Abstract

Purpose

   To investigate the clinicopathological value and potential roles of
   microRNA-198 (miR-198) in hepatocellular carcinoma (HCC).

Methods

   Ninety-five formalin-fixed paraffin-embedded HCC and the para-cancerous
   liver tissues were gathered. Real-time reverse transcription
   quantitative polymerase chain reaction was applied to determine the
   miR-198 expression. The association between the miR-198 expression and
   clinicopathological features was examined. Meanwhile, potential target
   messenger RNAs of miR-198 in HCC were obtained from 14 miRNA prediction
   databases and natural language processing method, in which we pooled
   the genes related to the tumorigenesis and progression of HCC and
   classified them by their frequency. The selected target genes were
   finally analyzed in the Gene Ontology analysis and Kyoto Encyclopedia
   of Genes and Genomes pathway.

Results

   miR-198 expression was significantly lower in HCC than that in adjacent
   noncancerous liver tissues (1.30±0.72 vs 2.01±0.58, P<0.001). Low
   miR-198 expression was also correlated to hepatitis C virus infection
   (r=−0.48, P<0.001), tumor capsular infiltration (r=−0.43, P<0.001),
   metastasis (r=−0.26, P<0.010), number of tumor nodes (r=−0.25,
   P=0.013), vaso-invasion (r=−0.24, P=0.017), and clinical tumor node
   metastasis stage (r=−0.23, P=0.024). Altogether, 1,048 genes were
   achieved by the concurrent prediction from at least four databases and
   natural language processing indicated 1,800 genes for HCC. Further, 127
   overlapping targets were further proceeded with for pathway analysis.
   The most enriched Gene Ontology terms in the potential target messenger
   RNAs of miR-198 were cell motion, cell migration, cell motility, and
   regulation of cell proliferation in biological process; organelle
   lumen, membrane-enclosed lumen, and nuclear lumen in cellular
   component; and enzyme binding, protein domain-specific binding, and
   protein kinase activity in molecular function. Kyoto Encyclopedia of
   Genes and Genomes analysis showed that these target genes were
   obviously involved in focal adhesion and pathways in cancer.

Conclusion

   Lower expression of miR-198 was related to several clinicopathological
   parameters in HCC patients. miR-198 might play a regulatory role
   through its target genes in the development of HCC.

   Keywords: miR-198, hepatocellular carcinoma, metastasis, RT-qPCR, in
   silico

Introduction

   Hepatocellular carcinoma (HCC) is one of the most frequent malignancies
   in the world. The estimate incidence of liver cancer ranked fourth and
   its mortality ranked third in People’s Republic of China, 2015.[43]^1
   Currently, several genes have been identified to be related with the
   tumorigenesis and progression of HCC, but the molecular mechanisms of
   their functions and interactions have not been clearly studied.
   Therefore, it is urgent to find the potential function of these genes
   during the development of HCC.

   MicroRNA (miRNA) is classified as endogenous noncoding RNA, repressing
   protein translation by binding to their target genes.[44]^2 From nearly
   all biological processes, miRNA can exert the function as
   posttranscriptional regulation.[45]^3 Aberrant expression of
   microRNA-198 (miR-198) has been found to be correlated to the
   carcinogenesis and disease progression of several classes of cancers,
   including lung adenocarcinoma,[46]^4 esophageal cancer,[47]^5 squamous
   cell carcinoma of tongue,[48]^6 retinoblastoma,[49]^7 pancreatic
   adenocarcinoma,[50]^8 multiple myeloma,[51]^9 colorectal cancer,[52]^10
   and prostate cancer.[53]^11 However, there were only three research
   papers studying the expression of miR-198 in HCC. Varnholt et al first
   reported the lower expressed miR-198 in Caucasian patients with
   hepatitis C virus (HCV) related HCC.[54]^12 Tan et al identified the
   suppressing role of miR-198 in HGF/c-MET pathway that influenced
   migration and invasion of HCC cells.[55]^13 Elfimova et al found
   another pathway that was repressed by miR-198 in vitro which could
   negatively regulate cell growth and migration.[56]^14 However, whether
   miR-198 is related to clinicopathological parameters has not been
   studied, there still remains a mystery regarding miR-198’s role in the
   key processes of hepatocellular carcinogenesis.

   Thus, in this study, we assessed the expression of miR-198 in tumor
   tissues of HCC patients with their paired noncancerous liver. In
   addition, the relationship of miR-198 with several clinicopathological
   parameters was also investigated. Moreover, we predicted the target
   genes of miR-198 via 14 miRNA databases and filtered in the natural
   language processing (NLP) to achieve the specific messenger RNAs (mRNA)
   profiles of miR-198 targets for HCC. Gene Ontology (GO) terms and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway annotation were
   further undertaken to explore the function of these mRNAs. We attempted
   to pioneer the use of real-time reverse transcription quantitative
   polymerase chain reaction (RT-qPCR) and in silico method to clarify the
   potential molecular mechanism of miR-198, providing guidelines for the
   following research on miR-198 in HCC.

Materials and methods

Tissue samples

   There were 95 cases of HCCs and their paired paraneoplastic liver
   formalin-fixed paraffin-embedded (FFPE) tissues included in this
   retrospective study. The age of the HCC patients ranged from 29 to 82
   years old, with a mean age of 52 years. All the clinicopathological
   features are provided in [57]Table 1. Adjacent noncancerous liver
   tissues were obtained from the location at least 2 cm away from the
   tumor node. The adjacent tissue was from the same HCC patient. We
   designed these paired sample groups in order to eliminate the
   individual difference. Tumor sizes of HCC ranged from 1 to 11 cm (mean
   size, 6.4 cm). All cases were from patients who were under initial
   hepatectomies without any treatment and they were randomly chosen from
   hepatectomy surgeries in the First Affiliated Hospital of Guangxi
   Medical University, People’s Republic of China between March 2010 and
   December 2011. The research protocol was approved by the Ethical
   Committee of the First Affiliated Hospital of Guangxi Medical
   University. Written informed consent was signed by the patients for the
   treatment and sample usage in this study. All samples were diagnosed by
   three pathologists independently (Dian-zhong Luo, Zhen-bo Feng, Gang
   Chen).

Table 1.

   Relationship between the expression of miR-198 and clinicopathological
   features in HCC
   Clinicopathological feature n miR-198 relevant expression (2^−ΔCq)
     __________________________________________________________________

   Mean ± SD t P-value
   Tissue
    Adjacent noncancerous liver 95 2.01±0.58 7.53 <0.001[58]^***,[59]^a
    Noncirrhotic liver 50 1.96±0.57 −0.76 0.449[60]^b
    Cirrhotic liver 45 2.06±0.59 5.67 <0.001[61]^***,[62]^c
    HCC 95 1.30±0.72 6.25 <0.001[63]^***,[64]^d
   Age, years
    ≥50 46 1.42±0.74 1.66 0.10
    <50 49 1.18±0.68
   Sex
    Male 75 1.30±0.69 0.04 0.97
    Female 20 1.29±0.83
   Differentiation
    High 6 1.87±0.52 10.20 <0.001[65]^***,[66]^e
    Moderate 60 1.45±0.74
    Low 29 0.87±0.45
   Size (cm)
    <5 18 1.48±0.72 0.99 0.33
    ≥5 77 1.26±0.72
   Tumor nodes
    Single 52 1.47±0.76 2.72 0.008[67]^**
    Multi 43 1.08±0.61
   Metastasis
    Without metastasis 46 1.49±0.73 2.62 0.01[68]^*
    With metastasis 49 1.12±0.66
   Clinical TNM stage
    I–II 22 1.60±0.72 2.34 0.021[69]^*
    III–IV 73 1.21±0.69
   Portal vein tumor embolus
    − 63 1.35±0.71 1.03 0.31
    + 32 1.19±0.72
   Vaso-invasion
    − 59 1.42±0.69 2.23 0.028[70]^*
    + 36 1.09±0.71
   Tumor capsular infiltration
    With complete capsule 45 1.64±0.77 4.92 <0.001[71]^***
    No capsule infiltration 50 0.99±0.50
   HCV
    − 63 1.53±0.71 5.89 <0.001[72]^***
    + 32 0.83±0.44
   HBV
    − 17 1.42±0.78 0.77 0.44
    + 78 1.27±0.70
   AFP
    − 41 1.40±0.79 1.36 0.18
    + 38 1.17±0.71
   Cirrhosis
    − 50 1.21±0.71 −1.30 0.20
    + 45 1.40±0.72
   nm23
    − 20 1.29±0.67 −0.07 0.95
    + 75 1.30±0.73
   P53
    − 40 1.29±0.75 −0.04 0.97
    + 55 1.30±0.69
   P21
    − 62 1.34±0.78 0.81 0.42
    + 33 1.22±0.58
   VEGF
    − 25 1.15±0.65 −1.21 0.23
    + 70 1.35±0.73
   Ki-67LI
    Low 47 1.40±0.72 1.37 0.18
    High 48 1.20±0.70
   MVD
    Low 47 1.24±0.67 −0.73 0.47
    High 48 1.35±0.76
   [73]Open in a new tab

   Notes:
   ^*

   P<0.05,
   ^**

   P<0.01,
   ^***

   P<0.001.
   ^a

   Adjacent noncancerous liver (including liver with and without
   cirrhosis) vs HCC.
   ^b

   Noncirrhotic liver vs cirrhotic liver.
   ^c

   Noncirrhotic liver vs HCC.
   ^d

   Cirrhotic liver vs HCC.
   ^e

   Differentiation group pairwise comparison: high vs low differentiation
   (t=4.851, P<0.001) and moderate vs low (t=4.610, P<0.001) have
   significant differences, high vs moderate (t=1.336, P=0.186) has no
   difference.

   Abbreviations: AFP, alpha fetal protein; HBV, hepatitis B virus; HCC,
   hepatocellular carcinoma; HCV, hepatitis C virus; miR-198,
   microRNA-198; MVD, microvessel density; SD, standard deviation; TNM,
   tumor node metastasis; VEGF, vascular endothelial growth factor.

RT-qPCR

   Total RNA including miRNA was extracted from tumor and nontumorous
   liver sections and the miRNeasy FFPE kit (QIAGEN, KJ Venlo, the
   Netherlands) was applied according to our previous
   reports.[74]^15^–[75]^17 RNA concentrations were measured by NanoDrop
   2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The OD260/OD280
   ratio of the total mRNA (including miRNA) isolated from the FFPE
   tissues ranged from 1.84 to 2.06, and OD260/OD230 from 1.90 to 2.04.
   The PCR amplification efficiency of all the quantitative real-time PCR
   (RT-qPCR) reactions ranged from 91.0% to 95.2%. A combination of RUN6B
   and RUN48 was chosen as the house-keeping reference for detection of
   miR-198 expression. The primers of miR-198, as well as RNU6B and RNU48,
   were provided by TaqMan^® MicroRNA Assays (4427975, Applied Biosystems,
   Life Technologies, Grand Island, NY, USA). Sequence of miRNA and
   references in the paper were as follows: miR-198 (Applied Biosystems