Author Affiliations: Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China (Wu LM, Yang Z, Xing CY, Xie HY, Zhang F, Zhuang L, Zhou L and Zheng SS); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health; Key Laboratory of Organ Transplantation of Zhejiang Province; Division of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Wu LM, Yang Z, Xing CY, Pan TT, Xie HY, Zhang F, Zhuang L, Zhou L and Zheng SS); Lishui Hospital of Zhejiang University, Lishui 323000, China (Ji JS)

Corresponding Author: Shu-Sen Zheng, MD, PhD, FACS, Division of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571- 87236570; Fax: 86-571-87236466; Email: shusenzheng@zju.edu.cn)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60038-8

Published online November 18, 2015.

 

Contributors: ZSS proposed the study. WLM and JJS performed research and wrote the first draft. YZ, XCY and PTT collected and analyzed the data. WLM and JJS contributed equally to the article. All authors contributed to the design and interpretation of the study and to further drafts. ZSS is the guarantor.

Funding: This study was supported by grants from the Fundamental Research Funds for the Central Universities (2015FZA7013), the Natural Science Foundation of Zhejiang Province (LY15H160021), the National Natural Science Foundation of China (81101840 and 81302074), the Medical Science and Technology Project of Zhejiang Province (201472813), the Medical and Health Platform Backbone Personnel Plan of Zhejiang Province (2012RCB015 and 2013KYB107), the Innovative Research Group of the National Natural Science Foundation of China (81421062).

Ethical approval: This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: It has been found that microRNA-423-5p (miR423-5p) is an oncogenic factor and frequently upregulated in gastric carcinoma. However, the involvement of miR423-5p in hepatocellular carcinoma (HCC) has been rarely reported. The aim of this study was to assess whether miR423-5p is aberrantly expressed in HCC tissues, and to characterize its roles in the cancerous biology of HCC.

 

METHODS: HCC and corresponding nonmalignant tissues were obtained from 115 patients during liver transplantation to detect the expression level of miR423-5p. The miR423-5p mimic and inhibitor were transfected into LM3 cell line. Cell viability assay, cell cycle analysis, transwell invasion and migration experiments were used to evaluate the oncogenic role of miR423-5p.

 

RESULTS: miR423-5p was significantly upregulated in HCC compared with nonmalignant tissues, and this upregulation was negatively associated with recurrence-free survival. For patients beyond the Milan criteria, low expression of miR423-5p was correlated with better prognosis. Functional analysis showed that miR423-5p enhanced the proliferative, invasive and migratory capacity of HCC cells.

 

CONCLUSIONS: miR423-5p contributed to the tumorigenesis and progression of HCC. It could be a new predictor in HCC patients beyond the Milan criteria and would help to improve patient outcomes and enlarge recipient pools of liver transplantation.

 

(Hepatobiliary Pancreat Dis Int 2015;14:613-618)

KEY WORDS: hepatocellular carcinoma; miR423-5p; proliferation; invasion; oncogene

 

Hepatocellular carcinoma (HCC) is among the most common cancers worldwide, which leads to an estimate of 695 900 deaths annually.^{[1, 2]} Although the treatment of HCC has been dramatically improved, its prognosis remains dismal. Early diagnosis is crucial to the treatment and new biomarkers are helpful for diagnosis and clinical treatment of HCC patients.

 

MicroRNAs (miRNAs) are about 22 nucleotides, noncoding RNAs.^[3] They act mainly as negative regulators of gene expression through sequence-specific base pairing with target mRNAs^[4] and involve in various of biological pathways.^{[5, 6]} Although the molecular mechanism of miRNAs remains far from completely understood, growing evidences have shown that there is a strong association between cancer and miRNAs.^{[7, 8]} It is well documented that miRNAs play important roles in oncologic related processes, such as proliferation, apoptosis, invasion and differentiation.^[9-12]

 

MicroRNA-423 (miR423) is a recently identified new member of the miRNAs family that was first cloned from neuroblastoma.^[13] Liu et al^[14] demonstrated that miR423 inhibited trefoil factor 1 (TFF1) expression, and regulated proliferative and invasive ability of gastric cancer cells in a TFF1-dependent manner. In breast cancer, it has found that CC homozygote in rs6505162 of miR423 is a protective factor for carcinogenesis of breast cancer.^[15] The high miR423-5p expression increased the response to sorafenib treatment in HCC patients involving in autophagy regulation in HCC cells.^[16] Though miR423 has been identified as an oncogene in gastric cancer, its role in HCC is rarely studied. The aim of this study was to assess whether miR423-5p was aberrantly expressed in HCC tissues, and characterize its roles in the cancerous biology of HCC.

 

 

HCC and corresponding nonmalignant tissues were obtained from 115 patients who underwent liver transplantation from 2001 to 2010 in our hospital. Written consent was obtained from patients enrolled in this study. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

 

Cell culture was performed as we previously described.^[17] Briefly, human HCC cell line MHCC-LM3 was grown in DMEM medium (Gibco-Invitrogen, California, USA) containing 10% FBS (SAFC Biosciences, Victoria, Australia), and incubated at 37 �� in a 5% carbon dioxide incubator. Cells were passaged with trypsin (Gibco-Invitrogen) when they reached 90% confluency.

 

To transfect miRNA mimic (Qiagen, Hilden, Germany) and miRNA inhibitors (Qiagen) into HCC cells, we used Lipofectamine 2000 (Invitrogen) according to the manufactuer's instructions. Briefly, 1×10⁶ cells were added into each well of a 6-well plate filled with complete culture medium and grown to 50% confluency. Then the culture medium was replaced with transfection medium (Opti-MEM containing Lipofectamine 2000 and 200 pmol of miR423-5p inhibitor or 5 pmol of miR423-5p mimic). Six hours later, the transfection medium was changed to complete culture medium. The cells were then incubated for another 72 days before use.

 

Trizol (Invitrogen) and miScript II RT Kit (Qiagen) were used to extract RNA from cells. Quantitative real-time PCR was conducted using miScript SYBR Green Kit (Qiagen) on Applied Biosystems ABI 7700 System according to the manufacturer's instructions. ΔΔCt analysis was used to analyze the PCR data.

 

CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) was used to measure the cell growth as we previously described.^[17] Briefly, 24 hours after transfection with miR423-5p mimic or inhibitor, 2×10³ cells were plated in each experimental well of the 96-well plates and culture for 24, 48, 72 and 96 hours. Then the culture media was replaced with DMEM containing 10% CCK-8 and cultured at 37 �� for 1 hour. The absorbance was detected at 450 nm.

 

Cell cycle analysis was conducted as previously reported.^[17] Briefly, 72 hours after transfected with miR423-5p mimic or inhibitor, cells were collected and treated with DNA PREP kit (Beckman Coulter, Florida, USA) according to the instruction. FC-500 flow cytometer (Beckman Coulter) was used to detect the percentage of cells in each phase.

 

Transwell invasion and migration experiments were conducted as previously reported.^[18] Briefly, the polycarbonate membrane of the upper chamber was coated with Matrigel for invasion experiments. Seventy-two hours after transfection with miR423-5p mimic or inhibitor, cells were collected for transwell experiments. Cells (1×10⁴) were plated in the upper chamber containing DMEM without FBS. DMEM with 10% FBS was added into the lower compartment. After 18 (for migration experiments) or 36 (for invasion experiments) hours, the cells attached to the upper side of the membrane were cleaned, and the rest cells were counted using microscopy.

 

Pearson's product-moment correlation coefficient and the Chi-square test were used to assess the association of miR423-5p expression with clinical parameters. To compare difference between the two groups, independent Student's t test was used. The Kaplan-Meier method was used to analyze the association of miR423-5p expression and recurrence-free survival. A P value <0.05 was considered statistically significant.

 

 

To evaluate the expression level of miR423-5p in 115 HCC patients, quantitative real-time PCR was performed. Patients were segregated into miR423-5p low/high expression groups based on receiver operating characteristics analysis in terms of overall survival [cut-off point 1.7, which can achieve the most optimal predictive sensitivity (71.7%) and specificity (60.0%)]. Our results showed that compared with corresponding nonmalignant tissues, 65 (56.5%) of the 115 HCC tissues showed higher miR423-5p expression. However, this overexpression of miR423-5p in HCC was not associated with any tumor features such as vascular invasion, tumor differentiation, tumor size and so on (Table 1). The Kaplan-Meier method was used to assess whether miR423-5p could influence the recurrence-free survival of HCC patients. The results showed that miR423-5p was negatively associated with recurrence-free survival (P<0.002, Fig. 1A). Since the Milan criteria are most frequently used to predict the survival of HCC patients who received liver transplantation and select candidates with HCC for liver transplantation,^[20] we analyzed the prognostic utility of miR423-5p for patients based on the Milan criteria. We found that miR423-5p expression level had no significant effect on recurrence-free survival for patients within the Milan criteria (P=0.158, Fig. 1B). But for patients beyond the Milan criteria, lower miR423-5p expression was correlated with longer recurrence-free survival (P=0.001, Fig. 1C). We thus evaluated the risk indicators for the tumor recurrence (Table 2). Cox-multivariate analysis revealed that portal vein tumor thrombus, tumor size, AFP level and miR423-5p expression were prognostic factors independent of the Milan criteria (Table 3).

 

Quantitative real-time PCR analysis showed that miR423-5p mimic and inhibitor altered the expression of miR423-5p effectively (Fig. 2). Transient overexpression of miR423-5p increased the rate of cell growth and vice versa (Fig. 3). Cell cycle experiments showed overexpression of miR423-5p decreased G1 and increased G2/M cells. Reverse result was obtained by depressing miR423-5p expression with miR423-5p inhibitor, further consolidate our finding that miR423-5p has the ability to promote the growth of HCC cells by facilitating G1/S and S/G2 transition (Fig. 4).

 

The expression of miR423-5p was positively associated with the growth of HCC cells; overexpression of miR423-5p significantly increased the invasion of LM3 cells, while miR423-5p inhibition impaired the invasion of LM3 cells (Fig. 5A). Compared with the control group, cell migration ability was significantly promoted after overexpression of miR423-5p and vice versa (Fig. 5B).

 

 

Though miR423-5p was found to be involved in the carcinogenesis of gastric cancer,^[14] its role in HCC has rarely been studied. The present study showed that miR423-5p was commonly upregulated in HCC tissues, and this upregulation was associated with a lower recurrence-free survival rate of patients after liver transplantation.

 

The Milan criteria are frequently used for patient selection of liver transplantation. More recently, many researchers have argued that these criteria are too strict, and may leave out some patients with a favorable prognosis.^{[19, 20]} Thus, it is important to find new molecular indicators to classify HCC patients out of the Milan criteria. The results of our study indicated that for patients beyond the Milan criteria, lower miR423-5p contributed to longer recurrence-free survival, indicating that miR423-5p could be a new predictor in HCC patients out of the Milan criteria.

 

To evaluate the molecular functions of miR423-5p in HCC, we investigated whether altered miR423-5p expression affected the malignant behaviors of HCC. The pro-proliferative effects of miR423-5p have been well established in gastric cancer.^[14] In this study, we found that miR423-5p could enhance the proliferation of HCC cells. Furthermore, our results demonstrated that overexpression of miR423-5p facilitated G1/S and S/G2 transition, matched well with the previous study in gastric cancer.^[14] The underlying mechanism of miR423-5p on cell proliferation is still not clear. miR423-5p may play its role through the TFF1 dependent pathway. It has been reported that miR423-5p could negatively regulated the expression of TFF1. Liu et al^[14] found that the inhibition of miR423-5p expression increased the mRNA expression of TFF1, and suppressed cyclin D1 and cyclin D3 expression in a TFF1 dependent manner, which are major regulators of the cell cycle transition.^{[21, 22]} Cyclin D could form complexes with CDK4 and CDK6, and phosphorylate retinoblastoma to the inactive form, causing the upregulation of genes (like cyclin E) involved in cell cycle progression.^{[23, 24]}

 

Metastasis is a hallmark of malignant process of HCC.^[25] The clinical data of our study showed a positive association between miR423-5p expression and metastatic ability of HCC cells. We found that cell invasion was significantly increased after overexpression of miR423-5p and vice versa. This finding could also be explained by the negative regulation of TFF1 by miR423-5p. It has been reported that TFF1 increased the invasive ability of human HCT8/S11 cells,and the overexpression of TFF1 showed a migratory and invasive phenotype in epithelial cells.^{[26, 27]} There was a negative correlation between the TFF1 and β-catenin, and transfection with the miR423-5p mimics caused the inhibition of TFF1 expression and the increase of β-catenin expression,^[14] which plays an important role in both cell adhesion and proliferation. However, whether miR423-5p plays its role through other pathways independent of TFF1 awaits further studies.

 

In conclusion, miR423-5p commonly upregulated in HCC cells is associated with poor prognosis. It plays an important role in the proliferation and invasion of HCC cells, and inhibition of miR423-5p reverts the malignant phenotype of HCC. 

 

 

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