High expression of long non‑coding HOTAIR correlated with hepatocarcinogenesis and metastasis

  • Authors:
    • Da‑Ni Zhong
    • Yi‑Huan Luo
    • Wei‑Jia Mo
    • Xin Zhang
    • Zhong Tan
    • Na Zhao
    • Si‑Min Pang
    • Gang Chen
    • Min‑Hua Rong
    • Wei Tang
  • View Affiliations

  • Published online on: November 7, 2017     https://doi.org/10.3892/mmr.2017.7999
  • Pages: 1148-1156
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Abstract

HOX transcript antisense RNA (HOTAIR), a newly discovered long noncoding RNA (lncRNA), has been reported to be a poor prognostic marker in many types of cancers. The current study attempted to investigate the biological roles and clinicopathlogical implications of HOTAIR in hepatocellular carcinoma (HCC), as well as understand the molecular mechanisms of HOTAIR in HCC progression. HOTAIR expression in 95 HCC patients with paired HCC tissues and adjacent non‑cancer tissues were investigated using quantitative reverse transcription‑polymerase chain reaction. The association between HOTAIR expression and clinicopathological features was assessed. The effects of HOTAIR were examined in vitro assays by silencing the lncRNA. Pathway analyses were performed to illustrate the biological functions of the HOTAIR and coexpression genes. The expression level of HOTAIR was observed significantly higher in the HCC tissue than the adjacent non‑tumor tissue. HOTAIR expression levels were significantly higher in tumor samples from patients with distant metastasis, advanced stage, portal vein tumor embolus, vasoinvasion, tumor capsular infiltration or positive nm23 expression than those from patients without these conditions, correspondingly. The silencing of HOTAIR in liver cancer cells induced the inhibition of cell proliferation and promotion of apoptosis. Several pathways such as extracellular matrix‑receptor interaction, focal adhesion, pathways in cancer were annotated with the HOTAIR and coexpression genes. In summary, the present analysis indicates that HOTAIR might be an oncogene in HCC. It functions though promoting tumor cell growth and inhibiting apoptosis. HOTAIR may potentially be involved in HCC metastatic progression by several pathways correlated to cell adhesion, and may be a therapeutic target in future.

Introduction

Hepatocellular carcinoma (HCC) is a commonly diagnosed cancer worldwide, with ~782,500 newly diagnosed cases and >700,000 HCC-related deaths in 2012 (1). Therapeutic strategies have been improved greatly including liver surgical resection, liver transplantation, chemotherapy and other modalities for HCC (2). However, the prognosis remains extremely dismal following surgical resection or liver transplantation in HCC patients, of which a long-term 5-year survival was reported ranging from 25–39%, and a 5-year recurrence rate for whom had surgical treatment was reported ranging from 65–80% (35). Identification of a reliable oncogene is essential to predict the propensity of metastasis, prognosis and suggest further therapeutic decisions. Besides, integrated assessment for the underlying mechanisms has potential to identify novel therapeutic targets.

Long noncoding RNAs (lncRNAs) with >200 nucleotides are transcripts non-coding for proteins. LncRNAs have recently been found to involve in the development and progression of various kinds of cancer as key regulators of multiple biological processes (6). Typically, the lncRNA HOX transcript antisense intergenic RNA (HOTAIR) has been reported as a potential prognostic biomarker for cancers since it was discovered in 2007 (7). HOTAIR as a long and polyadenylated RNA that does not code for protein, and is expressed from the development HOXC locus located on chromosome 12q13.13 (7). HOTAIR overexpression has been extensively observed in multiple cancers and is reported to predict poor prognosis in esophageal carcinoma, gastric cancer, colorectal cancer, HCC, breast cancer and other types of cancer (8,9). There have been plenty of investigations carried out to study the potential molecular mechanisms of HOTAIR in cancer progression. Likewise, efforts were made to evaluate the expression pattern of HOTAIR in HCC and research its clinical implications (10,11). However, the sample size ranged from 60 to 64 HCC patients and clinicopathological parameters of each individual previous study were relatively limited. The absence of bioinformatics analysis of the previous studies might have missed essential information for functional evaluation.

Therefore, the current study incorporating a larger HCC sample size, comprehensive clinicopathological parameters, and combined analyses of in vitro assays and functional assessments was designed to: 1) Evaluate the expression of HOTAIR in HCC tissues and adjacent non-tumor tissues, 2) evaluate the association between HOTAIR expression and clinical, histological, pathological and other biological features in HCC patients, 3) test the effects of HOTAIR on HCC using liver cancer cell lines, 4) find out genes associated or coordinated with HOTAIR and enrich in pathways.

Materials and methods

Patients

Eligible HCC participants were enrolled from the First Affiliated Hospital of Guangxi Medical University (Nanning, China) from March 2010 to December 2011. All participated samples were pathologically diagnosed with HCC by two independent pathologists. In brief, the clinicopathological parameters were collected and summarized in Table I, including age, gender, differentiation, tumor size, tumor nodes, clinical TNM stages, portal vein tumor embolus, metastasis, capsular infiltration and cirrhosis, vasoinvasion as well as other biomarkers, such as serum alpha fetal protein level detected by ELISA, nm23, P53, P21, vascular endothelial growth factor, microvessel density stained by CD34 through immunohistochemistry. The Ethical Committee of First Affiliated Hospital, Guangxi Medical University (Nanning, China) approved the current study. Informed consents were obtained from all the enrolled patients. Related research procedure was conducted based on the Helsinki Declaration.

Table I.

Relationship between the expression of HOTAIR and clinicopathological features in HCC.

Table I.

Relationship between the expression of HOTAIR and clinicopathological features in HCC.

Relative HOTAIR expression

Clinicopathological featuresn (patients)Mean ± standard deviationtP-value
Tissue
  Adjacent liver95 4.7600±1.789415.823a<0.001
  HCC95 6.3618±1.99692
Age
  <5046 6.1169±1.992931.2370.219
  ≥5049 6.6226±1.98960
Gender
  Male75 6.3641±1.961630.0220.982
  Female20 6.3530±2.17753
Differentiation
  High  6 7.1333±1.58072 F=1.259b0.289
  Moderate60 6.4928±2.19732
  Low29 5.9310±1.55267
Size
  <5 cm18 6.3317±2.177440.0710.944
  ≥5 cm77 6.3688±1.96758
Tumor nodes
  Single52 6.0300±1.84271−1.8050.075
  Multiple43 6.7630±2.12144
Metastasis
  No46 5.8957±1.88997−2.2520.027
  Yes49 6.7994±2.01415
Clinical TNM stage
  I~II22 5.4091±1.16452−3.5410.001
  III~IV73 6.6489±2.10944
Portal vein tumor embolus
  No63 6.0397±1.72295−2.0390.047
  Yes32 6.9959±2.35131
Vasoinvasion
  No59 6.0231±1.69528−2.0000.050
  Yes36 6.9169±2.33170
Tumor capsular infiltration
  No45 5.8644±1.73548−2.3580.020
  Yes50 6.8094±2.12455
HBV
  −17 7.0106±1.836621.4880.140
  +78 6.2204±2.01345
AFP
  −41 6.2466±2.162530.4910.625
  +38 6.4779±2.01384
Cirrhosis
  No50 6.6014±1.97817−1.2360.219
  Yes45 6.0956±2.00590
NM23
  −20 5.5600±1.10043−2.9200.005
  +75 6.5756±2.12943
P53
  −40 6.3548±1.91948−0.0290.977
  +55 6.3669±2.06894
P21
  −62 6.3227±1.92007−0.2600.795
  +33 6.4352±2.16279
VEGF
  −25 5.8720±1.74464−1.4370.154
  +70 6.5367±2.06297
Ki-67
  Low47 6.2809±1.94552−0.3890.689
  High48 6.4410±2.06347
MVD
  Low47 6.2894±2.21763−0.3480.728
  High48 6.4327±1.77532

a Paired t test

b ANOVA test was performed. AFP, alpha fetal protein; HBV, hepatitis B virus; HCV, hepatitis C virus; MVD, microvessel density; SD, standard deviation; TNM, tumor node metastasis; VEGF, vascular endothelial growth factor; HCC, hepatocellular carcinoma; MTDH, expression of metadherin; HOTAIR, HOX transcript antisense intergenic RNA.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of HOTAIR expression levels

RNA isolation and RNA normalization were performed by the method descried in our previous reports (12,13). Extracted RNA was analyzed by RT-qPCR using Applied Biosystems PCR7900 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The expression of HOTAIR was assessed by reverse transcription RT-qPCR kits (Qiagen GmbH, Hilden, Germany) based on the instructions described previously (14,15). Human GAPDH was selected as the housekeeping reference for HOTAIR expression analysis. Primer sequences used in the PCR were as follows: HOTAIR 5′-GAGGGAGCCCAGAGTTACAGA-3′ (sense), 5′-TCCTCCATTTCAGCCTTTCT-3′ (antisense) and GAPDH: 5′-TGACTTCAACAGCGACACCCA-3′ (sense), 5′-CACCCTGTTGCTGTAGCCAAA-3′ (antisense). The HOTAIR expression was calculated with the formula 2−∆∆Cq (16).

Cell line and culture

The liver cancer cell line SMMC-7221 was purchased from Shanghai Institute of Cell Biology (Shanghai, China). The cell line was cultured at 37°C in 5% carbon dioxide in a humidified incubator following the recommended culture conditions (14,15).

Lentiviral infection and gene transfection

Lentivirus containing HOTAIR shRNA segments (HOTAIR shRNA sequence was 5′-GAACGGGAGUACAGAGAGAUU-3′) was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). SMMC-7721 cells were infected with the viral suspension. HOTAIR 3′ domain (nucleotides 1 to 300 of HOTAIR) and 5′ domain (nucleotides 1500 to 2146 of HOTAIR) were inserted into pcDNA3.1 (+) plasmid. pcDNA3.1 (+)-3′ domain and pcDNA3.1 (+)-5′ domain plasmids were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The authors collected the transfected cell samples at 24 and 48 h points following transfection.

Analysis of cell line

After the SMMC-7721 cells were transfected with HOTAIR shRNA stably, the following steps to investigate the role and molecular mechanisms of HOTAIR in HCC progression were conducted at different time points (24 and 48 h).

i) Cell viability was evaluated using fluorimetric detection of resorufin according to the manufacturer's protocols (CellTiter-Blue Cell Viability Assay, G8080, Promega Corporation, Madison, WI, USA). FL600 fluorescence plate reader was used for fluorimetry (ex: 560 nm/em: 590 nm; Bio-Tek Instruments, Inc., Winooski, VT, USA).

ii) Cell growth and apoptosis were evaluated with Hoechst 33342 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and propidium iodide (Sigma-Aldrich; Merck KGaA) double fluorescent chromatin staining. The number of cells in different states (necrotic, apoptotic and viable) were counted under ×200 magnification, and each type of cell was calculated in 10 diverse fields in each well. The mean values compared with the mock control group were shown as the final results.

iii) A microplate reader was used to measure the cell proliferation ability (Scientific Multiskan MK3, Thermo Fisher Scientific, Inc.) at 490 nm after the liver cancer cells were processed by colorimetric tetrazolium (MTS) assay based on instruction (CellTiter96 Aqueous One Solution Cell Proliferation Assay G3580, Promega Corporation).

iv) The SMMC-7221 cells were plated into 96-well plate and treated as indicated. The cell lysates were harvested for caspase activity assays with caspase-Glo3/7 reagents (Promega Corporation). The luminescence of each sample was measured with a FL600 fluorescence plate-reading luminometer.

Construction of HOTAIR co-expression network and biological function analysis

HOTAIR coexpression genes were calculated with EBcoexpress package of R (17). The gene network map was drawn in Cytoscape Web (18), thereby providing figures to visually understand the relationship among coexpression genes. To clarify the functions of the HOTAIR and coexpression genes, the authors used the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.ad.jp/kegg/) and Gene Ontology (GO) (http://www.geneontology.org) analysis. GO can assign genes into hierarchical categories based on the associated aspects: Biological process, cellular component and molecular function (19). P<0.05 was considered to indicate a statistically significant difference and was set as the threshold in GO and KEGG analyses.

Statistical analysis

SPSS software (version, 20.0; IBM SPSS, Armonk, NY, USA) was used for statistical analyses. For the analysis of the significance of two groups, the authors performed Student's test. Accordingly, they also applied one-way analysis of variance followed by Dunnett's post hoc test to analysis data from experiments in vitro. The associations between HOTAIR expression and clinicopathological parameters were tested using the Spearman rank correlation. The value of HOTAIR for differentiating the HCC from noncancerous tissues was tested by receiver operating characteristic (ROC) curve. All tests were two-sided, and P<0.05 was considered to indicate a statistically significant difference.

Results

HOTAIR expression in HCC

A total of 95 eligible patients with diagnosed HCC, available cancer and adjacent non-cancer tissues were finally enrolled in the study. The age of the included individuals ranged from 29 to 82 years old, and the mean age was 52 years. HOTAIR expression was evaluated in 95 paired FFPE HCC and adjacent non-tumor tissues using RT-qPCR. The HOTAIR expression was significantly higher in HCC tissues, compared with the adjacent non-tumor tissue (at least 2 cm away from the tumor node; Fig. 1). All the clinicopathological parameters are presented in Table I.

HOTAIR expression and clinicopathological features of HCC

The relative expression of HOTAIR was 6.7994±2.01415 in tissues with distant metastasis, significantly higher than those without distant metastasis (5.8957±1.88997, P=0.027). In patients with advanced (III–IV) stage, higher HOTAIR expression (6.6489±2.10944) was observed than patients in early (I~II) stage (5.4091±1.16452, P=0.001). Compared with those without portal vein tumor embolus (6.0397±1.72295), the expression level of HOTAIR was obviously higher in patients with portal vein tumor embolus (6.9959±2.35131, P=0.047). Similarly, HOTAIR expression was remarkably higher in patients with vasoinvasion (6.9169±2.33170) than those without vasoinvasion (6.0231±1.69528, P=0.005). In addition, the level of HOTAIR distinctly upregulated in patients with tumor capsular infiltration (6.8094±2.12455) than those with complete liver capsule (5.8644±1.73548, P=0.020). In addition, HOTAIR expression was visibly higher in positive nm23 expression group of patients (6.5756±2.12943) than negative nm23 expression group (5.5600±1.10043, P=0.005). In Spearman analysis, the current result indicated that the relative expression of HOTAIR was significantly positive correlated with distant metastasis (r=0.210, P=0.041), clinical TNM stage (r=0.295, P=0.004), tumor capsular infiltration (r=0.235, P=0.022) and nm23 (r=0.228, P=0.027). Nevertheless, no association was identified between HOTAIR expression and others clinicopathological features (P>0.05), including age, gender, differentiation, size and other parameters (Table I). Collectively, the results above demonstrated that the expression level of HOTAIR was associated with specific clinicopathological features that related to tumor deterioration and HOTAIR may play a vital role in promoting HCC progression.

Diagnostic significance of HOTAIR in HCC

The authors took advantage of ROC analysis to calculate the potential value of HOTAIR for diagnosing HCC. ROC analysis demonstrated that the Area Under The Curve (AUC) of HOTAIR was 0.715 [95% confidence interval (CI): 0.643–0.787] with a sensitivity of 45.3% and a specificity of 86.3% in distinguishing HCC and the cut off was 6.45. The ROC analysis of HOTAIR and clinicopathological features revealed that HOTAIR levels remarkably discriminated HCC patients with distant metastasis, advanced TNM stage, tumor capsular infiltration and positive nm23 expression and the AUC was 0.621 (95%CI: 0.509–0.734, sensitivity=24.5%, specificity=97.8%), 0.701 (95%CI: 0.595–0.808, sensitivity=58.9%, specificity=81.8%), 0.636 (95%CI: 0.524–0.747, sensitivity=74.0%, specificity=51.1%) and 0.661 (95%CI:0.552–0.770, sensitivity=49.3%, specificity=90.0%), respectively.

Expression of HOTAIR in the cell line and transfected cells

The behavior alterations of SMMC-7221cells on viability, proliferation, caspase-3/7 and apoptosis are presented in Fig. 2. The viability and proliferation of HOTAIR siRNA cells were significantly inhibited compared to that in control cells in 48 h. Both levels of the caspase3/7 activity and apoptosis in the transfected cells exhibited higher than those in the control cells. The efficacy of the apoptosis measurement with SMMC-7221 cell line is presented in Fig. 3.

GO and KEGG pathway annotation

A total of 150 genes were identified in the coexpression analysis. The coexpression genes were then annotated through GO and KEGG pathway analyses. GO can organize genes into hierarchical categories and show the gene functions based on regulatory network of biological process, cellular component and molecular functions (Table II) (19). The genes showed dominant enrichments in the KEGG pathways of ECM-receptor interaction, focal adhesion and pathways in cancer (Table III).

Table II.

Gene ontology terms enriched in HOTAIR and coexpression genes (P<0.05, FDR<0.1).

Table II.

Gene ontology terms enriched in HOTAIR and coexpression genes (P<0.05, FDR<0.1).

GO analysisCountP-valueFDR
Biological process
  GO:0001501~skeletal system development26 2.78×1018 4.37×1015
  GO:0048598~embryonic morphogenesis20 3.54×1012 5.57×109
  GO:0048705~skeletal system morphogenesis13 8.17×1011 1.29×107
  GO:0048706~embryonic skeletal system development11 4.84×1010 7.61×107
  GO:0003002~regionalization14 5.47×109 8.60×106
  GO:0009952~anterior/posterior pattern formation12 1.44×108 2.27×105
  GO:0007389~pattern specification process15 2.58×108 4.07×105
  GO:0030326~embryonic limb morphogenesis10 2.87×108 4.52×105
  GO:0035113~embryonic appendage morphogenesis10 2.87×108 4.52×105
  GO:0035108~limb morphogenesis10 8.95×108 1.41×104
  GO:0035107~appendage morphogenesis10 8.95×108 1.41×104
  GO:0060173~limb development10 1.26×107 1.99×104
  GO:0048736~appendage development10 1.26×107 1.99×104
  GO:0048704~embryonic skeletal system morphogenesis  8 3.23×107 5.09×104
  GO:0006355~regulation of transcription, DNA-dependent35 5.25×107 8.26×104
  GO:0051252~regulation of RNA metabolic process35 8.87×107 1.40×103
  GO:0048562~embryonic organ morphogenesis10 1.12×106 1.77×103
  GO:0048568~embryonic organ development11 1.12×106 1.77×103
  GO:0043009~chordate embryonic development14 2.27×106 3.57×103
  GO:0009792~embryonic development ending in birth or egg hatching14 2.50×106 3.94×103
  GO:0006357~regulation of transcription from RNA polymerase II promoter20 4.27×106 6.73×103
  GO:0060348~bone development  9 6.12×1006 9.64×103
  GO:0045893~positive regulation of transcription, DNA-dependent15 2.55×105 4.01×102
  GO:0051216~cartilage development  7 2.68×105 4.23×102
  GO:0051254~positive regulation of RNA metabolic process15 2.79×105 4.40×102
  GO:0031328~positive regulation of cellular biosynthetic process18 2.84×105 4.46×102
  GO:0009954~proximal/distal pattern formation  5 3.03×105 4.76×102
  GO:0009891~positive regulation of biosynthetic process18 3.41×105 5.36×102
  GO:0001503~ossification  8 3.67×105 5.77×102
  GO:0007155~cell adhesion18 3.73×105 5.87×102
  GO:0022610~biological adhesion18 3.80×105 5.98×102
  GO:0045944~positive regulation of transcription from RNA polymerase II promoter13 3.95×105 6.22×102
  GO:0045941~positive regulation of transcription16 4.02×105 6.33×102
  GO:0021515~cell differentiation in spinal cord  5 4.27×105 6.72×102
  GO:0051173~positive regulation of nitrogen compound metabolic process17 4.94×105 7.78×102
  GO:0010628~positive regulation of gene expression16 5.64×105 8.88×102
  GO:0010557~positive regulation of macromolecule biosynthetic process17 5.94×105 9.34×102
Cellular component
  GO:0031012~extracellular matrix18 4.61×1010 5.51×107
  GO:0005578~proteinaceous extracellular matrix16 1.07×108 1.28×105
  GO:0044420~extracellular matrix part10 1.83×107 2.20×104
  GO:0044421~extracellular region part22 4.77×106 5.713×103
Molecular function
  GO:0043565~sequence-specific DNA binding25 5.21×1011 6.68×108
  GO:0003700~transcription factor activity29 1.59×109 2.04×106
  GO:0030528~transcription regulator activity33 1.36×107 1.74×104
  GO:0005201~extracellular matrix structural constituent  7 6.40×105 8.21×102

[i] FDR, False Discovery Rate; HOTAIR, HOX transcript antisense intergenic RNA.

Table III.

KEGG pathways enriched in HOTAIR and coexpression genes (P<0.05).

Table III.

KEGG pathways enriched in HOTAIR and coexpression genes (P<0.05).

PathwayCountP-valueFDRGenes
KEGG
  hsa04512:ECM-receptor interaction5 7.85×104 7.28×101TNC, COL1A2, COL6A1, LAMB1, COL5A2
  hsa04510:Focal adhesion5 1.79×102 1.55×101TNC, COL1A2, COL6A1, LAMB1, COL5A2
  hsa05200:Pathways in cancer6 2.32×102 1.96×101FGFR1, HDAC2, TGFB3, LAMB1, GLI3, MMP2

[i] KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, False Discovery Rate; HOTAIR, HOX transcript antisense intergenic RNA.

Discussion

HCC is a critical health problem worldwide and is an aggressive disease. The high morbidity and mortality rates of this malignance are not arbitrary but raise the researcher's attention. In the present study, the HOTAIR expression level was detected significantly higher in the HCC tissues than the adjacent non-tumor tissues. The HOTAIR expression levels were observed significantly higher in tumor samples from patients with distant metastasis, advanced stage, portal vein tumor embolus, vasoinvasion, tumor capsular infiltration or positive nm23 than those from patients without distant metastasis, advanced stage, portal vein tumor embolus, vasoinvasion, tumor capsular infiltration or positive nm23 correspondingly. In vitro assays, the silencing of HOTAIR in liver cancer cells induced the promotion of apoptosis and inhibition of cell proliferation. ECM-receptor interaction, focal adhesion and pathways in cancer were annotated with the HOTAIR and coexpression genes.

HOTAIR has been evaluated extensively within the context of cancer prognosis (20). A meta-analysis for patients with solid tumors reported that higher HOTAIR expression could significantly predict worse overall survival (21). In gastric cancer, high expression of HOTAIR was significantly associated with the depth of tumor invasion, lymph node metastasis, vessel invasion, lymphatic vessel involvement and TNM stage (22). The incidence of lymph node metastasis was illustrated to be higher in cancer patients with high HOTAIR expression compared to patients with low HOTAIR expression (23). Likewise, HOTAIR was indicated to be a potential predictor of poor relapse-free survival, metastasis-free survival and disease-free survival in cervical, ovarian, breast and endometrial cancer patients (24).

To the best of the authors' knowledge, the current study contained a larger HCC sample than the previous investigations for HCC and HOTAIR. Compared with adjacent non-HCC tissue, the analysis yielded that HOTAIR expressed higher in the HCC tissue, which was observed in several previous reports, implying the diagnosis value of HOTAIR in HCC patients (11,2527). Ishibashi et al (11) found that patients with HOTAIR expression had significantly larger primary tumor size than those without HOTAIR expression. A vast majority of samples from study of Geng et al (26) showed higher levels of HOTAIR in tumor tissues than in adjacent non-tumor tissues, and HOTAIR expression were significantly higher in tumor tissues from patients with lymph node metastasis. The HOTAIR expression was reported increased at least two-fold in HCC tumor tissues relative to that in non-tumor tissues (27). These evidences were robust to illustrate that HOTAIR might be an oncogene for HCC.

In the current study, a Spearman's rank correlation coefficient analysis showed that the higher HOTAIR expression level was significantly correlated with metastasis, advanced TNM stage and portal vein tumor embolus. Besides, HOTAIR expression was remarkably higher in patients with vasoinvasion than those without vasoinvasion, as well as in capsular infiltration than those with complete liver capsule. The clinicopathological characteristics of vasoinvasion, capsular infiltration always correlated to invasion and progression, suggesting HOTAIR might be involved in HCC invasion and serve as poor prognosis predictor. These findings allowed HOTAIR to be a potential molecular for risk classification of HCC patients, and that molecular-based tumor risk stratification was important for the decision of individual diagnosis and therapy (25,28). ROC analysis demonstrated that the AUC of HOTAIR was 0.715 (95%CI: 0.643–0.787) with a sensitivity of 45.3% and a specificity of 86.3% in distinguishing HCC. The authors speculated that an individual biomarker was insufficient to diagnosis malignant disease, and a combined analysis of the well-studied diagnosis biomarkers might provide novel insight into the diagnosis method for HCC.

Given the clinical implications of HOTAIR upon HCC and the currently poor interpretation of its mechanisms, a series of in vitro assays were conducted to uncover the effects of HOTAIR on HCC. After silencing HOTAIR expression, the proliferation and viability of SMMC-7221 cells were depressed, while the significant promotion of apoptosis and caspase ability was observed. Furthermore, the level of the depressed and promoted effects was associated with the transfection processing time, indicating the high HOTAIR expression level was related to the increased HCC progression risk. Taken the in vitro results together, it demonstrated that HOTAIR involved in promoting HCC cell growth and inhibiting apoptosis, explaining the phenomenon that the increased HOTAIR expression was found in HCC tissues and the significant associations with the advanced TNM stage (26). Moreover, the ROC analysis of HOTAIR and distant metastasis, advanced TNM stage and tumor capsular infiltration also indicated expression of HOTAIR might be used for molecular based tumor staging or progressive risk stratification. Since increased HOTAIR expression was not exclusively specific in one particular cancer, and observed promoting cell proliferation, invasion and migration in variant types of tumor cells, the authors speculated high HOTAIR expression could be a potential marker for predicting poor prognosis in multiple types of cancers (20,29,30).

It was well recognized that HOTAIR interacted with multiple genes in a manner dependent on polycomb repressive complex 2 (PRC2), which could induce histone H3 lysine 27 (H3K27) methylation and thus silence transcription of the HOXD locus, and eventually cause epigenetic silencing of metastasis suppressor genes (3133). Knockdown of HOTAIR was associated with reductions in levels of vascular endothelial growth factor protein and matrix metalloproteinase-9, which were vital for cell motility and metastasis (26). Besides, HOTAIR was found to promote cell migration and invasion via inhibiting RNA binding motif protein 38 (RBM38) in HCC cells, since knockdown of HOTAIR raised the expression RBM38 both on mRNA levels and protein levels, and the increased expression of RBM38 could restrain cell motility (10). A previous study reported HOTAIR enhanced cell viability and escaped G1-phase arrest through suppressing miRNA-218 expression and inhibiting P14 and P16 signaling. Suppressing oncogene Bmi-1 was shown to be a functional target of miR-218, and the main downstream targets signaling, P16(lnk4a) and P14(ARF), were inactivated in HOTAIR tumorigenesis (34). In HepG2 cells, the phenomenon that microRNA miR-125a-5p decreased and released caspase 2 to promote HCC cell apoptosis was reported after HOTAIR knockdown (35). Therefore, HOTAIR might control HCC cell proliferation through interacting with microRNAs. The identification of microRNAs and target genes of HOTAIR in HCC is of great importance to understand HCC pathogenesis.

Although accumulating evidence has revealed substantial biological functions of HOTAIR, the precise mechanisms remained largely to be explored. Coexpression network analysis, which was used to find genes having similar or coordinated expression patterns, was carried out to identify HOTAIR coexpression genes (36,37). GO enrichment analysis revealed that a majority of the HOTAIR and coexpression genes were involved in pathways related to cell adhesion, biological adhesion, transcription regulator activity, transcription factor activity and sequence-specific DNA binding. These data supported that most lncRNAs worked with DNA-binding proteins, and epigenetically regulated the expression of multiple genes (7,31). It is noteworthy that in HCC patients with positive nm23, which has also been reported to contribute to metastasis (38), HOTAIR expression level was higher than that in nm23 negative patients. To some extent, the significant correlation between HOTAIR expression and nm23 status suggested that they might function in a manner of cooperation. However, a previous study reports that the absence of nm23 in HCC is significantly correlated with extrahepatic metastasis (39). In mice models, nm23 has been also observed overexpressed in the HCC, whereas lung metastasis was found to be promoted in the transgenic nm23-null mice (40). The oncogenic and metastasis role of the nm23 is still debated. The contradiction between the results suggests future studies with larger samples are needed to illustrate the function of nm23.

In addition, three pathways were screened out using KEGG analysis for the HOTAIR and coexpression genes, such as ECM-receptor interaction, focal adhesion and pathways in cancer. Most of these pathways were reported involving in cancer progression (41). Typically, pathways linked to cell spread and migration was both annotated in GO and KEGG, such as cell adhesion, biological adhesion and focal adhesion (42,43). It strongly suggests that HOTAIR might be a therapeutic target to decrease metastasis risk, which remained a field to be investigated (44).

Concerning with the limitations, the absence of survival data hindered this study to gain prognosis value of HOTAIR in HCC. As for control tissue origin, only the adjacent noncancerous tissues including cirrhotic and noncirrhotic liver tissues were available. Given the multi-stages hepatocarcinogenesis process of HCC (45), the authors suggested that the combined analysis of normal tissues might reveal more in-depth mechanisms of HOTAIR in HCC progression. A future study to validate HOTAIR mechanisms in HCC patients is necessary.

The current study suggested that HOTAIR was an oncogene in HCC. It functioned though promoting tumor cell growth and inhibiting apoptosis. HOTAIR potentially regulated HCC metastatic progression by several pathways correlated cell adhesion, and might be a therapeutic target in future.

Acknowledgements

The present study was supported by grants from the Fund of Future Academic Star of Guangxi Medical University (grant no. WLXSZX16001) and the Scientific Research Project of the Department of Education in Guangxi Zhuang Autonomous Region (grant nos. LX2014064 and 201204LX044).

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January-2018
Volume 17 Issue 1

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Copy and paste a formatted citation
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Spandidos Publications style
Zhong DN, Luo YH, Mo WJ, Zhang X, Tan Z, Zhao N, Pang SM, Chen G, Rong MH, Tang W, Tang W, et al: High expression of long non‑coding HOTAIR correlated with hepatocarcinogenesis and metastasis. Mol Med Rep 17: 1148-1156, 2018
APA
Zhong, D., Luo, Y., Mo, W., Zhang, X., Tan, Z., Zhao, N. ... Tang, W. (2018). High expression of long non‑coding HOTAIR correlated with hepatocarcinogenesis and metastasis. Molecular Medicine Reports, 17, 1148-1156. https://doi.org/10.3892/mmr.2017.7999
MLA
Zhong, D., Luo, Y., Mo, W., Zhang, X., Tan, Z., Zhao, N., Pang, S., Chen, G., Rong, M., Tang, W."High expression of long non‑coding HOTAIR correlated with hepatocarcinogenesis and metastasis". Molecular Medicine Reports 17.1 (2018): 1148-1156.
Chicago
Zhong, D., Luo, Y., Mo, W., Zhang, X., Tan, Z., Zhao, N., Pang, S., Chen, G., Rong, M., Tang, W."High expression of long non‑coding HOTAIR correlated with hepatocarcinogenesis and metastasis". Molecular Medicine Reports 17, no. 1 (2018): 1148-1156. https://doi.org/10.3892/mmr.2017.7999