Yonsei Med J.  2019 Mar;60(3):267-276. 10.3349/ymj.2019.60.3.267.

MicroRNA-370 Regulates Cellepithelial-Mesenchymal Transition, Migration, Invasion, and Prognosis of Hepatocellular Carcinoma by Targeting GUCD1

• He Y
• He X

• KMID: 2441304
• DOI: http://doi.org/10.3349/ymj.2019.60.3.267

Abstract

PURPOSE
Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor, the prognosis of which remains poor. Recently, microRNAs have been reported to play crucial functions in multiple tumors, including HCC. However, the molecular mechanisms of miR-370 in HCC still remain largely unknown. The present study focused on the effects of miR-370 on HCC migration, invasion, and epithelial-mesenchymal transition (EMT).
MATERIALS AND METHODS
We investigated the key roles and possible regulatory mechanism of miR-370 in regulating HCC metastasis with functional assays, such as transwell assay. Quantitative real-time PCR (qRT-PCR) was used to detect miR-370 and guanylylcyclase domain containing 1 (GUCD1) expression in HCC tissues and cells. Subsequently, we performed transwell assays to determine the functions of miR-370 in HCC cell invasion and migration. Western blot was used to determine protein expressions of relevant genes. Luciferase reporter assays were conducted to confirm the target gene of miR-370.
RESULTS
qRT-PCR analysis demonstrated that miR-370 was dramatically downregulated in HCC. Moreover, downregulated miR-370 was found to be associated with poor survival and adverse clinicopathologic characteristics of HCC patients. Transwell assays revealed that miR-370 overexpression dramatically suppressed HCC invasion and migration. Meanwhile, miR-370 restoration prominently inhibited EMT progression in HCC cells. Luciferase reporter assays confirmed GUCD1 as a downstream target gene of miR-370. GUCD1 expression in HCC tissues was prominently increased and inversely correlated with miR-370 expression. Furthermore, GUCD1 was verified as mediating the suppressive influence of miR-370 on cell metastasis and EMT in HCC.
CONCLUSION
Taken together, our study confirmed that miR-370 suppressed HCC cell metastasis and EMT via regulating GUCD1. Accordingly, the miR-370/GUCD1 axis may potentially acts as attractive therapeutic targets and novel biomarkers for HCC treatment.

Keyword

Hepatocellular carcinoma; epithelial-mesenchymal transition; migration; invasion; prognosis; miR-370; GUCD1

MeSH Terms

Biomarkers
Blotting, Western
Carcinoma, Hepatocellular*
Epithelial-Mesenchymal Transition
Humans
Luciferases
MicroRNAs
Negotiating
Neoplasm Metastasis
Prognosis*
Real-Time Polymerase Chain Reaction
Biomarkers
Luciferases
MicroRNAs

Figure

• 

  Fig. 1 miR-370 was downregulated, and GUCD1 was upregulated in HCC. (A and B) miR-370 expression in HCC tissues and cells was measured using qRT-PCR. (C) GUCD1 expression in HCC and normal liver cells was detected by qRT-PCR. (D) Spearman's correlation analysis between the miR-370 expressions and GUCD1 mRNA expressions in HCC tissues.*p<0.05, †p<0.01. GUCD1, guanylylcyclase domain containing 1; HCC, hepatocellular carcinoma.

• 

  Fig. 2 Prognostic value of miR-370 and GUCD1 for HCC patients was analyzed by Kaplan-Meier analysis. (A) OS and (B) DFS of HCC patients with low and high miR-370 expression. (C) OS and (D) DFS of HCC patients with low and high GUCD1 expression. HCC, hepatocellular carcinoma; GUCD1, guanylylcyclase domain containing 1; OS, overall survival; DFS, disease-free survival.

• 

  Fig. 3 miR-370 overexpression suppressed cell migration and invasion in HCC cells. (A) miR-370 expression in miR-370 overexpressed HCCLM3 cells were detected by qRT-PCR. (B) The expression levels of miR-370 in miR-370 suppressed Hep3B cells were examined using qRT-PCR. (C) Cell invasion and (D) migration abilities of miR-370 overexpressed or suppressed HCC cells were determined by transwell assays. *p<0.05, †p<0.01. HCC, hepatocellular carcinoma.

• 

  Fig. 4 miR-370 restoration suppressed EMT progression in HCC cells. (A) Western blot analysis and (B) qRT-PCR results of miR-370 overexpression in EMT of HCCLM3 cells. (C) Western blot and (D) qRT-PCR results indicated that inhibition of miR-370 in Hep3B cells downregulated E-cadherin expression and enhanced N-cadherin and Vimentin expression. *p<0.05, †p<0.01. EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma.

• 

  Fig. 5 GUCD1 was a direct target of miR-370 in HCC. (A) The putative binding sites of miR-370 in the GUCD1 3′-UTR. (B) Luciferase activity was detected by luciferase reporter gene assays in HCC cells cotransfected with wild-type or mutational GUCD1 3′UTR and miR-370 mimics, respectively. (C and D) GUCD1 expression in HCC cells transfected with miR-370 mimics or inhibitor were examined by Western blot (left) and qRT-PCR (right). *p<0.05. GUCD1, guanylylcyclase domain containing 1; HCC, hepatocellular carcinoma.

• 

  Fig. 6 Alteration of GUCD1 expression partially reversed the miR-370-mediated effect on HCC cell migration and invasion. (A) Western blot (up) and qRT-PCR (down) analysis of GUCD1 expression in miR-370-overexpressed HCCLM3 cells cotransfected with GUCD1 overexpression plasmid. (B) Transwell assays were conducted to examine cell migration and invasion abilities of miR-370-overexpressed HCCLM3 cells cotransfected with GUCD1 overexpression plasmid. (C) GUCD1 expression in miR-370-suppressed Hep3B cells cotransfected with GUCD1 siRNA was measured by Western blot (up) and qRT-PCR (down) analysis. (D) Transwell assays were performed to measure cell invasion and migration abilities of miR-370-suppressed Hep3B cells cotransfected with GUCD1 siRNA. *p<0.05. GUCD1, guanylylcyclase domain containing 1; HCC, hepatocellular carcinoma.

• 

  Fig. 7 Altering GUCD1 expression reversed the effect of miR-370 on cell EMT process in HCC. (A) Western blot and (B) qRT-PCR results showed that GUCD1overexpression in miR-370-overexpressed HCCLM3 cells decreased E-cadherin expression and increased N-cadherin and Vimentin expression. (C)Western blot and (D) qRT-PCR results showed that GUCD1 knockdown in miR-370-suppressed Hep3B cells upregulated E-cadherin and downregulated N-cadherin and Vimentin significantly. *p<0.05. GUCD1, guanylylcyclase domain containing 1; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma.

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