106b-5p.

Fig. 1

CircMEMO1 Expression Was Identified to Be Significantly Downregulated in HCC Tissue and Related to Patient Prognosis. a A heatmap of differentially expressed circRNAs in our HCC tissue samples compared with paratumour DN samples is shown. b Schematic illustration of circMEMO1. c qRT-PCR revealed that circMEMO1 expression was significantly downregulated in HCC tissue samples. d The circMEMO1 level in HCC tissue samples was related to vascular invasion in HCC patients. e,f Kaplan-Meier analysis showed that the level of circMEMO1 was predictive of overall survival and DFS in HCC. g Multivariate Cox analysis revealed that the circMEMO1 level in HCC tissue was an independent prognostic factor in HCC patients

Fig. 2

CircMEMO1 Inhibits HCC Cell Proliferation and Invasion In Vitro and In Vivo. a sh-circMEMO1 was overexpressed and knocked down in HCCLM3 and Huh-7 cells by lentivirus-mediated transduction, respectively. b, c Cell Counting Kit-8 (CCK-8) assays were used to determine the role of circMEMO1 in the proliferation of HCC cells. * P < 0.05; **, P < 0.01. d, e Colony formation assays showed that circMEMO1 inhibited the colony formation activity of HCC cells. f, g Transwell assays showed that circMEMO1 inhibited the migration and invasion of HCC cells. h-j Xenograft tumours composed of circMEMO1-OE HCCLM3 cells were significantly smaller than those composed of control cells. k, l circMEMO1 inhibited the lung metastasis of HCCLM3, and representative images of haematoxylin-eosin (HE) staining of metastatic nodules in the lungs of animals in different groups are shown

Fig. 3

CircMEMO1 Regulates the Level of the TET1/5hmC Axis by Sponging MiR-106b-5p in HCC Cells. a, b miR-106b-5p was abundantly pulled down by a circMEMO1 probe, and miR-106b-5p captured large amounts of circMEMO1 in both Huh-7 and HCCLM3 cells. c Representative seed sequences for miR-106b-5p in the human TET family are shown: 9 base pairs (green), 8 base pairs (red) and 7 base pairs (blue). d HCC cancer cells with different miR-106b-5p expression levels were subjected to western blot analyses for the indicated proteins. e A luciferase assay with the luciferase gene linked to the 3′-UTR of TET1/2 was performed. HEK293 cells were transiently transfected with a combination of pGL3 firefly luciferase reporter plasmids encoding the wild-type 3′-UTR sequences of human TET genes, miR-106b-5p, and a Renilla luciferase reporter for normalization. The data are represented as the mean ± SD (n = 3). f 5hmC-, 5-mC- and DAPI-stained HCC cells expressing different miR-106b-5p levels are shown. Scale bars, 100 μm. g Genomic DNA purified from HCC cells with different miR-106b-5p levels was denatured and neutralized. Global 5hmC levels were then measured by using a dot blot assay with an anti-5hmC antibody. h HCC cancer cells with different circMEMO1 expression levels were subjected to western blot analyses for the indicated proteins. i A dot blot assay with an anti-5hmC antibody was used to detect changes in the 5hmC level in HCC cells with different circMEMO1 expression levels

Fig. 4

The circMEMO1 Level Correlates with the miR-106b-5p/TET1/5hmC Axis and Predicts a Poor Prognosis in HCC Patients. a, b The correlations between the level of circMEMO1 and mRNA levels of the TET1 and TET2 genes were analysed using qRT-PCR analysis of HCC patient samples. c, d HCC patient samples were subdivided into two groups according to the expression status of TET1. Kaplan-Meier plots representing the overall survival (c) and cumulative recurrence rate (d) of the stratified patients are shown. e Representative HCC cases in tissue microarrays (serial sections) were analysed by an in situ hybridization assay for miR-106b-5p and immunohistochemical staining for 5hmC. f, g Positive miR-106b-5p expression with a low level of 5hmC correlated with the worst survival (f) and highest recurrence rate (g) in HCC patients. I, miR-106b-5p (+); II, low 5hmC level; III, miR-106b-5p (−); IV, high 5hmC level

Fig. 5

CircMEMO1 Inhibits HCC Progression by Regulating the EMT Process via the miR-106b-5p/TET1/5hmC Axis. a Huh-7 cells transduced with sh-circMEMO1 or an empty vector were subjected to immunofluorescence. Scale bars, 100 μm. b Western blotting was used to detect the levels of the indicated proteins in Huh-7 and HCCLM3 cells with different circMEMO1 levels. c sh-circMEMO1 expression increased the ability of Huh-7 cells to form tumoursphere structures, while overexpression of circMEMO1 expression decreased the ability of HCCLM3 cells to form tumoursphere structures. d Huh-7 cells infected with a combination of sh-circMEMO1, miR-106b-5p, and TET1-expressing vectors were subjected to a cell invasion assay. Scale bars, 100 μm. e Cell lysates from Huh-7 cells expressing a combination of sh-circMEMO1, miR-106b-5p, and TET1 were subjected to western blot analysis for the indicated proteins. f Genomic DNA purified from Huh-7 cells expressing a combination of sh-circMEMO1, miR-106b-5p, and TET1 was denatured and neutralized. Global 5hmC levels were then measured by a dot blot assay using an anti-5hmC antibody. g Representative HCC cases in tissue microarrays were analysed by an in situ hybridization assay for miR-106b-5p and immunohistochemical staining for TET1, E-cadherin and Vimentin

Fig. 6

CircMEMO1 Inhibits HCC progression by Regulating the DNA demethylation and expression of TCF21. a, b The relationship of the TCF21 mRNA level with miR-106b-5p or CDH1 level in TCGA database. c The TCF21 mRNA level was downregulated in a series of cancers, including HCC, compared with normal tissues. d, e The TCF21 gene promoter methylation level between the HCC tissues and the controls was significantly higher in HCC patients than in controls from the same research. f, g circMEMO1 and miR-106b-5p can regulate TCF21 expression. h hMeDIP-qPCR assays showed that TCF21 genes had decreased 5hmC in gene bodies in Huh-7 and HCCLM3 cells with different circMEMO1 levels. i, j TCF21 overexpression diminished the metastasis properties and EMT process induced by inhibition of circMEMO1 or activation of miR-106b-5p

Fig. 7

CircMEMO1 Regulated the Antitumour Activity of Sorafenib Treatment in HCC Cells. a Overall survival curves were compared between HCC patients with a high or low TCF21 level treated with sorafenib in TCGA database. b Comparison of overall survival curves between 40 advanced-stage recurrent HCC patients receiving combined sorafenib and TACE treatment who underwent liver resection before combined therapy. c MTT assays were used to detect the growth of Huh-7 and HCCLM3 cells with different circMEMO1 levels treated with sorafenib. d Matrigel invasion assays were used to investigate the migration ability of HCC cells with different circMEMO1 levels treated with sorafenib. e, f Overall survival curves were compared between recurrent HCC patients with a high or low circMEMO1 or miR-106b-5p level treated with sorafenib. g Schematic depiction of the mechanisms underlying circMEMO1 regulating HCC progression via the miR-106b-5p/TET1/5hmC axis