MicroRNA-644a promotes apoptosis of hepatocellular carcinoma cells by downregulating the expression of heat shock factor 1

Abstract

In this study, we investigated the role of microRNA-644a (miR-644a) in the growth and survival of hepatocellular carcinoma (HCC) cells. MiR-644a levels were lower in HCC tissues than in adjacent peri-cancerous tissues (n = 135). MiR-644a expression was inversely correlated with heat shock factor 1 (HSF1) expression, tumour diameter and TNM stage. Moreover, HepG2 and SMMC-7721 cell lines showed lower miR-644a expression than normal L-O2 hepatocytes. MiR-644a overexpression in HepG2 and SMMC-7721 cells increased apoptosis by downregulating HSF1. Dual luciferase reporter assays confirmed the presence of a miR-644a binding site in the 3'-untranslated region (3'-UTR) of HSF1. Xenograft tumours derived from SMMC-7721 cells transfected with a miR-664a mimic showed less growth than tumours derived from untransfected controls. Protein chip analysis revealed that miR-644a-overexpressing SMMC-7721 and HepG2 cells strongly expressed pro-apoptotic BH3-only proteins, such as BID, BAD, BIM, SMAC, Apaf-1 and cleaved caspases-3 and -9. These findings suggest miR-644a promotes apoptosis in HCC cells by inhibiting HSF1.

Keywords: Apoptosis; BH3-only protein; HSF1; Hepatocellular carcinoma; miR-644a.

Fig. 1

Low expression of miR-644a in HCC tissues negatively correlates with HSF1 expression and a poor prognosis. (a) Representative images (× 400) show in situ hybridization (ISH) of miR-644a in HCC and adjacent peri-cancerous liver tissues. (b) Quantitative analysis of miR-644a expression in HCC and adjacent peri-cancerous liver tissues (n = 135/group) based on ISH. Note: *** denotes P < 0.001 when compared to adjacent tissues. (c) Representative IHC images (× 400) show HSF1 expression in HCC and adjacent peri-cancerous tissues (n = 135/group). (d) Quantitative analysis of HSF1 expression in HCC and adjacent peri-cancerous liver tissues (n = 135/group) based on IHC. Note: *** denotes P < 0.001 when compared to adjacent tissues. (e) Quantitative RT-PCR analysis of miR-644a expression in HCC and adjacent peri-cancerous liver tissues (n = 135/group). Note: *** denotes P < 0.001 when compared to adjacent tissues. (f) Spearman’s test results show low miR-644a expression that negatively correlates with high HSF1 expression in HCC tissues (R² = 0.304, P < 0.05). (g-h) Kaplan-Meier survival analysis demonstrates a correlation between miR-644a expression in HCC patients and their overall survival (g) and recurrence-free survival (h)

Fig. 2

Correlation between miR-644a and HSF1 expression in HCC cells. (a) Quantitative RT-PCR analysis of miR-644a expression in HCC cell lines, HepG2, SMMC-7721, sk-Hep1, MHCC-97 L, QGY-7701 and the normal liver cells, L-O2. (b) Representative western blot shows HSF1 protein expression in the HCC cell lines HepG2, SMMC-7721, sk-Hep1, MHCC-97 L, and QGY-7701 and the normal liver cells, L-O2. (c) Quantitative RT-PCR analysis of HSF1 mRNA expression in the HCC cell lines, HepG2, SMMC-7721, sk-Hep1, MHCC-97 L, and QGY-7701 and the normal liver cells, L-O2. (d) Spearman’s analysis shows a correlation between miR-644a and HSF1 expression in the HCC cell lines (R² = 0.522, P < 0.05). Note: Data are represented as the mean ± SD of three independent experiments and were analysed by a paired t-test

Fig. 3

MiR-644a upregulation inhibits proliferation and promotes apoptosis of HCC cells. (a) Quantitative RT-PCR analysis of miR-644a expression in control and miR-644a mimic-transfected HepG2 and SMMC-7721 cells. (b) The CCK8 assay shows proliferation in the control and miR-644a mimic-transfected HepG2 and SMMC-7721 cells. (c) Flow cytometry analysis of percent apoptosis (AnnexinV⁺ PI⁺) in the control and miR-644a mimic-transfected HepG2 and SMMC-7721 cells. Note: * denotes P < 0.05 and ** denotes P < 0.01 compared to controls

Fig. 4

MiR-644a downregulates HSF1 expression by binding to its 3’UTR. (a) Targetscan and miRanda analysis show putative miR-644a binding sites in the 3’UTR of HSF1. (b) Quantitative RT-PCR analysis of HSF1 mRNA expression in HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild type or mutant HSF1. (c) Representative western blot shows HSF1 protein expression in HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild type or mutant HSF1. (d–e) Relative luciferase activity in HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild type or mutant HSF1. Note: * denotes P < 0.05 and ** denotes P < 0.01 compared to controls

Fig. 5

HSF1 silencing decreases proliferation and increases apoptosis of HCC cells. (a) CCK8 assay shows proliferation of HepG2 and SMMC-7721 cells transfected with control or HSF1-siRNA. (b) Colony formation assay shows the total number of colonies formed by HepG2 and SMMC-7721 cells transfected with control or HSF1-siRNA. (c) Flow cytometry analysis shows apoptosis of HepG2 and SMMC-7721 cells transfected with control or HSF1-siRNA. Note: * denotes P < 0.05 and ** denotes P < 0.01 compared to controls

Fig. 6

HSF1 overexpression promotes proliferation and inhibits apoptosis of HCC cells. (a) CCK8 assay shows proliferation of HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild-type or mutant HSF1 (wild type or mutant 3’UTR). (b) Colony formation assay shows the total number of colonies in HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild-type or mutant HSF1. (c) Flow cytometry analysis of percent apoptosis (AnnexinV⁺ PI⁺) in HepG2 and SMMC-7721 cells co-transfected with miR-644a mimic and wild-type or mutant HSF1. Note: * denotes P < 0.05 and ** denotes P < 0.01 compared to controls

Fig. 7

MiR-644a/HSF1 axis regulates HCC cell apoptosis via the BH3-only protein signalling pathway. (a) Protein chip clustering analysis shows differential protein expression in control and HSF1 siRNA transfected SMMC-7721 cells. (b) Protein chip enrichment analysis shows signalling pathways differentially regulated by silencing HSF1 in SMMC-7721 cells. (c–d) Representative western blots show expression of apoptosis-related proteins in SMMC-7721 cells transfected with miR-644a mimic or control miRNA. All experiments were repeated three times

Fig. 8

MiR-644a overexpression inhibits xenograft HCC tumour growth. (a) Representative images of xenograft tumours from nude mice subcutaneously injected with SMMC-7721 cells stably transfected with miR-644a mimic or control miRNA. (b) Xenograft tumour weights from nude mice subcutaneously injected with SMMC-7721 cells stably transfected with miR-644a mimic or control miRNA. (c) Representative images (400×) of IHC staining of HSF1 in xenograft tumours derived from SMMC-7721 cells stably transfected with miR-644a mimic or control miRNA. (d) Representative western blot shows HSF1 expression in xenograft tumours derived from SMMC-7721 cells stably transfected with miR-644a mimic or control miRNA. Note: * denotes P < 0.05 and ** denotes P < 0.01 compared to controls

Fig. 9

Schematic diagram of apoptosis regulation in HCC cells by miR-644a