Edited by: Tao Liu, University of New South Wales, Australia
Reviewed by: Francesca Lovat, The Ohio State University, United States; Deborah Stroka, University of Bern, Switzerland
This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology
†These authors have contributed equally to this work
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Hepatocellular carcinoma (HCC) is one of the most common gastro-intestinal cancer and one of the leading causes of cancer-related mortality worldwide (
MicroRNAs (miRNAs) represent small non-coding RNAs that bind to the 3′ untranslated regions (UTRs) of mRNA transcripts to negatively regulate target gene expression. miRNAs participate in various crucial biological processes such as cell proliferation, apoptosis, development, and differentiation (
hsa-miR-210 has been widely studied in many diseases. It has been reported that the role of hsa-miR-210 in HBG2 regulation and induction of HbF makes it valuable for the diagnosis and treatment of Sickle cell disease (
In this study, we analyzed the dataset GSE6857 using the R package “limma,” and found 7 significantly up-regulated miRNAs in venous metastasis positive HCC tissues. To explore whether these miRNAs are significant for the prognosis of HCC patients, the survival analysis was conducted and results showed that high hsa-miR-210 expression was correlated with a poor outcome of HCC patients. We then verified the expression of hsa-miR-210 in clinical HCC tissues using RT-qPCR, result showed that hsa-miR-210 was significantly high expressed in tissues with portal vein metastasis. In the analysis with clinicopathological variables, hsa-miR-210 showed a close correlation with AFP, pathological grade, TNM stage, tumor stage and vascular invasion. Furthermore, we also identified the hsa-miR-210 target genes and their biological functions. Our data help to understand the crucial role of hsa-miR-210 in HCC.
We downloaded one dataset from GEO database to screen the venous metastasis-related miRNAs. High throughput miRNA data and clinical information of 375 HCC tissues were downloaded from The Cancer Genome Atlas (TCGA) database to identify miRNAs which are significant for the prognosis of HCC patients. RT-qPCR was conducted to verify the expression of miRNA selected from public database. Other potential functions of selected miRNAs were also analyzed using bioinformatic analysis. The research strategy is embodied in the flowchart (Figure
The Overview of the strategy.
Primary data and platform annotation file of dataset GSE6857 (
The high throughput miRNA data of 375 HCC tissues and corresponding donors' information were downloaded from TCGA (
Aiming to verify the expression of miRNAs selected using public databases, we collected 37 HCC samples with portal vein metastasis and 29 HCC samples without portal vein metastasis. All the clinical specimens were obtained from patients who received liver cancer resection in Zhongnan Hospital of Wuhan University from March of 2018 to October of 2018. Each patient was diagnosed with a histopathological examination and didn't receive any treatment before surgery. Fresh samples were first incubated in RNA stable liquid (RNAlater®) at 4°C for one night and then stored at −80°C for further use. All the work was under approval of Institutional Review Board, and written informed consent was obtained from all participants.
Total RNA was isolated using TRIzol reagent (Invitrogen, Life Technologies) and the quality of RNA was assessed by a Thermo Scientific™ Nanodrop 2000 platform (Thermo Fisher Scientific, Waltham, MA, United States). Then reverse transcription was performed using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, United States). The U6 was used as the endogenous control and was amplified simultaneously with target genes. Designed PCR primers were as follows: U6: Forward, 5′-CTCGCTTCGGCAGCACA-3′ and Reverse, 5′-AACGCTTCACGAATTTGCGT-3′; hsa-miR-210: Forward, 5′-CTGTGCGTGTGACAGC-3′ and Reverse, 5′-GTGCAGGGTCCGAGGT-3′. The reactions started at 95°C for 5 min, followed by 42 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s. All experiments were carried out in duplicate for each data point. The relative expression level of hsa-miR-210 was calculated using the comparative Ct method formula 2−ΔCt.
Clinical characteristics including age, gender, AFP level, pathological grade, TNM stage, tumor stage, lymph node metastasis, distant metastasis, child-pugh score, liver fibrosis, vascular invasion and race of the donors of 375 HCC tissues were downloaded from TCGA. Spearman's rank correlation was employed to analyze the relationship between miRNAs with clinicopathological variables.
miRNA target genes were predicted using 2 prediction databases including miRanda (Good mirSVR score, Conserved miRNA) (
To comprehensively investigate the potential roles of indicated miRNAs in HCC, GO (
After confirming the miRNA target genes, to identify the chief mRNAs underlying the regulation mechanism of these miRNAs, high-throughput mRNA sequencing data from the LIHC project of TCGA database were used (data downloaded on March 6th, 2018). LIHC project contains 374 human HCC samples and 50 normal liver samples, in which the expression of 60244 genes was detected. Gene annotation file was downloaded from Ensembl (GRCh38) (
Given that miRNAs negatively regulate their target genes, we took the intersection of the down-regulated genes in DEGs with the target genes of those up-regulated miRNAs, while the up-regulated genes in DEGs were compared with the target genes of the down-regulated miRNAs. The mutual ones were considered hub target genes.
Significantly differentially expressed genes in microarray data were analyzed using R package “limma,” and the high throughput miRNA data of TCGA were normalized using Trimmed Mean of M-values (TMM) method by R package “edgeR.” The version of R software was 3.2.5.
Kaplan-Meier survival analysis was conducted for univariate survival analysis, while Cox proportional hazards regression analysis was used for multivariate survival analysis (the analysis method is Forward:LR). The log-rank test was used to determine the difference in survival rates between two or more groups. Spearman's rank correlation was employed to analyze the relationship between miRNAs and clinicopathological variables. Differences between two group of samples were compared using Student's
To identify the miRNAs critical for venous metastasis in HCC, we analyzed the differentially expressed miRNAs between HCC tissues with or without venous metastasis. Based on the pre-set screening threshold, the expression of 15 miRNAs was notably different between two groups. hsa-miR-301, hsa-let-7e, hsa-let-7b, hsa-miR-31, hsa-miR-210, hsa-miR-371, and hsa-miR-183 were upregulated while hsa-miR-367, hsa-miR-22, hsa-miR-182, hsa-miR-100, hsa-miR-1b-1, hsa-miR-101, hsa-miR-124a-1, and hsa-miR-101 were down-regulated in HCC tissues with venous metastasis, in comparison with those in HCC tissues without venous metastasis (Table
Significantly differentially expressed miRNAs in venous metastasis positive HCC tissues.
hsa-miR-301 | 0.842786 | 0.000267078 | Up |
hsa-let-7e | 0.847988 | 9.04E-11 | Up |
hsa-let-7b | 0.850236 | 2.94E-12 | Up |
hsa-miR-31 | 0.853136 | 5.34E-07 | Up |
hsa-miR-210 | 0.868472 | 1.68E-06 | Up |
hsa-miR-371 | 0.991324 | 1.92E-10 | Up |
hsa-miR-183 | 1.027867 | 1.14E-05 | Up |
hsa-miR-367 | −1.08021 | 1.81E-06 | Down |
hsa-miR-22 | −0.9947 | 0.00055601 | Down |
hsa-miR-182 | −0.94816 | 0.00023209 | Down |
hsa-miR-100 | −0.94307 | 9.38E-07 | Down |
hsa-miR-1b-1 | −0.87813 | 0.00015521 | Down |
hsa-miR-101 | −0.84328 | 0.000209509 | Down |
hsa-miR-124a-1 | −0.81885 | 2.27E-08 | Down |
hsa-miR-101 | −0.80452 | 9.15E-10 | Down |
To identify the miRNAs associated with the prognosis of HCC patients, we used normalized expression of 7 up-regulated miRNAs and the survival data (overall survival time and living state) of 375 HCC patients in TCGA. We found that the overall survival rate of patients with high hsa-miR-210 expression was significantly lower than the survival rate of patients with low hsa-miR-210 expression. Other miRNAs such as hsa-let-7e, hsa-let-7b, hsa-miR-31, hsa-miR-371 and hsa-miR-183 had no significant influence on the survival rate of HCC patients (Figure
The correlation of 6 up-regulated miRNAs with overall survival of HCC patients. Survival analysis of hsa-miR-210
A multivariate survival analysis was conducted to explore whether hsa-miR-210 is an independent prognostic factor for HCC. At first, a univariate survival analysis was conducted. Clinical characteristics including age, gender, AFP level, pathological grade, TNM stage, tumor stage, lymph-node metastasis, distant metastasis, child-pugh score, liver fibrosis, vascular invasion and race were subject to Kaplan-Meier analysis to identify the factors influencing the survival rate of HCC patients. As a result, AFP, TNM stage, tumor stage and distant metastasis were significantly associated with the survival rate of HCC patients (Table
Univariate analysis of factors affecting overall survival of HCC patients.
Age | < 50 | 71 | 0.173 |
≥50 | 301 | ||
Gender | Male | 253 | 0.309 |
Female | 119 | ||
AFP | < 20 | 150 | |
≥20 | 130 | ||
Grade | 1 | 55 | 0.858 |
2 | 176 | ||
3 | 123 | ||
4 | 13 | ||
TNM stage | I | 173 | |
II | 85 | ||
III | 85 | ||
IV | 5 | ||
Tumor stage | T1 | 183 | |
T2 | 93 | ||
T3 | 80 | ||
T4 | 13 | ||
Lymph-node metastasis | N0 | 254 | 0.317 |
N1 | 4 | ||
Distant metastasis | M0 | 269 | |
M1 | 4 | ||
Child-pugh | A | 219 | 0.357 |
B | 21 | ||
C | 1 | ||
Liver fibrosis | No | 75 | 0.316 |
Yes | 139 | ||
Vascular invasion | No | 206 | 0.162 |
Yes | 110 | ||
Race | Asian | 160 | 0.129 |
Others | 202 |
Cox proportional hazards regression analysis was then conducted to carry out the multivariate survival analysis. Five variates, which were demonstrated to be significant in the univariate analysis, entered the Cox hazard model to test their independent impact on HCC. Based on this analysis, high hsa-miR-210 level was identified as an independent prognostic factor for HCC (HR 2.627; 95% CI 1.516–4.551;
Cox proportional hazards regression analysis of factors affecting overall survival of HCC patients.
hsa-miR-210 | 0.966 | 0.280 | 0.001 | 2.627 | 1.516–4.551 |
Distant metastasis | 1.638 | 0.602 | 0.007 | 5.146 | 1.580–16.760 |
Based on the previous analysis using public database, we found high hsa-miR-210 expression in venous metastasis HCC tissues. Aiming to verify this expression trend, we conducted RT-qPCR assay on 37 HCC samples with portal vein metastasis and 29 HCC samples without venous metastasis. The results showed that the mean value of hsa-miR-210 in portal vein metastasis positive tissues and negative HCC tissues were 0.00098 vs. 0.00032, respectively (
The expression of hsa-miR-210 in HCC samples with positive and negative portal vein metastasis. The relative expression was determined using RT-qPCR. hsa-miR-210 levels in the portal vein metastasis positive tissues were higher than those in portal vein metastasis negative tissues.
To clarify the relationship between HCC and hsa-miR-210, we analyzed the correlation of hsa-miR-210 with HCC clinicopathological variables.
Notably, our analysis showed that hsa-miR-210 expression was positively associated with vascular invasion. What's more, high expression of has-miR-210 portends high AFP level, pathological grade, TNM stage and tumor stage, which verified the essential role of hsa-miR-210 in influencing the degree of tumor malignant. But its expression had no significant correlation with age, gender, Lymph-node metastasis, distant metastasis, child-pugh, liver fibrosis and race (Table
The relationship between hsa-mir-210 and the clinicopathological variables of HCC.
Age | < 50 | 71 | 0.072 | 0.164 |
≥50 | 300 | |||
Gender | Male | 253 | 0.093 | 0.074 |
Female | 119 | |||
AFP | < 20 | 150 | 0.095 | 0.112 |
≥20 | 131 | |||
Grade | 1 | 55 | ||
2–4 | 313 | |||
TNM stage | I–II | 258 | ||
III–IV | 90 | |||
Tumor stage | T0–T1 | 182 | ||
T2–T4 | 187 | |||
Lymph-node metastasis | N0 | 254 | 0.061 | 0.332 |
N1 | 4 | |||
Distant metastasis | M0 | 269 | −0.005 | 0.934 |
M1 | 4 | |||
Child-pugh | A | 220 | 0.028 | 0.67 |
B–C | 22 | |||
Liver fibrosis | No | 75 | −0.087 | 0.206 |
Yes | 140 | |||
Vascular invasion | No | 206 | ||
Yes | 111 | |||
Race | Asian | 160 | −0.024 | 0.647 |
Others | 202 |
To provide deep insight into the role of hsa-miR-210 in the HCC initiation and progression, hsa-miR-210 target genes were predicted by two prediction databases. 684 target genes and 4,059 target genes were found from miRanda database and TargetScan database, respectively. Among them, a total of 168 common genes were identified as hsa-miR-210 target genes (Table
Regarding the function and pathway enrichment analysis of the target genes, the GO analysis of the hsa-miR-210 target genes was summarized in Figure
GO analysis of hsa-miR-210 target genes. GO enrichment of target genes in biological process ontology
KEGG analysis of hsa-miR-210 target genes. The circle represents target genes; the diamonds represent signaling pathways. Circles with the same color with diamonds indicate the target genes participating in the corresponding signaling pathways. The relationship between each signaling pathway and each gene is represented by a gray line. The network was generated by Cytoscape_3.5.1.
To identify the chief mRNAs that participate in the regulation of hsa-miR-210, the high-throughput mRNA sequencing data from TCGA was introduced. The differential expression analysis using R package “edgeR” revealed 4895 differentially expressed genes. Among them, 3847 genes were up-regulated while 1048 genes were down-regulated (Figure
The aberrantly expressed mRNAs in HCC tissues. Aberrantly expressed miRNAs calculated by edgeR R. 3847 up-regulated and 1048 down-regulated miRNAs were found
The intersection between the differentially expressed genes and the predicted hsa-miR-210 target genes was set according to the negative correlation between miRNAs and their target genes. hsa-miR-210, which was up-regulated in HCC, significantly down-regulated 7 target genes (JDP2, SAA1, CR1, SHMT1, KMO, NOL4, and EHD3) in HCC tissues.
Metastasis is one of the main malignant characters of cancers. It is responsible for up to 90% of cancer-related mortality (
As a hypoxia-inducible factor, hsa-miR-210 has been confirmed by multiple studies to play a crucial role in tumor proliferation, metastasis and invasion. High hsa-miR-210 expression promotes the metastasis and indicates poor prognosis of gastric cancer and colorectal cancer (
In this study, through analyzing the miRNA expression profile of HCC tissues with or without venous metastasis, we found that hsa-miR-210 was significantly increased in venous metastasis positive samples. Moreover, high hsa-miR-210 expression was closely correlated with poor HCC outcome in both univariate and multivariate survival analysis. Additionally, hsa-miR-210 expression was positively correlated with AFP, pathological grade, TNM stage, tumor stage and vascular invasion, suggesting that hsa-miR-210 indicate the extent of HCC malignancy. It has been proposed that hsa-miR-210 promotes the metastasis of hypoxic HCC cells by directly targeting the metastasis suppressor vacuole membrane protein 1 (VMP1) (
To explore the biological function of hsa-miR-210 and the signaling pathways it regulates, we used two miRNA target gene prediction databases to obtain hsa-miR-210 target genes. Among these genes, the expression of BDNF, ZNF462, GIT2, GPD1L, ISCU, NFIC, FGFRL1, MNT, EFNA3, and E2F3 have been shown to be regulated by hsa-miR-210 in previous studies (
In conclusion, this study demonstrates that hsa-mir-210 promote venous metastasis in HCC. hsa-mir-210 may be a promising biomarker for evaluating the prognosis of HCC patients.
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by Science and Technology Innovation Fostering Foundation of Zhongnan Hospital of Wuhan University (cxpy20160025, cxpy2017080), and National Natural Science Foundation of China (No. 81672114). This work was also funded by Applied Basic Research Program of Science and Technology Bureau Foundation of Wuhan (No. 2016060101010054) and Wuhan City health and family planning medical talented youth development project.
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