In China, chronic hepatitis B virus (HBV) infection is the main cause of hepatocellular carcinoma (HCC), and the prevalence of HBV infection in patients with HCC is approximately 80% , , . Once the diagnosis is definite, the patients with HCC often proceed to the progressive stage or occurrence in distal metastasis, precluding the opportunity for rational surgery for these patients. Currently, the most commonly used marker for HCC is serum α-fetal protein (AFP); however, approximately 50% of HCC patients are negative for this marker. Hence, it is important for clinical scientists to identify ideal HCC markers, particularly for HBV-related HCC.
Exosomes are small vesicles of approximately 30–100 nm in diameter, which selectively pack mRNA, non-coding RNA, DNA, protein, etc. and can be secreted by a wide variety of cells. In functional studies, exosomes play an important role in signaling between the cells including physiological and pathological cells , , . Reflecting these characteristics, exosomes could potentially be applied in the diagnosis and prognosis assessment of tumors. In a previous study, we pooled four pairs of sera from untreated-HCC patients and healthy subjects into one pair of sample, respectively, extracted total exosomal RNA, and used RNA sequencing (RNA-seq) to screen the differentially expressed transcripts (unpublished data). In the exosomal mRNAs data (see Supplementary Table S1) produced by RNA-seq, the results revealed heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) mRNA as a significantly upregulated transcript in HCC patients. HnRNPH1 is a type of RNA binding protein and splicing factor, which stimulates the dissociation and polyadenylation of mRNA precursors and plays a decisive role in alternative splicing . Studies have shown that hnRNPH1 is highly expressed in esophageal squamous cell carcinoma, prostate cancer and other tumor tissues and is associated with cell differentiation , , . These findings indicate that serum exosomal hnRNPH1 mRNA could represent a novel “liquid biopsy” assay for HCC.
Therefore, in the present study, we detected the relative expression levels of serum exosomal hnRNP H1 mRNA in HCC, liver cirrhosis (LC) and chronic hepatitis B (CHB) patients and healthy subjects. A receiver operating characteristics (ROC) curve was constructed to evaluate the effectiveness of the diagnosis for HCC. In addition, we discuss the relationship between the hnRNPH1 mRNA and the clinicopathological characteristics and overall survival (OS) of patients with HCC.
Materials and methods
A total of 223 patients, including 88 cases of HCC, 67 cases of LC and 68 cases of CHB among patients who visited Hangzhou First People’s Hospital, were enrolled in the present study between January 2015 and June 2016. HCC was diagnosed based on ultrasound, computed tomography (CT), serum AFP and histopathological examination. LC was diagnosed based on ultrasound and CT examination and further characterized according to patients with or without portal hypertension and hypersplenotrophy. CHB was diagnosed based on patients with HBV surface antigen (HBsAg)-positive serum for more than 6 months. Sixty-eight cases of healthy subjects were collected from the physical examination center of Hangzhou First People’s Hospital. The participants with autoimmune hepatitis, alcoholic liver disease, Wilson’s disease, other types of viral hepatitis and other major diseases were excluded. All participants were age- and gender-matched.
The present study was approved through the Medical Ethics Committee of Hangzhou First People’s Hospital, and all participants completed an informed consent process.
Four milliliters of blood were drawn from each participant in the fasting state on the first visiting day. Two milliliters of serum were separated after centrifugation at 3000×g for 5 min within 8 h, and 2 mL of serum was subsequently obtained after centrifugation at 10000×g for 5 min. The prepared serum samples were stored at −70°C until further use.
Serum exosome separation and total RNA extraction
A 1-mL aliquot of serum was transferred to a new tube, and 200 μL of total exosome isolation reagent (Thermo Fisher Scientific Co., Ltd, Lithuania) was added. The serum/reagent mixture was mixed well by pipetting up and down until a homogenous solution was obtained. The sample was incubated at 4°C for 30 min, followed by centrifugation at 10,000×g for 10 min at room temperature. The obtained pellet was resuspended in 200 μL of 1× PBS solution. Total exosomal RNA was extracted and purified using a commercial kit (Thermo Fisher Scientific Co., Ltd, Lithuania), and eventually 50 μL of total RNA was acquired. The above procedures were strictly performed according to the manufacturer’s instructions.
TaqMan real-time PCR
The primers and probes for exosomal hnRNPH1 mRNA and GAPDH mRNA amplification were designed using Express v3.0 software (Applied Biosystems Co., Ltd, USA), and synthesized at Shanghai Hui-rui Biological Technology Co., Ltd. The following primer and probe sequences were used: forward primer for hnRNPH1 5′-CGCCCGGCCTGTTTG-3′, reverse primer for hnRNPH1 5′-TGCAGTGAGCCGAGATTGTG-3′, and probe for hnRNPH1 5′-FAM-ACAGTCTCACTTTATCGCCCAGGCTGG-TAMRA-3′; forward primer for GAPDH 5′-GAGCTGAACGGGAAGCTCAC-3′, reverse primer for GAPDH 5′- GCCTGCTTCACCACCTTCTT-3′, and probe for GAPDH 5′-FAM-ACAGTCTCACTTTATCGCCCAGGCTGG-TAMRA-3′. The reaction system comprised 10 μL of 2× PCR buffer, 0.4 μL of DNA polymerase II, 0.4 μL of reverse transcriptase mixture, 0.4 μL of each primer, 0.8 μL of probe, 2 μL of RNA template and deionized water to the total volume of 20 μL (TaKaRa Co., Ltd, China). The ABI Prism® 7500 PCR instrument (Applied Biosystems Co., Ltd, USA) was used for detection. The following reaction conditions were used: the first stage (50°C for 15 min), the second stage (95°C for 5 min), and the third stage, including 45 cycles (95°C for 15 s, 60°C for 40 s). The relative expression level of hnRNPH1 mRNA was calculated using the 2−ΔCt method as previously described . Each sample was assessed in duplicate, with an average of the two duplicates used for analysis.
Non-parametric variables were described as median and interquartile range (IQR) and examined using the Mann-Whitney U or Kruskal-Wallis H-test. Furthermore, exosomal hnRNPH1 mRNA relative expression levels were illustrated as box plots. Categorical variables were compared using the χ2-test. ROC curves were constructed to determine the feasibility of hnRNPH1 mRNA alone and in combination with AFP in the diagnosis of HCC. OS curves were estimated using the Kaplan-Meier method. Differences were considered significant at the α level of 0.05 for two-sided tests. All data analyses were performed using SPSS software, version 21.0 (SPSS Inc., Chicago, IL, USA).
Serum exosomal hnRNP H1 mRNA relative expression levels
Serum exosomal hnRNPH1 mRNA relative expression levels in the HCC group at 0.970 (0.764, 1.386) were significantly higher than those in the LC group at 0.671 (0.393, 1.346), the CHB group at 0.354 (0.175, 0.637) and the healthy control group at 0.390 (0.226, 0.495) (p=0.012, p=0.006 and p=0.004, respectively). Compared with the CHB group and healthy control, the serum exosomal hnRNPH1 mRNA levels in the LC group were also obviously higher (p=0.009 and p=0.008, respectively). Notably, no difference between the CHB group and healthy control was observed (p=0.591) (Figure 1).
Effectiveness of exosomal hnRNPH1 mRNA relative expression levels in identifying HCC
To determine whether exosomal hnRNPH1 mRNA could be a potential diagnostic marker for HCC in high HBV prevalence areas, ROC curve analyses were performed. The exosomal hnRNPH1 mRNA discriminated HCC patients from CHB patients with an AUC of 0.865 (95% CI=0.808–0.922, p=0.003). At a cut-off value of 0.670, the sensitivity and specificity of detection was 85.2% and 76.5%, respectively. Serum AFP at the cut-off value of 20 ng/mL yielded an AUC of 0.785 (95% CI=0.694–0.845, p=0.007) with 69.3% sensitivity and 87.9% specificity. Furthermore, the AUC for exosomal hnRNPH1 mRNA in combination with AFP was 0.891 (95% CI=0.873–0.939, p=0.005) with 87.5% sensitivity and 84.8% specificity (Figure 2A).
Similarly, exosomal hnRNPH1 mRNA discriminated HCC patients from LC patients with an AUC of 0.647 (95% CI=0.553–0.741, p=0.002) with 86.4% sensitivity and 54.0% specificity. Serum AFP yielded an AUC of 0.674 (95% CI=0.590–0.758, p=0.006) with 46.6% sensitivity and 88.3% specificity. The AUC for exosomal hnRNPH1 mRNA in combination with AFP was 0.749 (95% CI=0.674–0.825, p=0.001) with 50.3% sensitivity and 91.0% specificity (Figure 2B).
Relationship between exosomal hnRNPH1 mRNA relative expression levels and the clinicopathological features of HCC patients
We examined the clinicopathological features, including gender, age and other clinical parameters, according to the exosomal hnRNPH1 mRNA relative expression status. Significant associations between the Child-Pugh classification, portal vein tumor emboli, lymph node metastasis and TNM stage and exosomal hnRNPH1 mRNA relative expression levels (p<0.05) were observed, and gender, age, HBsAg, histological grade and cirrhosis showed no associations with exosomal hnRNPH1 mRNA (p>0.05) (Table 1).
Association between exosomal hnRNPH1 mRNA relative expression levels and HCC survival
In the present study, the clinical cases for 88 HCC patients were followed for 48 months. According to the median exosomal hnRNPH1 mRNA level (0.970), HCC patients were divided into two subsets. Patients whose exosomal hnRNPH1 mRNA levels were higher than 0.970 were classified into the high-level subset (n=58), and patients whose exosomal hnRNPH1 mRNA levels were lower than 0.970 were classified into the low-level subset (n=30). The results of the Kaplan-Meier analysis demonstrated that the exosomal hnRNPH1 mRNA level is a prognostic factor in HCC patients. High exosomal hnRNPH1 mRNA levels were associated with worse OS in HCC patients (χ2=5.206, p=0.023) (Figure 3).
The exchange of exosomal packages between the cells could influence the tumor microenvironment and contribute to the tumor development , , , . Exosomes are easy to obtain from nearly all body fluids, including blood, urine, saliva, semen and ascites. From the perspective of exploring novel tumor markers, exosomes could assuredly undertake this role, as these vesicles are stable, can easily be used for dynamic tracking, and particularly, selectively pack bioactive molecules. Exosomal microRNAs (miRNAs) have been recommended as diagnostic and prognostic markers for lung cancer, esophageal squamous cell carcinoma, prostate cancer, breast cancer and glioblastoma , , , , , . Comparison of the exosomal proteins and non-coding RNAs in the diagnosis of cancers showed that the application of exosomal mRNAs was limited.
The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins that complex with heterogeneous nuclear RNA and play key roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Moreover, the dysregulation of alternative pre-mRNA splicing has been implicated in carcinogenesis , , . HnRNPA1 is a major factor associated with various types of cancer and metastasis. This molecule is markedly overexpressed in lung cancer tissues and is associated with tumor proliferation . Similarly, hnRNPA2/B1 is significantly upregulated in patients suffering from lung cancer and may be a marker for the early detection of lung cancer . Sun et al.  suggested that the levels of hnRNPH1 mRNA were obviously increased in esophageal squamous cell carcinoma tissues and decreased in non-tumorous tissues (p=0.0026); the high levels of hnRNPH1 mRNA were associated with poorly differentiated tumors (p=0.0287). A recent study showed that hnRNPH1/H2 mediated the switch in expression from high-activity fructokinase (KHK)-C to the low-activity KHK-A isoform. Above all, KHK-A acts as a protein kinase that phosphorylates and activates phosphoribosyl pyrophosphate synthetase 1 (PRPS1) to promote pentose phosphate pathway-dependent de novo nucleic acid synthesis and HCC occurrence . So far, the molecular mechanism of exosomal hnRNPH1 mRNA participation in the formation and development of cancer remains obscure.
In the present study, we screened exosomal hnRNPH1 mRNA as a candidate marker for HCC diagnosis using the TaqMan real-time PCR method. The results revealed that the levels of serum exosomal hnRNPH1 mRNA in HCC patients were remarkably higher than those in other groups. Exosomal hnRNPH1 mRNA discriminated HCC from CHB with an AUC of 0.865 with 85.2% sensitivity and 76.5% specificity at a cut-off value of 0.670. The effectiveness of hnRNPH1 mRNA combined with AFP (≥20 ng/mL) was further improved.
In addition, we examined the relationship between serum exosomal hnRNPH1 mRNA levels and the clinicopathological characters in HCC. The results indicated that high levels of serum exosomal hnRNPH1 mRNA were associated with Child-Pugh classification, portal vein tumor emboli, lymph node metastasis and TNM stage, which represent more progressive clinical phenotypes. Moreover, the association of serum exosomal hnRNPH1 mRNA levels with the prognosis of HCC patients was assessed, and the results showed that patients with high serum exosomal hnRNPH1 mRNA levels exhibited worse OS than those with low hnRNPH1 mRNA levels, suggesting that the level of serum exosomal hnRNPH1 mRNA has important predictive value in HCC prognosis classification.
Although the differences of serum exosomal hnRNPH1 mRNA levels have been described in detail among HCC, LC, CHB patients and healthy subjects, there are some limitations in this study because of a lack of clinical data. For example, it is need to compare the results between patients with chronic active hepatitis B and those with inactive infection; the difference between patients with compensated cirrhosis and those with decompensated cirrhosis is worth to be discussed also, and it would be of interest to evaluate the differences in CHB patients with various fibrosis stages.
In conclusion, these findings suggest that serum exosomal hnRNPH1 mRNA may be a novel biomarker for HCC patients in high HBV prevalence areas.
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The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2017-0327).
About the article
Published Online: 2017-12-18
Published in Print: 2018-02-23
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by a study on exosomal long noncoding RNA in serum as markers for hepatocellular carcinoma, Hangzhou Scientific and Technological Commission (20140733Q14), Zhejiang Province, China.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.