Detection of DNA methylated microRNAs in hepatocellular carcinoma

Accepted Manuscript Detection of DNA methylated microRNAs in hepatocellular carcinoma

Wafaa M. Ezzat, Khalda Said Amr, Yasser A. Elhosary, Abdelfattah E. Hegazy, Hoda H. Fahim, Noha H. Eltaweel, Refaat R. Kamel PII: DOI: Reference:

S0378-1119(19)30229-X https://doi.org/10.1016/j.gene.2019.02.080 GENE 43669

To appear in:

Gene

Received date: Revised date: Accepted date:

19 November 2018 13 January 2019 15 February 2019

Please cite this article as: W.M. Ezzat, K.S. Amr, Y.A. Elhosary, et al., Detection of DNA methylated microRNAs in hepatocellular carcinoma, Gene, https://doi.org/10.1016/ j.gene.2019.02.080

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ACCEPTED MANUSCRIPT Detection of DNA methylated microRNAs in hepatocellular Carcinoma Wafaa M. Ezzat,a Khalda Said Amr,b Yasser A. Elhosary,a Abdelfattah E. Hegazy,cHoda H. Fahim,d Noha H. Eltaweel,b and Refaat R. Kamele aDepartment

of Internal Medicine, National Research Centre, Cairo, Egypt of Medical Molecular Genetics, National Research Centre, Cairo, Egypt cDepartment of Surgery, Elsahel Teaching Hospital, Cairo, Egypt dDepartment of Anesthesia, Elsahel Teaching Hospital, Cairo, Egypt eDepartment of Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt bDepartment

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Abstract:

Irregular methylation, including DNA hypomethylation and/or promoter gene CpG

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hypermethylation, is involved in the pathogenesis of several solid tumors, including

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liver cancer. miRNAs are small, endogenous, noncoding RNAs that serve as posttranscriptional regulators of gene expression. Previous research has focused on identifying the factors that regulate the expression of miRNAs in hepatic

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carcinogenesis. The factors that regulate miRNA expression are not clear; in particular, the role of DNA methylation, an epigenetic regulatory factor that controls

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miRNA transcription, has not been clarified. The goal of this study is to explore our understanding of the mechanism by which HCC may develop and progress through identification of the role of epigenetically regulated miRNAs influences in the liver

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carcinogenesis. The current study included 60 patients who were well diagnosed as

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HCC patients. 60 patients who suffer from liver cirrhosis were also enrolled in the current study and 30 healthy control subjects who serve as control group. All patients will be subjected to: full clinical assessment, abdominal ultrasound, Blood sample will be withdrawn from every patients for both biochemical and serum detection of

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microRNAs (191-203 -335) by real time PCR. We found that all studied microRNAs were down regulated among HCC patients when compared to cirrhotic patients and

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controls (p value: 0.005, 0.005 and 0.001 for microRNAs 191, 203 and 335 respectively). Moreover, these microRNAs can discriminate cases of HCC from risky cirrhotic patients. We can conclude that downregulated microRNAs among HCC cases proposed a pattern to explain the role of DNA methylation on miRNA and gene expression in HCC. Key words: epigenetic-DNA methylation-miRNAs-HCC Introduction:

Several studies have demonstrated that various miRNAs are deregulated in HCC tissue samples compared with noncancerous liver tissue controls. Other researches

ACCEPTED MANUSCRIPT have focused on identifying the factors that regulate the expression of miRNAs in hepatic carcinogenesis; for example, DNA methylation or transcription factors could regulate the expression of miR-1 (1) and miR-124/miR-203 (2). The factors that regulate miRNA expression are not clear; in particular, the role of DNA methylation, an epigenetic regulatory factor that controls miRNA transcription (3). An important feature of miRNAs is that a single miRNA can regulate multiple target mRNA genes.

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For instance, miR- 122 has 32 validated cellular mRNA targets. This property enables miRNAs to exert wide control on a network of genes that play a critical role in cell division, cell death, and DNA repair. Alterations in these genes results in unrestrained

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cell proliferation that predisposes to liver cancer. Functional and target association

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studies on dysregulated miRNAs in HCC have enabled us to gain a more comprehensive understanding of their roles in HCC carcinogenesis(4).

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As hypermethylation can be reversed by specific drugs, the application of DNA demethylation has been investigated to explore its therapeutic ability to “reactivate”

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genes whose subsequent gene expression leads to the inhibition of established tumors (5,6). A number of DNA methylation inhibitors are currently under trial, including azacytidine and decitabine. Epigenetic alterations in cancer cells affect virtually every

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cellular pathway involved in cell-cycle progression ,angiogenesis, apoptosis, cell

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survival, and immunogenicity. Naturally, it is believed that “epigenetic drugs” also demonstrate versatile abilities (7,8). Our hypothesis is that DNA methylation in tumor suppressive miRNA genes is a

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common event influencing relevant mature miRNA expression, and contributes to pathogenesis of HCC. Detection of epigenetic mechanism that underlying

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pathogenesis of HCC is not yet known among Egyptian patients. In this study, we sought to identify miRNAs that are regulated by DNA methylation. Certain challenges including the development of safe and reliable personalized miRNAs delivery systems become a reality. However, our previous data with the finding accumulated to date, in conjunction with newly emerging results, are expected to yield novel insights into the pathogenesis of HCC and may eventually lead to the identification of improved therapeutic targets for treatment of this aggressive malignancy. Patients and methods: Selection of the patients:

ACCEPTED MANUSCRIPT The current study included 60 patients who were well diagnosed as HCC patients. The diagnosis of HCC depends on detection of hepatic focal lesion(s) by abdominal ultrasonography which is confirmed by spiral triphasic abdominal CT to prove the vascular criteria for HCC. 60 patients who suffer from liver cirrhosis were also enroller in the current study. The current research also included 30 healthy subjects who serve as control group. Both sexes and age ˃ 18 years were enrolled in

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the current research. Patients with prior treatment of HCC (for cases); clinical evidence of significant hepatic diseases (for controls); detection of HCC at initial evaluation (for controls); clinical or pathological evidence of metastatic tumor inside

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the liver other than HCC; clinical or pathological evidence of other malignant diseases

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or patients with HCC due to hepatitis B virus (HBV) were excluded from the study. Every patient signed an informed consent after approval of medical ethical committee

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in National Research Center.

Clinical and biochemical assessment of the patients: All enrolled subjects were subjected to:1- Detailed history taking including history

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of smoking, alcohol intake, exposure to schistosomiasis, occupational exposure to chemicals.2- Thorough clinical assessment including general signs of liver

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cirrhosis as ascites, bruising,jaundice, clubbing and hypertrophic osteoarthropathy;

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palmar erythema, gynecomastia and signs of hepatic encephalopathy.3- Laboratory examination: Blood sample were withdrawn from every patients for assessment of: Routine laboratory tests: complete blood count, fasting blood sugar, blood urea ,serum creatinine, and conventional liver biochemical tests [aspartate aminotransferase

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(AST), alanine aminotransferase (ALT), total bilirubin, serum albumin, prothrombin time and concentration]. For diagnosis and exclusion of HCV we pursued HCV

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antibody by a third-generation enzyme immunoassay and HCV RNA by PCR. - HB surface antigen (HB s Ag) and HBcAb. Antischistosomal antibody by enzyme linked immunoassay (ELISA). Alpha fetoprotein was done by enzyme linked immunoassay (ELISA). Abdominal ultrasound was done to detect focal lesions in liver and to diagnose associated conditions as ascites . Patients with HCC underwent triphasic abdominal CT examination to confirm diagnosis. Blood sampling: Five milliliters of venous blood were collected from each participant. To harvest cellfree serum, the blood was drawn into a sterile tube without anticoagulant. samples

ACCEPTED MANUSCRIPT were centrifuged at4˚C 4500 rpm for 10 minutes, and the supernatant serum was quickly removed and stored immediately at -80˚C until analysis.

Detection of serum miRNAs: Total RNA extraction was carried out using QiAzol and miRNeasy mini kit (Qiagen, Germany) according to the manufacturer’s protocol designed for plasma/serum

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samples. Quantitative RT-PCR was carried out in 2 steps. First, the reverse transcription of RNA extracts was carried out using miScript II RT kit (Qiagen, Germany) followed by qPCR using miScript SYBR Green Kit and miScript primer

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assays (Qiagen, Germany) according to the manufacturer’s protocol. Cts were

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obtained and the differential expression of each target miRNA of the patients verus healthy controls was calculated using ∆∆Ct method, and normalized using SNORD68

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as an endogenous control. Statistical Analyses:

Data analyses was conducted using Statistical Package for the Social Sciences (SPSS)

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software (SPSS Inc., Chicago, USA), version 19.0. Results are expressed as mean ± standard deviation (SD). Statistical differences were considered significant at P <

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0.05. MiRNA expression levels were compared using Mann-Whitney’s U test. The Spearman's rank correlation coefficient (r) was calculated, and testing the significance

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of r was also carried out to determine the correlation between each miRNA expression and other variables. ROC curves were constructed and AUCs were calculated. Results:

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The general characteristics of the studied groups of patients were presented in table 1. It was found that most of the studied patients were males. Chronic HCV is the most

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prevalent among the studied patients of two groups. About HCC lesions, it was noticed that most of these lesions were single (66.7%), within right lobe (50%), of grade 2 differentiation (80 %) and liver background showed Mixed cirrhosis (90%) with moderate activity.

Median serum values for for microRNAs 191, 203 and 335 were significantly decreased among patients with HCC, (p value: 0.05, 0.0001 and 0.001 for microRNAs 191, 203 and 335 respectively) as shown in table 2. MiR-191expression level showed inverse correlation with SGOT (r=-0.48, p=0.019), SGPT (r=-0.58, p=0.004), bilirubin (r=-0.5, p=0.022), and number of HCC lesions (r=-0.39, p=0.05).

ACCEPTED MANUSCRIPT While altered expression of miR-203 showed inverse correlation with SGPT (r=-0.5, p=0.049) and number of HCC lesions (r= -0.88, p=0.0001). Inverse correlation was detected statistically between MiR-335 expression and AFP (r= -0.5, p=0.03), SGOT (r= -0.66, p=0.005), SGPT (r= -0.68, p=0.004), albumin (r= 0.58, p=0.015), bilirubin (r= -0.5, p=0.037) and number of HCC lesions (r= -0.558, p=0.019)

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To detect diagnostic accuracy of the studied microRNAs to diagnose HCC cases among patients with liver cirrhosis, ROC curve was applied. It was found that studied

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miRs miR-191, miR203 and miR-335 could help diagnose HCC with sensitivity

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(80%, 92.3%and 62.5% respectively) and specificity of (87.8%, 93.7% and 63%respectively)

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Table 1: General characteristics of the two studied groups of patients. HCC group

Chronic hepatitis group

N= 60

N= 60

55 (40-67)

47.7(25-96)

55 (91.7)

45(75)

5 (8.3 )

15 (25)

Negative No (%)

9 (39.1)

9 (52.9)

Positive No (%)

14 (60.9)

8(47.1)

mean± SD.

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Variables

Sex

No (%)

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Male

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Age in years average (range).

Female No (%)

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PH OF anti-Sch.ttt

AST

IU/L

average (range).

67(25-321)

66.9(19-207)

ALT IU/L

average (range)

35(16-250)

59.7(10-204)

Albumin gm/dl

average (range)

1.8(0.9-3.1)

3.2(1.4-4.7)

Proth. Conc.%:

average (range)

58(44-75)

73.8(35-100)

Total bilirubin mg/dl average (range)

2.5(0.5-5.1)

1.8(0.6-5.6)

AFP

65(8.1-7497)

17.5(1.60-84)

ng/ml

average (range).

ACCEPTED MANUSCRIPT Cause of chronic liver disease: HCV

No (%)

HBV

No (%)

No (%)

57 (95) 3

(5)

PBC

PSC

53 (88.3) 5 (8.3) 1 1

(1.7) (1.7)

No (%)

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HCC lesions Number:

40 (66.7)

Multiple No (%)

20(33.3)

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1

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No (%)

Site:

Lt. lobe No (%) No (%)

Size cm

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Both

30 (50)

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Rt. lobe No (%)

5 (8.3) 25(41.7)

3.75±1.38

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Grade :

No (%)

12(20)

П No (%)

48 (80)

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І

Type:

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Trabecular No (%) Mixed

24 (40)

No (%)

36(60)

Liver background :

1-Mixed cirrhosis with mild activity

No

6 (10)

15 (25)

54(90)

45 (75)

(%) 2-Mixed cirrhosis with moderate activity No (%)

PH OF anti-Sch.ttt: past history of antischistosomal treatment.

ACCEPTED MANUSCRIPT AST:aspartate transaminase. ALT: alanine transaminase AFP: α-fetoprotein Proth. Conc.: prothrombin concentration

Table 2. Comparison between HCC group and chronic hepatitis group as regards

Controls

N= 60

N= 30

.01(.00-1.6)

.68(.04-11.59)

1.26 (1.23-1.59)

P value 0.05

.28 (.00-5.0)

13.48(.45-46.69) 1.00 (1.00-1.05)

0.000

.12(.00-1.00)

1.06(.03-7.08)

0.001

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Cirrhosis

N= 60

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miR-191 median (range) miR-203 median (range) miR-335 median (range)

HCC

1.15 (1.00-1.75)

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variables

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serum expression of miR-191; miR-203 and miR-335

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AFP

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Spearman's rho

AST

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Tabe 3: correlation of studied microRNAs to the biochemical variables.

miR-191 miR-203 miR-335 Correlation -.288Coefficient Sig. (1-tailed) .116 Correlation -.480-* Coefficient Sig. (1-tailed) .019

ALT

Correlation -.582-** Coefficient Sig. (1-tailed) .004

PC

Correlation .031 Coefficient Sig. (1-tailed) .455

-.374-

-.514-*

.116

.030

-.452-

-.664-**

.070

.005

-.501-*

-.679-**

.049

.004

-.532-

-.201-

.070

.289

ACCEPTED MANUSCRIPT Correlation .258 Coefficient

.403

.580*

Sig. (1-tailed) .143

.097

.015

-.368-

-.492-*

.119

.037

Correlation -.467-* Coefficient

bilirubin

Sig. (1-tailed) .022 Correlation -.385Coefficient

-.883-**

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Lesion no.

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albumin

Sig. (1-tailed) .052

.019

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

.000

-.558-*

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*: p≤0.05 , significant

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** :p≤0.001 , highly significant.

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risky cirrhotic patients.

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Table 4: accuracy of studied microRNAs to diagnose patients with HCC among

Std. Errora

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Test Result Variable(s) AUC

Pvalue

Cut-off value

sensitivity

specificity

.000

1.486

80%

78.8%

miR-203 .959 .033

.000

1.579

92.3%

93.7%

.004

0.2358

62.5%

63%

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miR-191 .859 .051

miR-335 .755 .074

Discussion:

The deregulated miRNAs and their contribution with epigenetic regulators in malignancy make them attractive biomarkers and prospective therapeutic targets in clinical applications. The use of miRNAs as therapeutic target in malignancy includes two strategies:supression of oncogenic miRNAs by using miRNA antagonists, such as anti-miRs or antagomiRs; or adminstration of tumor suppressor miRNAs through

ACCEPTED MANUSCRIPT either synthetic miRNA mimics or by stable and vector-based transfection of genes coding for miRNAs (9). deeper realization of these epigenetically controlled miRNAs might lead to a novel therapeutic strategy based on using of epigenetic drugs to control the expression of both onco-miRNAs and tumor- suppressor-miRNAs for prevention or treatment of human cancers.

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Most of the HCC cases in the current study were due to HCV infection. Pedersen and his colleagues demonstrated that interferon B (IFN-B) stimulates several specifically eight cellular miRNAs, that have sequence-expected goals within the HCV genomic

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RNA(10). Up regulation of these miRNAs in infected liver cells considerably

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inhibited viral replication. Bala and his colleagues reported that host miRNAs have released to aim viral genes and prevent their replication, and thus might play as host

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antiviral immune response (11). In the same context, Peng and his colleagues have found differential profiles of cellular miRNAs that aim the genes contributed in chemokine, B-cell receptor, PTEN, IL-16, ERK MAPK and JAK STAT signaling

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pathways, exploring an essential role of miRNAs in the replication, propagation, and latency of virus in the host cell(12). They have also concluded that miR-122, miR-

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320 and miR-191 were downregulated, whereas miR-215, miR-16, miR-26, miR-130, miR-199 and miR-155 are upregulated.43 These findings support the concept that

malignant disease.

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miRNAs have the potential to become a targeted in virally induced infectious or

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In our cases, miR-191 was found to be downregulated in the HCC

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patients relative to the healthy controls. This result was in contrast to He and his colleagues, who posted that 58.9% of hsa-miR-191 expression was higher in HCC tissues than in adjacent noncancerous tissues(3). This may be because our results were compared to healthy controls while his results were compared to adjacent noncancerous tissues of the same patients. Another study was carried out by Nagpal and Kulshreshtha, who reported that miR-191 is over-expressed in 16 type of cancers and down regulated in other 6 types (13). He and his co workers investigated hsamiR-191 locus methylation in the 11 pairs of primary liver tumors. They

ACCEPTED MANUSCRIPT found that the entire DNA was hypomethylated in these HCC samples and hypermethylated in their adjacent tissues. The miR-191 expression was correlated with methylation status in these samples. They also reported that miR-191 expression is accompanied with poor prognosis in HCC and that DNA methylation is the factor that causes increased miR-

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191 expression in HCC(3). The discrepancies between our results and that of other studies may be referred to other circumstances which may

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affect serum expression of miR-191.Environmental carcinogens also control expression of miR-191, for example, cigarette smoke and TCDD

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(a dioxin family carcinogen) was found to control miR-191 expression in

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rat lungs and liver cancer, (14,15). Thus, these findings provide evidence for hormonal, environmental and dietary control of miR-191 expression.

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Regards to miR-203, was revealed down-regulation in our HCC cases relative to the controls. Mayuko and his colleagues showed that miR-203 is a silenced tumor suppressor miRNA in HCC (16).Wei and his

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colleagues found that lower expression of miR-203 contributes to the

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progression of HCC via targeting survivin and increasing its expression and might thus promote HCC proliferation (17). The reason of the low

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expression of miR-203 in HCC tissue, however, is still unknown and needs further investigation. Epigenetic silencing of miR-203 in HCC was

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reported by Furuta and his colleagues (18), CDK6 and ABCE1 were shown as direct target genes of miR-203 supporting the role of miR-203 as tumor suppressor. In hepatocellular tumor, significantly decreased expression of miR-203 was reported in benign tumors when compared with non-tumorous liver tissue and HCC (19). Restoration of miR-203 in HCC cell lines lacking its expression of this miRNA and inducing apoptosis instead of cell cycle arrest, specific regulation of the expression status of miR-203 may be an alternative approach to molecular targeting therapy for some malignancies, including HCC. So that, they concluded

ACCEPTED MANUSCRIPT that the tumor-specific hypermethylation-mediated silencing of miR-124 and miR-203 was a relatively frequent molecular event in primary HCCs also , miR-124 and miR-203 can have cell growth-inhibitory effect with the downregulation of their potential targets, resulting in cell cycle arrest at the G 1 –S checkpoint and apoptosis, respectively, suggesting that the

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epigenetic silencing of these miRNA may contribute to hepatic carcinogenesis(19). Our study was supported with previously published

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work conducted on HCC and cirrhotic patients for different five miRs revealed that miR-203 among them due to HCV ,Khairy and his

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colleagues compared the expression of 5 target microRNAs between

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Egyptian cirrhotic HCV cases and HCV-related HCC cases and they found that among the measured 5 targets, miR-203 showed the highest

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potential as a non-invasive biomarker for HCC(20). These findings matched our results that miR-203 can predict HCC with highest

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sensitivity and specificity , 92.3% and 93.7% respectively.

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Liu and his colleagues(21) agreed Wang and his colleagues(22) who revealed that altered miR-335 expression could inhibit the proliferation and migration invasion of HCC cells via regulating ROCK1, suggesting

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that miR-335 may be used as a prognostic biomarker of HCC in the future. Cui and his colleagues found decreased miR-335 expression

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levels in patients with HCC(23) these findings were supported by Li L and his co workers᾽ study which indicate that miR-335 is reduced in HCC via aberrant promoter hypermethylation (24). Moreover, Cui and his colleagues found that HCC patients with low miR-335 levels depicted an unfavorable clinicopathologic features (high AFP values, vascular invasion, cirrhosis and large tumor volume), and those patients also had a poor response to TACE(23). These findings alone could explain the

ACCEPTED MANUSCRIPT worse clinical long-term outcome in patients with lower miR-335 expresser status. Suzuki and his colleagues concluded that aberrant DNA methylation is one of the major mechanisms underlying miRNA dysregulation in malignancy, and methylation of a subset of miRNA genes could be

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promising biomarkers for diagnosing cancer and/or expecting prognosis. Moreover, replacement of silenced tumor-suppressive miRNAs in cancer

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cells may pave the way for tailored therapy for malignant diseases(25) .

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Anwar and Lehmann posted that Concurrent hypermethylation at several genes in HCC also leads to the emerging concept of CpG island

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methylator phenotype (“CIMP”) (26). This concept was found in gastric cancer and colorectal demonstrating carcinogenesis through inactivation

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of tumor suppressor and DNA repair genes by DNA methylation(27). It was found that CIMP can be used as an autonomous prognostic marker

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(27). Though this concept is still under research in HCC provided that

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gene group for rating and determination for the phenotype are not yet universally accepted, CIMP positive HCCs have been generally related to poor prognosis (28,29) Several studies have also reported that

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DNA methylation profiles can be useful in molecular sub-classification

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of HCC to ameliorate the prognosis and the foretelling of therapeutic outcomes. The definite challenges for rote implementation in patientbased service will be not only the determination of a assent gene panel but furthermore the optimization of the methodology for methylation analysis(30,31). Conclusion: microRNAs are differentially regulated among patients with HCC. The studied microRNAs are DNA methylated. On account of the reversibility of DNA methylation and the amplified effects of miRNAs, these

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27. Issa JP. CpG island methylator phenotype in cancer. Nat Rev Cancer 2004; 4: 988-993 [PMID: 15573120 DOI: 10.1038/nrc1507] 28. Zhang C, Guo X, Jiang G, Zhang L, Yang Y, Shen F, Wu M, Wei L. CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma. Int J Cancer 2008; 123: 998-1004 [PMID:

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methylation and environmental exposures in human hepatocellular carcinoma. J Natl Cancer Inst 2002; 94: 755-761 [PMID: 12011226 DOI: 10.1093/jnci/94.10.755]

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31. Zhang C, Li Z, Cheng Y, Jia F, Li R, Wu M, Li K, Wei L. CpG island methylator phenotype association with elevated serum alpha-fetoprotein level

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in hepatocellular carcinoma. Clin Cancer Res 2007; 13: 944-952

ACCEPTED MANUSCRIPT List of abbreviation: HCC: hepatocellular carcinoma PCR: polymer chain reaction miR: micro RNA mRNA: messenger Ribonucleic acid

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HBV: hepatitis B virus AST: aspartate aminotransferase

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ALT: alanine aminotransferase

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ELISA: enzyme linked immunoassay ROC: receiving operating curve

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AUC: area under curve.

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H OF anti-Sch.ttt: past history of antischistosomal treatment. P AFP: α-fetoprotein

Proth. Conc.: prothrombin concentration

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IL-16: interleukin 16

JAK: Janus kinases

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PTEN: Phosphatase and tensin homolog

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STAT: signal transducer and activator of transcription

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TACE: Transarterial chemoembolization

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DNA methylation in tumor suppressive miRNA genes is a common event influencing relevant mature miRNA expression, and contributes to pathogenesis of HCC.

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Certain challenges including the development of safe and reliable personalized miRNAs delivery systems become a reality.

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On account of the reversibility of DNA methylation and the amplified effects of miRNAs, these differentially methylated miRNAs may provide clues for

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the diagnosis and therapy of HCC

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