Obesity-associated miR-27a upregulation promotes hepatocellular carcinoma metastasis through suppressing SFRP1

Abstract

Background: Obesity was a recognized risk factor for the development and progression of hepatocellular carcinoma (HCC). However, the effects and mechanisms by which obesity promotes HCC metastasis remain poorly understood.

Materials and methods: We cultured adipocyte induced by preadipocyte 3T3-L1 in vitro and established HCC metastasis model in obesity mouse in vivo to mimic the tumor microenvironment in obese status. The mechanisms underlying obesity-associated miR-27a upregulation promoting HCC metastasis were investigated.

Results: In this study, we showed that miR-27a was upregulated in adipocytes, obese mouse model and clinical samples, and the increased miR-27a level promoted migration and invasion in HCC cells, increased the number of metastasis nodes in obese mouse model, and was associated with poor clinical outcomes. Overexpressed secreted frizzled-related protein 1 in HCC cells and tissues significantly alleviated the upregulation of β-catenin and matrix metalloproteinase-7 induced by high level of miR-27a. Meanwhile, the E-cadherin expression decreased and Vimentin expression increased, linking with high level of β-catenin in high-fat group.

Conclusion: Taken together, our results have elucidated the critical role of extracellular miR-27a as a pro-metastatic factor in HCC and revealed that obesity-associated miR-27a upregulation promoted HCC metastasis through activated Wnt/β-catenin signaling by suppressing secreted frizzled-related protein 1. Our findings shed light on the novel mechanism underlying HCC metastasis and provided miR-27a as a promising target for obese liver cancer therapy.

Keywords: SFRP1; hepatocellular carcinoma; miR-27a; obesity; β-catenin.

Figure 1

The establishment of co-culture transwell chamber system of differentiated adipocyte 3T3-L1 cells with HepG2 cells. Notes: (A) qRT-PCR analysis of miR-27a expression in 3T3-L1 cells without transfection or transfected with miR-27a overexpression plasmid, miR-27a inhibitor plasmid, or empty vector after differentiation. (B, C) qRT-PCR analysis of miR-27a expression in cell culture medium (B) in the lower chamber or HepG2 cells (C) in the upper chamber alone, or with 3T3-L1, miR-27a overexpressing 3T3-L1, miR-27a inhibitor 3T3-L1 and 3T3-L1 transfected with empty vector co-culture. Each bar represents the mean ± SD of 3 replicates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviation: qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 2

Secretory miR-27a promotes migration and invasion of HCC cells in vitro. Notes: (A) Transwell assays were employed to evaluate the migratory and invasive ability of HepG2 cells under co-culture alone, or with 3T3-L1, miR-27a overexpressing 3T3-L1, miR-27a inhibitor 3T3-L1 and 3T3-L1 transfected with empty vector co-culture. The original magnification is ×40, with a scale of 1:200 μm. (B, C) Quantitative results for the migratory and invasive ability of each group are shown as the number of cells. Each bar represents the mean ± SD of 3 replicates. *p<0.05 and **p<0.01. Abbreviation: HCC, hepatocellular carcinoma.

Figure 3

miR-27a activated wnt/β-catenin signaling by suppressing SFRP1 in vitro. Notes: (A) Western blot analysis of SFRP1 protein expression in HepG2 cells transfected with empty vector or SFRP1 overexpressed plasmid. β-actin was used as an internal control. (B) Transwell assays were employed to evaluate the migratory and invasive ability of HepG2 cells transfected with empty vector or SFRP1-overexpressed plasmid under co-culture with 3T3-L1 cells alone, miR-27a-inhibited 3T3-L1 cells, or miR-27a-overexpressed 3T3-L1 cells. The original magnification is ×40, with a scale of 1:200 μm. Quantitative results for the migratory and invasive ability of each group are shown as the number of cells. (C) Protein expressions of SFRP1, β-catenin, MMP7 in HepG2 cells transfected with empty vector or SFRP1-overexpressed plasmid under co-cultured with 3T3-L1 cells alone, miR-27a-inhibited 3T3-L1 cells, or miR-27a-overexpressed 3T3-L1 cells. β-actin was used as an internal control. Each bar represents the mean ± SD of 3 replicates. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: MMP7, matrix metalloproteinase-7; SFRP1, secreted frizzled-related protein 1.

Figure 4

Obesity promoted HCC metastasis in vivo with upregulation of miR-27a. Notes: (A) The livers and lungs were excised and metastasis nodes on lung surface were quantified in the control and the HF groups. (B) qRT-PCR analysis of miR-27a expression in adipose tissue, serum, liver, and lung from the control and the HF groups. Each bar represents the mean ± SD of 3 replicates. **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: Ctrl, control; HCC, hepatocellular carcinoma; HF, high fat; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 5

Wnt/β-catenin signaling was activated in HCC metastasis of obesity in vivo. Notes: (A) H&E staining and SFRP1 IHC staining of liver in the control and the HF groups. (B) Western blot analysis of SFRP1, β-catenin, MMP7, vimentin, and E-cadherin protein expression in tumor foci of liver from the control and the HF groups. β-actin was used as an internal control. Each bar represents the mean ± SD of 3 replicates. ***p<0.001, and ****p<0.0001. Abbreviations: Ctrl, control; HCC, hepatocellular carcinoma; HF, high fat; IHC, immunohistochemistry; MMP7, matrix metalloproteinase-7; SFRP1, secreted frizzled-related protein 1.

Figure 6

Expression of miR-27a and SFRP1 in HCC tissues of patients. Notes: (A) qRT-PCR analysis of miR-27a and Western blot analysis of SFRP1 in HCC tissues. β-actin was used as an internal control. N represents normal group and O represents obesity group. N1 was the negative control. Each bar represents the mean ± SD of 3 replicates. (B) Correlation analysis between miR-27a expression and SFRP1 expression in HCC tissues. (C) Kaplan–Meier overall survival curves for patients in normal and obesity groups. Abbreviations: BMI, body mass index; HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SFRP1, secreted frizzled-related protein 1.