Circulating microRNA-122 as a potential biomarker for Hepatitis C Virus-Induced Hepatocellular Carcinoma

doi:10.21203/rs.3.rs-1439497/v1

The features of translational repression by micro-RNA can cause various diseases in humans. The liver-specific microRNA (mir122) is primarily involved in tissue tropism during HCV infection. This study focuses on evaluating host serum miR-122 as a prognostic marker in HCV infection. A positive expression of miR-122 was observed in CHC patients followed by HCC patients compared to healthy controls. A difference in median levels of miR-122 expression in CHC and HCC patients (p < 0.000) was found in contrast to Cirrhosis patients (p = 0.511). The serum miR-122 expression was found threefold higher in liver cirrhosis patients than chronic hepatitis. Further, the AUROC analysis of miR-122 expression profile can efficiently distinguish CHC patients (AUROC = 0.978, p = 0.000, 95% CI = 0.958 to 0.998) and HCC from healthy controls (AUROC = 0.971, p = 0.000, 95% CI = 0.944 to 0.997). Moreover, ROC curve analysis significantly distinguished between CHC patients from Cirrhosis patients (AUROC = 0.955, p = 0.000, 95% CI = 0.925 to 0.986) but not CHC from HCC patients (AUROC = 0.584, p = 0.104, 95% CI = 0.485 to 0.684). This study revealed a substantial correlation of miR-122 with HCV viral load (r = 0.56, p = 0.000), ALT (r = 0.67, p = 0.000) and AST (r = 0.65, p = 0.000) levels. Serum miR-122 can potentially serve as a promising prognostic tool for HCV induced HCC.

HCV

HCC

miRNA

Liver Injury

Genotype 3

Hepatitis C Virus (HCV) is an enveloped, positive stranded RNA virus with a genome size of 9.6kb. It belongs to genus Hepacivirus in the family Flaviviridae. Persistent HCV infection is one of the leading causes of liver disease worldwide and leads to liver cirrhosis and Hepatocellular carcinoma (HCC) ^1–3. HCV genotype 3 is the most common in India and Northeast India has a similar prevalence rate as with the rest of the country ^4–6.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs that regulate up to 60% of the expression of cellular mRNAs. miRNAs mostly repress their targets by interaction with the 3' untranslated region (UTR), however, miRNAs may cause upregulation of their targets through interaction in a non-3' UTR region of the mRNA ⁷.The prospect of using miRNAs as prognostic markers is of interest and has been reported by various studies. MicroRNA changes in disease such as HCC and liver fibrosis, their sensitive detection by quantitative PCR, the non-invasive nature of its assessment make them attractive biomarkers for studying modulation of hepatotrophic virus infection ⁸. Also as miRNAs are not targeted by ribonucleases they remain extremely stable in clinical samples ^9–11. miR-122 is a liver specific miRNA whose biological functions are to maintain liver homeostasis, cholesterol metabolism and foetal liver development ⁷ and is hardly expressed in other tissues ¹². The abundance of miR-122 in the liver helps HCV to promote replication and translation of its genome ^2,13,14.

Circulating miR-122 in serum has previously been identified as biomarker for the detection of HCV induced liver injury ^9,15−17. Since the increasing trend of disease burden of HCV can only be tackled with easy and early diagnosis before treatment, the present study aimed to evaluate the diagnostic potential of serum miR-122 levels in HCV genotype 3 infected patients with chronic infection and cirrhosis from Northeast India.

Study subjects

Serum was collected from 192 patients attending the Outpatient Department of Department of Gastroenterology, Gauhati Medical College and Hospital, Guwahati India with informed consent. Patients with Anti-HCV antibodies and HCV RNA positive were included in the study. All patients were confirmed to have HCV genotype 3 infections. Patients with HIV or HBV co-infection, diabetes, alcoholic liver disease, autoimmune or haematological diseases and abnormal liver ultrasounds were excluded from the study.

72 patients were categorised as CHC (Chronic Hepatitis C), while 64 had liver cirrhosis and 56 were diagnosed with HCC. The presence of liver cirrhosis was ascertained by the occurrence of ascites, esophageal varices, splenomegaly, jaundice, imaging and liver biopsies (when available). HCC was diagnosed with 4-phase multi-detector computed tomography (CT) scan, dynamic contrast-enhanced magnetic resonance imaging (MRI) as mentioned elsewhere ¹⁸.

Serum from N=40 sex and age matched healthy individuals were taken as controls. All of the controls were selected on the basis of normal liver function tests, normal ultrasound of liver, and absence of viral markers, autoimmune hepatitis and diabetes. Informed consent was obtained from all individual participants included in the study. The study was approved by Institutional ethics committee of Gauhati Medical College and Hospital.

HCV RNA extraction, quantification and genotyping

QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) was used for extracting HCV RNA from serum of HCV infected patients according to manufacturer’s protocol and stored at −80 °C. HCV viral load was quantified using Artus HCV RG RT-PCR Kit (Qiagen, Valencia, CA, USA) on Rotor-Gene Q 5plex Platform (Qiagen, Valencia, CA, USA). For HCV genotyping 5’UTR-core region was PCR amplified with specific primers and 173bp product was sequenced to determine the HCV genotype.

miRNA isolation

Total RNA was isolated from whole blood samples using the miRNeasy Minikit (Qiagen, Valencia, CA, USA) followed by miRNA enrichment. Briefly, 100 µl of patient serum was taken with 500 µl of QIAzol lysis reagent and incubated at room temperature for 5 minutes. 140 µl of chloroform was added and vortex to mix for 15 s and incubated at room temperature for 2 minutes. Two separate phases were obtained by centrifuging at 12000 x g at 4⁰C for 15 minutes. For purification of only miRNA Upper aqueous phase was transferred into new sterile tubes and one volume of 70% ethanol was added and mixed by pipetting. Up to 700 µl of the mixture was added onto RNeasy mini spin column placed in a clean 2ml collection tube and centrifuged at 8000xg at room temperature for 15s. 450 µl of 100% ethanol was added to the flow through and mixed thoroughly by vortex. RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA) was used for the purification of miRNA fraction from total RNA. 700 µl of the mix was added onto the RNeasy MinElute spin column placed in a 2ml collection tube, and centrifuged at 8000xg at room temperature for 15s. The flow through was discarded and the process repeated for the whole mix volume. 700 µl buffer RWT was added onto the column and centrifuged at 8000xg for 15s. 500 µl of buffer RPE was added onto the column, centrifuged at 8000xg at room temperature for 15 Secs and the flow through discarded. 500 µl of 80% ethanol was added to the column, centrifuged at 8000xg at room temperature for 2 min and flow through was discarded. A new 2ml spin column was used and again centrifuged at 8000xg at room temperature for 5 min. 14 µl of RNase-free water was added onto the column and centrifuged at 8000xg to elute miRNA enriched fraction.

Reverse Transcription

10ng of miRNA was used in total volume of 20 µl to carry out reverse transcription by incubating for 60min at 37⁰C and 5min at 95⁰C using miScript PCR Starter Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol.

Quantitative real-time PCR

Mature miR-122 expression was carried out by quantitative real time PCR using miScript PCR Starter Kit and miScript SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) with Hs_miR-122a_1 miScript Primer Assay (Qiagen, Valencia, CA, USA) which targets hsa-miR-122-5p. Relative quantification was done using 2^-∆∆CT method using U6-snRNA as the internal reference.

Statistical analysis

Statistical calculations were carried out using Statistical Package for the Social Sciences version 16.0 software (SPSS Inc., Chicago, IL) or GraphPad Prism-5.0 (GraphPad Software, CA, USA). A Chi square test or Fischer exact test was performed to compare categorical data. Comparison of independent samples from two groups was performed using the Mann-Whitney U-test. Spearman’s nonparametric rank test was used to determine nonparametric statistical significance. The correlation coefficients (𝑟) were calculated using Spearman correlation. Receiver operating characteristic (ROC) analysis was performed to assess the diagnostic and prognostic accuracy and area under the curve (AUC) was calculated. AUROC less than 0.60 were considered unreliable for ROC curve. p < 0.05 was considered to be statistically significant.

Demographic and laboratory characteristics of the HCV patients and control group

The clinical and laboratory data of the patients are summarized in Table 1. The average age of Cirrhosis and HCC patients was found higher than both healthy controls and CHC patients (p = 0.0001). A significant difference was observed for liver enzyme ALT and AST level between controls and all other groups of patients (p = 0.0001). ALT and AST were also significantly elevated in HCC patients than CHC and Cirrhosis patients (p = 0.0001). Similarly, difference was observed for total bilirubin (p = 0.0001) and serum albumin level (p = 0.0001) in cirrhosis and HCC group upon comparison with healthy control. However, no difference was observed while comparing healthy controls with CHC patients. Also, no significant variation was observed between the occurrence of Ascites but Child Pugh score was significantly different among the Cirrhosis and HCC patients (p = 0.0112).

Serum levels of miR-122

Relative quantification of serum miR-122 was carried out using U6 snRNA as a reference. The highest expression of miR-122 was found in CHC patients followed by HCC patients with mean fold changes of 5.02 ± 1.48 and 4.46 ± 1.26 respectively in comparison to controls. Patients with Liver Cirrhosis had lesser miR-122 expression as evidenced by a fold change of 2.46±0.84 in comparison to healthy controls. A significant difference in median levels (calculated using Mann-Whitney U-Test) of miR-122 expression was observed in CHC and HCC patients (p < 0.000) upon comparison with healthy control in contrast to Cirrhosis patients (p = 0.511).

Mann-Whitney U-Test was also performed for serum miR-122 expression levels among different disease states in HCV infected patients. A significant difference was found between CHC and Liver Cirrhosis patients (p < 0.0001) but not with HCC patients (p = 0.0446). Also, while comparing miR-122 expression for Liver Cirrhosis and HCC patients (p < 0.0001) a significant difference was observed. Median levels of miR122 expression across the studied groups were compared by using Kruskal-Wallis test and found to be significantly different (p < 0.0001) Fig:1.

ROC curve analysis

The diagnostic ability of miR-122 as a non-invasive diagnostic biomarker for HCC in HCV infected patients was analysed by generating ROC curves. ROC analysis demonstrated that miR-122 expression profile can efficiently distinguish CHC patients (AUROC = 0.978, p = 0.000, 95% Confidence Interval = 0.958 to 0.998) and HCC from healthy controls (AUROC = 0.971, p = 0.000, 95% Confidence Interval = 0.944 to 0.997). However, miR-122 though had a reduced diagnostic ability to distinguish between Cirrhosis and healthy controls (AUROC = 0.632, p = 0.024, 95% Confidence Interval = 0.524 to 0.741). Further, ROC curve analysis also showed efficient discrimination of CHC patients from Cirrhosis patients (AUROC = 0.955, p = 0.000, 95% Confidence Interval = 0.925 to 0.986) but not CHC from HCC patients (AUROC = 0.584, p = 0.104, 95% Confidence Interval = 0.485 to 0.684). Also serum miR-122 level exhibit an important diagnostic value through its ability to distinguish Cirrhosis patients from HCC patients (AUROC = 0.935, p = 0.000, 95% Confidence Interval = 0.895 to 0.975) based on its altered expression profile in different disease state. (Fig 2)

miR-122 expression and its association with HCV viral load

High HCV viral load was not significantly associated with development of cirrhosis or HCC (p = 0.1908, Table No.1). We analysed the association of miR-122 expression with viral load in HCV infected patients. Spearman rank correlation (𝑟) analysis was used to determine association of miR-122 expression with Viral load in (r = 0.56, 95% confidence interval: 0.400 to 0.737, p = 0.000). Viral load was plotted against miR-122 expression as shown in Fig 3a thus establishing association of miR-122 with disease severity in terms of viral load.

miR-122 Fold change with Liver Function Tests in HCC patients

To evaluate whether serum miR-122 levels correlate with serum necro-inflammatory markers ALT, AST, AST/ALT, bilirubin, and albumin in HCC patients, Spearman rank correlation (𝑟) analysis was performed. miR-122 had significant positive correlation with serum ALT (r = 0.544, 95% confidence interval: 0.321 to 0.709, p< 0.0001) and AST (r = 0.532, 95% confidence interval: 0.306 to 0.701, p <0.0001) Fig: 3(a), 3(b), 3(c) and Table: 2.

The role of MicroRNAs are ascertain to be vital in virus–host interactions, pathogenesis and host resistance via regulation of post-transcriptional or translational modification. The abundance of hepatic microRNA-122 has made it the most commonly targeted biomarker of liver disease including CHC infection. A non-invasive technique to recognize the severity of liver disease by circulating serum miR-122 assessment essentially can become a preferable choice of diagnosis ^{2,10,15,19−21}.

MiR-122 expression levels of HCV patients are deregulated and distinct from healthy controls, thereby making miR-122 a possible biomarker ^14,16,17,22. Circulating miR-122 has been observed to be significantly high in serum of CHC patients in comparison to healthy controls but most of the studies have not included HCV genotype 3 at a large scale ²³. in a previous study by Cheng et al. 2014 higher miR-122 was reported in PBMCs of HCV patients ²⁴. Similar results were also reported in study conducted in HCV genotype 3 infected patients from North India, however, the number of patients analysed was low (n = 25) ⁶. In contrast, hepatic and blood expression levels of miR-122 was not found to be positively correlated among HCV genotype 3 patients ²⁵. Indeed, in the present study serum miR-122 was found two-fold higher in CHC patients in compared to normal controls in Northeast Indian population. ROC curve analysis revealed miR-122 in serum of HCV genotype 3 infected patients could be used as a diagnostic tool for HCV infection which is consistent with the previous research findings ²⁶.

The association of hepatic miR-122 levels and extent of liver fibrosis is not yet established. Hepatic miR-122 expression level is reduced significantly with the severity of liver fibrosis in patients with chronic HCV infection ^3,27,28 while in another study liver fibrosis stage was not significantly correlated with miR-122 ^2,29. The serum miR-122 has also been previously reported as a potent diagnostic marker in determining liver injury and distinguishing viral and non-viral etiology. ^30,31. In our study ROC analysis showed that serum miR-122 can be an extremely sensitive diagnostic tool for determining the disease status in CHC patients. MiR-122 has been widely implicated in the diagnosis of HCC, with higher miR-122 levels in comparison to controls ^10,11,32. Recently Motawi et al. have reported non-significant differences in miR-122 expression in HCC patients with that in controls ²⁰. In case of patients with liver cirrhosis serum miR-122 levels were significantly reduced in comparison to that of CHC patients with almost 3 fold reduction. This is in concurrence with previously reported studies and supports the notion that with hepatocyte destruction and replacement with fibrous tissue, miR-122 export from liver decreases ¹⁰.

In several studies on HCV, that have reported no correlation of miR-122 expression in serum with HCV viral load ^3,19,24,33. Hepatic miR-122 expression was also not correlated with serum HCV viral load ^34,35 However, few contrasting results have been reported with the association of decreased hepatic miR-122 with increased viral load ^27,36. In HCV genotype 3 infections no correlation was found between both hepatic and serum miR-122 levels with viral load ²⁵. Recently, Kumar et al reported a strong correlation of miR-122 in serum with viral load in HCV genotype 3 infections ⁶. We also observed significant positive correlation of miR-122 with serum HCV genotype 3 RNA levels.

Variations in miR-122 concentrations in serum or plasma have been established as more specific for liver diseases in comparison to serum transaminases, which potentially can originate from non-liver based sources ²⁴. Serum miR-122 concentrations were correlated with higher AST and ALT levels in Egyptian HCV genotype 4 patients and also in genotype 1 patients ^10,11. However, correlation between serum microRNA-122 and ALT levels was observed only in acute hepatitis patients and not in CHC patients in genotype 1 infection ³³. In another study serum miR-122 and miR-21 were weakly correlated with ALT and AST in Chronic Hepatitis B (CHB) and CHC patients of multiple genotypes ¹⁵. Previous studies focused on HCV genotype 3 have indicated that serum miR-122 levels were significantly higher with strong correlation with elevated ALT, AST and inflammatory scores ^2,6. Our study on HCV genotype 3 patients from Northeast India also followed a similar trend displaying a significant correlation of liver transaminases with serum miR-122 levels.

In conclusion, our study showed that serum miR-122 can be used to diagnose liver injury in CHC patients. No study has been previously reported the predictive ability of serum miR-122 levels in HCV genotype 3 infected patients from Northeast India.

Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Conflict of Interest: The authors declare that they have no conflict of interest.

Availability of Data and Materials: The data generated and/or analysed during the current study are not made publicly available as consent of sharing of raw data is not obtained from all patients, but the same will be available from the corresponding author on reasonable request.

Acknowledgements: The authors extend their appreciation to King Saud University for funding this work through research supporting project (RSP-2021/376), Riyadh, Saudi Arabia.

Chen, J. Y. et al. IL28B genetic variations are associated with high sustained virological response (SVR) of interferon-α plus ribavirin therapy in Taiwanese chronic HCV infection. Genes Immun. 12, (2011).
Butt, A. M. et al. Parallel expression profiling of hepatic and serum microRNA-122 associated with clinical features and treatment responses in chronic hepatitis C patients. Sci. Rep. 6, (2016).
El-Guendy, N. M. et al. The liver microRNA expression profiles associated with chronic hepatitis C virus (HCV) genotype-4 infection: A preliminary study. Hepat. Mon. 16, (2016).
Puri, P. et al. Consensus statement of HCV task force of the Indian National Association for Study of the Liver (INASL). Part I: Status report of HCV infection in India. Journal of Clinical and Experimental Hepatology vol. 4 (2014).
Firdaus, R. et al. Impact of host IL28B rs12979860, rs8099917 in interferon responsiveness and advanced liver disease in chronic genotype 3 hepatitis C patients. PLoS One 9, (2014).
Kumar, S., Chawla, Y. K., Ghosh, S. & Chakraborti, A. Severity of hepatitis C virus (genotype-3) infection positively correlates with circulating microrna-122 in patients sera. Dis. Markers 2014, (2014).
Szabo, G. & Bala, S. MicroRNAs in liver disease. Nature Reviews Gastroenterology and Hepatology vol. 10 (2013).
A, W. et al. Serum MicroRNAs as Biomarkers in Hepatitis C: Preliminary Evidence of a MicroRNA Panel for the Diagnosis of Hepatocellular Carcinoma. Int. J. Mol. Sci. 20, (2019).
Becker, P. P. et al. Performance of serum microRNAs-122,-192 and-21 as biomarkers in patients with non-Alcoholic steatohepatitis. PLoS One 10, (2015).
Zhang, Y. C., Xu, Z., Zhang, T. F. & Wang, Y. L. Circulating microRNAs as diagnostic and prognostic tools for hepatocellular carcinoma. World Journal of Gastroenterology vol. 21 (2015).
El-Garem, H. et al. Circulating microRNA, miR-122 and miR-221 signature in Egyptian patients with chronic hepatitis C related hepatocellular carcinoma. World J. Hepatol. 6, (2014).
Gerresheim, G. K. et al. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure. Cell. Mol. Life Sci. 74, (2017).
Masaki, T. et al. MiR-122 stimulates hepatitis C virus RNA synthesis by altering the balance of viral RNAs engaged in replication versus translation. Cell Host Microbe 17, (2015).
Thakral, S. & Ghoshal, K. miR-122 is a Unique Molecule with Great Potential in Diagnosis, Prognosis of Liver Disease, and Therapy Both as miRNA Mimic and Antimir. Curr. Gene Ther. 15, (2015).
Appourchaux, K. et al. MicroRNA-based diagnostic tools for advanced fibrosis and cirrhosis in patients with chronic hepatitis B and C. Sci. Rep. 6, (2016).
Amr, K. S., Elmawgoud Atia, H. A., Elazeem Elbnhawy, R. A. & Ezzat, W. M. Early diagnostic evaluation of miR-122 and miR-224 as biomarkers for hepatocellular carcinoma. Genes Dis. 4, 215–221 (2017).
El-Ahwany, E. G. E. et al. MicroRNA-122a as a non-invasive biomarker for HCV genotype 4-related hepatocellular carcinoma in Egyptian patients. Arch. Med. Sci. 15, 1454–1461 (2019).
Roberts, L. R. et al. Imaging for the diagnosis of hepatocellular carcinoma: A systematic review and meta-analysis. Hepatology 67, 401–421 (2018).
Cermelli, S., Ruggieri, A., Marrero, J. A., Ioannou, G. N. & Beretta, L. Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease. PLoS One 6, (2011).
Motawi, T. M. K., Sadik, N. A. H., Shaker, O. G. & Ghaleb, M. H. Elevated serum microRNA-122/222 levels are potential diagnostic biomarkers in Egyptian patients with chronic hepatitis C but not hepatic cancer. Tumor Biol. 37, (2016).
Kim, G. W. et al. Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2. PLoS Pathog. 12, (2016).
Coulouarn, C., Factor, V. M., Andersen, J. B., Durkin, M. E. & Thorgeirsson, S. S. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 2009 2840 28, 3526–3536 (2009).
Zhang, S. et al. Dysregulated serum microRNA expression profile and potential biomarkers in hepatitis C virus-infected patients. Int. J. Med. Sci. 12, (2015).
Chang, C. C. et al. MicroRNA expression profiling in PBMCs: A potential diagnostic biomarker of chronic hepatitis C. Dis. Markers 2014, (2014).
Oliveira, K. G. et al. Increased hepatic expression of miRNA-122 in patients infected with HCV genotype 3. Med. Microbiol. Immunol. 205, (2016).
Zhou, X. et al. Diagnostic value of circulating miRNA-122 for hepatitis B virus and/or hepatitis C virus-associated chronic viral hepatitis. Biosci. Rep. 39, 20190900 (2019).
Estrabaud, E. et al. Reduction of MicroRNA 122 Expression in IFNL3 CT/TT Carriers and during Progression of Fibrosis in Patients with Chronic Hepatitis C. J. Virol. 88, (2014).
Marquez, R. T. et al. Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans. Lab. Investig. 90, (2010).
Dubin, P. H. et al. Micro-RNA-122 levels in acute liver failure and chronic hepatitis C. J. Med. Virol. 86, (2014).
Van Der Meer, A. J. et al. Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte-derived microRNA-122. J. Viral Hepat. 20, (2013).
Wang, K. et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc. Natl. Acad. Sci. U. S. A. 106, (2009).
Zekri, A. R. N. et al. Serum microRNA panels as potential biomarkers for early detection of hepatocellular carcinoma on top of HCV infection. Tumor Biol. 37, (2016).
Wang, J. hua et al. Absolute quantification of serum microRNA-122 and its correlation with liver inflammation grade and serum alanine aminotransferase in chronic hepatitis C patients. Int. J. Infect. Dis. 30, (2015).
Sarasin-Filipowicz, M., Krol, J., Markiewicz, I., Heim, M. H. & Filipowicz, W. Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy. Nat. Med. 15, (2009).
Bihrer, V. et al. Serum mir-122 as a biomarker of necroinflammation in patients with chronic hepatitis C virus infection. Am. J. Gastroenterol. 106, (2011).
Morita, K. et al. Clinical significance and potential of hepatic microRNA-122 expression in hepatitis C. Liver Int. 31, (2011).

Table 1: Clinical characteristics of the patient groups recruited in the study

	Healthy controls (n = 40)	CHC (n=72)	Cirrhosis (n=64)	HCC (n=56)	p value
Age, years	41.9 ± 13.2	39.7 ± 15.1	51.5 ± 9.3^ab	53.2 ± 12.4^ab	0.0001
Male Gender, n (%)	30 (75.0)	48 (66.7)	53 (82.8)^b	43 (76.8)	0.0485
Leucocytes, x 10³/µL	8.1 ± 2.4	10.7 ± 5.3^a	6.1 ± 4.1^ab	6.1 ± 4.6^ab	0.0062
Haemoglobin, g%	13.2 ± 2.4	10.6 ± 1.2^a	9.3 ± 2.6^ab	8.5 ± 3.5^ab	0.0001
ALT, IU/L	43.2 ± 13.5	70.8 ± 15.6^a	87.3 ± 24.1^ab	116.3 ± 18.6^abc	0.0001
AST, IU/L	37.1 ± 17.8	72.8 ± 20.5^a	96.6 ± 28.2^ab	109.7 ± 14.9^abc	0.0021
ALP, IU/L	-	219.88 ± 102.39	317.62 ± 171.51	317.62 ± 171.51	-
Total Bilirubin, mg/dL	1.8 ± 1.2	2.2 ± 1.7	2.4 ± 1.6^ab	3.1 ± 1.8^abc	0.0001
Serum Albumin, g/dL	3.8 ± 1.8	2.9 ± 2.1^a	2.6 ± 1.2^a	2.2 ± 1.4^abc	0.0001
Ascites (Yes/No)	0/40	0/72	46/18	38/18	0.3891
Child score (A/B/C)	-	-	12/19/33	18/24/14	0.0112
Viral Load (<4x 10⁵, n (%)/ ≥4x 10⁵, n (%))	0	44 (61.1)/28 (38.9)	37 (55.2)/27 (44.8)	29 (51.8)/ 27 (42.2)	0.1902

Data are expressed as mean ± Standard Deviation

^adenotes significant difference from controls, ^bdenotes significant difference from CHC, ^cdenotes significant difference from Cirhossis

Table 2: Pearson correlation of serum miR-122 levels in liver cancer patients with routine laboratory parameters

Parameters	Cirrhosis		HCC
	r	P value	r	P value
ALT, IU/L	0.04	0.74	0.55	< 0.0001
AST, IU/L	0.07	0.53	0.53	< 0.0001
Viral Load	0.14	0.212	0.21	0.120

No competing interests reported.

Circulating microRNA-122 as a potential biomarker for Hepatitis C Virus-Induced Hepatocellular Carcinoma

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1