Abstract
Background/Aim: Hepatoma-derived growth factor (HDGF) is involved in the progression of hepatocellular carcinoma (HCC). The present study assessed the epigenomic changes in hepatoma-derived cells through HDGF stimulation. Materials and Methods: We used two hepatoma-derived cell lines (HepG2 and SK-Hep1) and searched for microRNAs whose expression commonly changed in response to HDGF administration. We further explored a genetic database to investigate the association of the candidate microRNAs with the survival of HCC patients. Results: Despite both HepG2 and SK-Hep1 cells being categorized as hepatoma-derived cells, the microRNA profile differed between these two lines. However, HepG2 and SK-Hep1 cells shared 30 up-regulated and 2 down-regulated microRNAs. Of these, miR-6072 and miR-3137 were significantly associated with a poor prognosis in HCC patients. Conclusion: We identified two candidate microRNAs whose expression increased in response to HDGF stimulation. Both these molecules were associated with a poor prognosis of HCC patients.
Hepatocellular carcinoma (HCC) is a common malignant disease with an unfavorable prognosis. Although several new drugs have been developed, there is no definitive therapy against HCC, and new treatment strategies for HCC are warranted (1, 2).
The hepatoma-derived growth factor (HDGF) was recently identified as a novel factor involved in HCC growth (3, 4). We have previously shown that HDGF promotes the proliferation of hepatoma cells in vitro and in vivo (4, 5). In addition, a high expression of HDGF was shown to be related to a poor clinical outcome for HCC patients (6-9). HDGF is considered to translocate to the nucleus and regulate the expression of target genes by binding to their promoter regions in the genomic DNA (10).
Recently, several non-genomic/epigenetic factors, such as microRNAs, have been suggested to play an important role in various diseases (11, 12). MicroRNAs are known to be non-coding short RNA molecules that bind to complementary sequences and influence the function of target genes.
Concerning HCC changes in microRNAs of hepatoma-derived cells and the importance of HDGF signaling have not been adequately clarified. For this reason, in the present study, we investigated which microRNAs showed a change in expression in response to HDGF stimulation and were associated with a poor prognosis of HCC patients.
Materials and Methods
Cell culture. We selected HepG2 and SK-Hep1 cells, as we previously confirmed the in vivo tumor growth promotion by HDGF in experiments with these cell lines (4). Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Sigma-Aldrich Japan, Tokyo, Japan). Cells were plated into 12 well-dishes (Corning International, Tokyo, Japan) at a density of 1×105 cells/well. Twenty-four hours after the inoculation, the cells were exogenously stimulated with the administration of recombinant HDGF (Abcam, Tokyo, Japan; product code: ab132259) for 48 h (final concentration: 100 ng/ml). At the same time, cells without HDGF administration were also cultured and used as the control cells.
RNA extraction and microRNA analysis. Total RNA was extracted from cultured cells using the miRNeasy Mini Kit (Qiagen-Japan, Tokyo, Japan) according to the manufacturer's instructions. The isolated RNA samples from the HDGF-stimulated HepG2 cells and control (untreated) HepG2 cells were used to search for the microRNAs whose expression changed in response to HDGF administration. We screened for microRNAs with an increased (≥1.5-fold) or a decreased expression (≤0.67-fold) following HDGF administration using the microarray system Toray 3D Gene, Human miRNA Ver. 21 (Toray, Kamakura, Japan) (13). We also used two RNA samples of SK-Hep1 cells with and without HDGF administration and compared the expression levels of microRNAs. Finally, we determined which microRNAs with changes in expression were commonly observed among the two RNA sets and considered them potentially functional microRNAs in response to HDGF (Figure 1).
Flowchart to determine the microRNAs whose expression changed in response to hepatoma-derived growth factor (HDGF).
Association of the microRNAs with the survival of primary liver cancer. We assessed the association of the microRNAs with the prognosis of patients with liver cancer in the UCSC Xena platform, which provides cancer genomics datasets (14). Based on the data from the system (GDC TCGA Liver Cancer), a total of 351 patients were divided into 2 groups according to the median copy number of each microRNA, and Kaplan-Meier survival curves were generated. MicroRNAs with a p-Value less than 0.05 according to the log-rank test was considered to have a significantly different prognosis.
Results
From the two RNA sample sets we assessed the changes in the microRNA expression in response to HDGF treatment (Figure 1). For HepG2 cells, we identified an increased expression of 632 microRNAs and a decreased expression of 37 microRNAs. For SK-Hep1 cells, we identified an increased expression of 45 microRNAs and a decreased expression of 213 microRNAs. Despite both HepG2 and SK-Hep1 cells being categorized as hepatoma-derived cells, the microRNA profile differed markedly between these two lines. However, HepG2 and SK-Hep1 cells shared 30 up-regulated microRNAs and 2 down-regulated microRNAs, suggesting that these microRNAs may be involved in the mechanism underlying cellular proliferation stimulated by HDGF. These 32 micro RNAs were considered potentially involved in HDGF-associated HCC growth (Table I).
MicroRNAs suggested to commonly increase or decrease in HepG2 and SK-Hep1cells in response to exogenously supplied HDGF.
Association of two HDGF-related microRNAs with the 10-year survival of patients with primary liver cancer. Using the UCSC Xena platform, we found that the copy numbers of two microRNAs in the cancerous tissues were related to the prognosis of the HCC patients. The Kaplan-Meier survival curves of miR-6072 (A) and miR-3137 (B) are shown.
We next assessed the association of these 32 candidates with the prognosis of patients with liver cancer. Among the microRNAs, increased copy numbers of miR-6072 and miR-3137 were related to the overall survival of HCC patients, suggesting that increases in these two microRNAs are associated with a poor clinical course in patients with a high HDGF expression (Figure 2).
Discussion
HCC is a major health concern worldwide, and we have previously reported that the HDGF protein as a novel mitogenic factor for HCC. In the present study, we identified 32 microRNAs whose expression was commonly changed in response to HDGF administration in two different hepatoma cell lines (HepG2 and SK-Hep1), suggesting that they may contribute to HDGF-related HCC growth.
MicroRNAs are considered to affect clinical features in various diseases (11, 12), and several of them have been proposed as candidates that are potentially involved in the malignant characteristics caused by the HDGF expression (15-20). However, the reported microRNAs vary between studies, depending on the cell lines used. In order to detect commonly observed changes in hepatoma cells, we used two different cell lines, as we previously confirmed the in vivo tumor growth promotion by HDGF in experiments with these cell lines (4). Since we identified 32 microRNAs commonly altered between the different hepatoma-derived cell lines, we hoped that some of these microRNAs would be relevant for the clinical course of HCC patients. Two of these microRNAs were in fact associated with a poor prognosis of HCC patients, suggesting that these commonly increased microRNAs might be involved in HDGF-related cellular growth. Interestingly, to our knowledge, the association of these two microRNA molecules with a malignant disease has not been previously reported, though recent studies showed that some microRNAs and their target genes were suggested to relate to the prognosis of the HCC patients (21-23). In addition, the use of two different cell lines detecting common changes in the expression of these microRNA molecules make are results reproducible. A high HDGF expression is known to be associated with various malignant diseases (24, 25), which highlights the importance of our approach as a research on this topic.
Several limitations associated with the present study warrant mention. First, we selected two microRNAs based on the in vitro study; however, the association of the prognosis in the HCC patients was evaluated in a cohort from a public genetic database (14). This evaluation using this type of patient cohort may have helped obtain independent and reliable results (26), however, this meant we were unable to include detailed clinical data in the current study. Second, our results were obtained using two cell lines and one cohort. A re-evaluation with additional cell lines and a different cohort should be conducted to confirm the results. Third, the function of the determined microRNAs and how these contribute to the poor survival of HCC patients remain unclear and should be explored in a subsequent study.
In conclusion, we identified two microRNAs that were induced by HDGF and were potentially associated with the poor survival of HCC patients.
Acknowledgements
The Authors thank Matsushita Y, Kanazawa N, Inui S, Higuchi Y, Fujii S, Kido H and Minemoto K (Hyogo College of Medicine) for their technical and secretarial assistance. This research was funded in part by grants from the Grants-in-Aid for Scientific Research (KAKENHI-16K09383).
Footnotes
Authors' Contributions
HE: Study design, data analysis, funding acquisition and the original draft writing; HN (Hideji Nakamura): data analysis and manuscript writing; HN (Hiroki Nishikawa), TN, YI: performed the experiments and data analysis; SN: study design, funding acquisition and supervising the study. HI: study design, data interpretation, review and editing the manuscript. All Authors read and edited the manuscript and approved the final version of the manuscript.
This article is freely accessible online.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received May 29, 2020.
- Revision received June 19, 2020.
- Accepted June 22, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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