Expression of miR - 122 plasma as a biomarker for hepatocellular carcinoma

In this study, we aim to evaluate the expression of microRNAs-122 plasma, and combination of microRNAs-122 with alpha-fetoprotein to diagnosis of hepatocellular carcinoma.

EXPRESSION OF miR-122 PLASMA AS A BIOMARKER FOR

HEPATOCELLULAR CARCINOMA

Dang Chieu Dương 1 ; Phan Quoc Hoan 2 ; Le Huu Song 2 Nguyen Linh Toan 3 ; Ngo Tat Trung 2

SUMMARY

Objectives: MicroRNAs participate in cell proliferation, apoptosis and transformation

However, the use of microRNAs-122 in combination with alpha-fetoprotein has not been

evaluated In this study, we aim to evaluate the expression of microRNAs-122 plasma, and combination of microRNAs-122 with alpha-fetoprotein to diagnosis of hepatocellular carcinoma Subjects and methods: Real-time PCR was employed to measure microRNAs-122 expression levels on 250 plasma samples of 101 patients with hepatitis B virus-related hepatocellular carcinoma, 46 chronic hepatitis B patients and 103 healthy controls Results: The relative expression levels of microRNAs-122 in hepatitis B virus-related hepatocellular carcinoma patients were higher than chronic hepatitis B and healthy controls (p < 0.001) The individual microRNAs-122 acquired high diagnostic accuracy for hepatocellular carcinoma surveillance (hepatocellular carcinoma vs chronic hepatitis B, AUC = 0.910; hepatocellular carcinoma vs healthy controls, AUC = 0.989) When alpha-fetoprotein levels were lower than 20 ng/mL, microRNAs-122 still preserves accuracy to distinguish hepatocellular carcinoma from other groups (chronic hepatitis B, AUC = 0.915; healthy controls, AUC = 0.991) When

microRNAs-122 were used in combination with alpha-fetoprotein levels, the diagnostic performance was significantly improved in discriminating hepatocellular carcinoma from group chronic hepatitis B (AUC = 0.914) Conclusions: MicroRNAs-122 is a potential biomarker to improve diagnostic in hepatitis B virus-related hepatocellular carcinoma patients, especially in patients with hepatocellular carcinoma with normal alpha-fetoprotein level

* Keywords: Hepatocellular carcinoma; MicroRNAs-122; Alpha-fetoprotein

INTRODUCTION

Liver cancer is currently the second

most common cause of cancer-related

death worldwide, and hepatocellular

carcinoma (HCC) accounts for more than

90% of liver cancers [1] One of the

reasons for the high mortality in HCC is

that the tumors are frequently detected

at a stage when curative resection is no

longer feasible because of intrahepatic and extrahepatic metastases Today, the diagnosis of HCC relies on the finding of

a liver mass in radiology imaging studies including ultrasonography, computed tomography (CT), and/or magnetic resonance imaging (MRI) [2] However, the diagnosis

of small lesions is relatively inaccurate One of the common approaches used

1 Viettiep Hospital

2 108 Military Central Hospital

3 Vietnam Military Medical University

Corresponding author: Dang Chieu Duong (dcduong@gmail.com)

for screening HCC in a high

risk-population is serum tumor markers such

as alpha-fetoprotein (AFP) and

Des-gamma-carboxy prothrombin (DCP) The

performance of the currently used serum

protein biomarkers for routine surveillance

of HCC is unsatisfied [3] Therefore, new

biomarkers with better accuracy or with

capabilities to complement for hepatic

imaging are in need to improve the

surveillance of HCC

MicroRNA (miR) is a small noncoding

RNA gene product known to

post-transcriptionally modulate gene expression

by negatively regulating the stability or

translational efficiency of its target

mRNAs MiRs control a wide array of

biological processes, such as cell

differentiation, proliferation, and apoptosis

[4] Expressions of miRs have been

widely reported in human cancers with

both up and down-regulation detected in

neoplastic cells compared with their

normal counterparts Several recent

studies reported that miRs are stably

detectable in plasma and serum The

finding also raised the possibility that

assaying miRs in plasma or serum may

serve as a novel approach for

blood-based detection of human cancers [5] In

this study, we focused on miR-122, which

is one of the first miRs detected

abundantly in HCC We aim to: Evaluate

the expression of miR-122 plasma, and

combination of miR-122 with AFP to

diagnosis of HCC

SUBJECTS AND METHODS

1 Subjects

Patients and sample: Plasma samples

from 101 patients with hepatitis B virus

(HBV)-related HCC, 46 chronic hepatitis

B (CHB), and 103 healthy control (HC) were collected for this study between

2014 and 2017 Biopsies were taken in all patients with HCC and CHB HCC and CHB were confirmed histologically in the groups Blood samples were obtained from all patients and healthy controls, plasma samples were immediately separated from blood cells and were stored

at -800C until use MiRNA extraction and cDNA synthesis: Total RNA, including miRNA fractions was isolated from 200 µL serum with TRIzol reagent and reconstituted in 50 µL water treated with diethylpyrocarbonate (DEPC) The quality

of total RNA preparations was assessed

by NanoDrop spectrometer at 260 and

280 nm (A260/280) Approximately 300 ng

of total RNA were used for reverse transcription (RT) by RevertAid First Strand cDNA Synthesis Kit following the manufacturer's instruction Primers used for cDNA synthesis were designed according to stem-loop Quantification of

miR-122 by real-time PCR: After reverse

transcription, cDNA was reconstituted in

100 µL 25 mM-Tris-HCl pH 8.0 The real-time PCR (qPCR) reaction mixtures consisted of 10 µL of 2x Sybr-Green I master mix, 5 µL of cDNA preparation,

5 pmoL of miR-122 universal reverse primer GTGCAGGGTCCGAGGT and

5 pmoL of forward primer specific for miR-122 The qPCR reaction was performed using the Stratagene M3000p device with a pre-incubation step at 500C for

15 minutes, initial denaturation at 950C for

5 minutes, followed by 45 cycles of 950C for 15 sec and 600C for 60 seconds The RT-PCR reactions were finalized by

amplicon melting dissociation The cycle of

threshold (Ct) values were recorded and

analyzed according to the comparative Ct

method, in which the Ct value of miR-16

was used as normalization factor [6]

Statistical analysis: The HCC staging

was performed according to the Barcelona

clinic liver cancer (BCLC) staging system

Data ware expressed as mean ± SD

Differences between groups were

assessed by the t-test, or the Mann-Whitney U test, p value < 0.05 denoted the presence of a statistically significant difference The diagnostic value for differentiating between HCC patients and the control was assessed by calculating the area under the receiver operator characteristic (ROC) curve (AUC) All statistical analyses were performed using SPSS (version 21.0)

RESULTS

The main clinical and demographic characteristics such as age, gender, viral loads

and the tumour marker AFP for HCC patients, CHB patients, and 103 healthy controls

are summarized in table 1

Table 1: Clinical characteristics of the studied patients

(n = 101)

CHB (n = 46)

HC (n = 103)

Tumor differentiation

(*: p < 0.05 for comparison HCC vs CHB; NA: Not applicable)

Levels of HBV loads were significantly higher in CHB compared to HCC group (p < 0.05) As expected, the levels of the tumour marker AFP were observed

significantly higher in HCC patients compared to CHB patients (p < 0.05)

Figure 1: Differential expression miR-122 levels in different groups

The plasma miR-122 expression in patients with HCC was significantly higher than

in patients with CHB and HC (p < 0.001, p < 0.001, respectively)

Figure 2: Diagnostic performance of miR-122 and miR-122 in combination with AFP in

differentiating HCC from other groups

(A: HCC vs CHB; B: HCC [AFP ≤ 20 ng/mL] vs CHB; C: HCC vs HC and D: HCC

[AFP ≤ 20 ng/mL] vs HC)

Table 2: Diagnostic performance of miR-122 and miR-122 in combination with AFP

in differentiating HCC against control subjects

AUC Variable(s)

vs CHB

HCC (AFP ≤ 20ng/mL)

vs HC

(*: p < 0.05 for comparison AFP vs miR-122; **: p < 0.05 for comparison AFP vs miR-122+AFP; A: Not applicable)

Diagnostic performance of miR-122

was significantly higher than AFP levels in

differentiating HCC from CHB (p < 0.05)

When miR-122 is combined with AFP

levels to form the so-called (miR-122 + AFP)

panel under the logistic regression model,

the diagnostic performance in discriminating

HCC from CHB obtained higher accuracy

(AUC = 0.914, p < 0.05) When the AFP

levels were lower than 20 ng/mL, miR-122

still preserved its diagnostic accuracy to

distinguish HCC from other groups

including CHB patients (AUC = 0.915),

with either HC (AUC = 0.991)

DISCUSSION

Cancer-specific alterations of miRs

expression are common in various

cancers and act as critical roles in cancer

progression More and more evidence

indicated that circulating plasma miRs

served as promising biomarker for

diagnosing and monitoring treatment

response Circulating miRs have been

found to be stable in serum and plasma,

suggesting its potential ability to be

diagnostic biomarkers Currently, AFP is

often criticized for its high false positivity

in distinguishing HCC with other liver

diseases In addition, it is estimated that

30 - 40% of all HCC patients are of AFP-negative status, making it difficult to diagnose and assess treatment response [7] Thus, identifying a new biomarker that could complement AFP is of great importance For now, several miRs have been identified as novel biomarkers for HCC [8]

In this study, we conducted analysis to evaluate whether plasma miR-122 had the potential ability to be a new biomarker for detecting HCC In pilot group, we assessed the expression of miR-122 on

250 plasma samples of 101 patients with HBV-related HCC, 46 chronic hepatitis B patients and 103 HC The present study demonstrated that plasma miR-122 expression in patients with HCC was significantly higher than in patients with CHB and HC (p < 0.001, p < 0.001, respectively) ROC analyses for the diagnostic power of plasma miR-122 yielded an AUC of 0.910 in differentiating patients with HCC from those with CHB, AUC of 0.989 in differentiating patients with HCC from HC

These results suggest that plasma miR-122 is a valuable biomarker for HCC Furthermore, the superiority of the

differentiating power of a single

measurement of plasma miR-122 compared

with AFP was statistically confirmed, and

the differentiating power of the combination

of plasma miR-122 and AFP was

significantly stronger than AFP alone,

suggesting that measurement of both

plasma miR-122 and AFP has a better

differentiating power than plasma

miR-122 and AFP alone Furthermore, plasma

miR-122 level was significantly elevated

even in patients with HCC who have AFP

levels under 20 ng/mL When the AFP

levels were lower than 20 ng/mL,

miR-122 still preserved its diagnostic accuracy

to distinguish HCC from other groups

including CHB patients (AUC = 0.915),

with either HC (AUC = 0.991) Whatever

the reason, that differentiating power of

plasma miR-122 was significantly superior

to that of AFP, we suggested that plasma

miR-122 is a useful diagnostic marker for

HCC At the same time, we must keep in

mind that, as even patients with advanced

HCC were included in the present study,

the enrollment of the patients was not

designed for the examination of

diagnostic markers, suggesting the

possibility that the aforementioned

sensitivity and specificity might be

over-estimated In addition, we did not examine

plasma miR-122 level in cirrhotic patients

who also have possibility for developing

HCC in the present study We should

investigate whether the miR-122

measurement is useful in differentiating

HCC patients from cirrhotic patients in the

future Furthermore, large-scale cohorts

and strict study design are needed

to further confirm the diagnostic role of

miR-122

CONCLUSIONS

Our findings suggest that plasma

miR-122 levels may help enhance the diagnosis of HCC, especially for AFP-negative HCC

REFERENCES

1 Omata M, Cheng A.L, Kokudo N et al

Asia-Pacific clinical practice guidelines on the management of HCC: A 2017 update Hepatol Int 2017, 11 (4), pp.317-370

2 Yu W.B, Rao A, Vu V et al

Management of centrally located HCC: Update 2016 World J Hepatol 2017, 9 (13), pp.627-634

3 Toyoda H, Kumada T, Tada T et al

Tumor markers for HCC: Simple and significant predictors of outcome in patients with HCC Liver Cancer 2015, 4 (2), pp.126-136

4 Tsuchiya N, Sawada Y, Endo I et al

Biomarkers for the early diagnosis of HCC World J Gastroenterol 2015, 21 (37), pp.10573-10583

5 Hayes C.N, Chayama K Micro-RNAs as

biomarkers for liver disease and HCC J Mol Sci 2016, 17 (3), p.280

6 Livak K.J, Schmittgen T.D Analysis of

relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method Methods 2001, 25 (4), pp.402-408

7 Bruix J, Sherman M American

Association for the study of liver, management

of HCC: An update Hepatology 2011, 53 (3), pp.1020-1022

8 Ding Y, Yan J.L, Fang A.N et al

Circulating miRNAs as novel diagnostic biomarkers in HCC detection: A meta-analysis based on 24 articles Oncotarget 2017, 8 (39), pp.66402-66413

9 This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number; 106-Ys.02-2016.20