Hepatocellular carcinoma (HCC) is the world’s second-leading cause of cancer-related deaths (De Toni et al., 2020), with chronic hepatitis C virus (HCV) infection being a key risk factor (Ozakyol, 2017). It is worth noting that HCC accounts for 13% of all malignancies in Egypt and is the second most common disease in men. In Egypt in 2010, chronic HCV accounted for 94 percent of HCC cases, with 6,000–7,000 fatalities per year owing to HCC (Fathy.Elmougy et al., 2019). Since they are protected from RNases, circulating miRNAs have been proven to have a high level of stability (Cui et al., 2019) even under extreme conditions like boiling, long storage, and many freeze–thaw cycles, with extremely low or high pH (Drees and Pegtel, 2020). These findings suggest that miRNAs are widely distributed and may be effective in the early detection of HCC (Shen et al., 2013).

MiRNA-122 (miR-122) expression is restricted to the liver, where it is estimated to account for 70% of all miRNA expression. miR-122 has been shown to have a role in hepatocarcinogenesis, and variations in the circulating amount of miR-122 were detected in patients with HCC compared to healthy people (Zhao et al., 2020). In addition, miR-122 was found to have a vital role in the infection of hepatitis C virus (Panigrahi et al., 2021). Deregulation of miR-122 has been linked to an aggressive kind of HCC in several investigations (Franck et al., 2020). miR-122 was first identified in HCC patients as a tumor suppressor gene (Fong et al., 2015). Also, miR-122 was detected in breast cancer patients as a tumor suppressor that targets the receptor of insulin-like growth factor-1 (IGF1R) (Wang et al., 2012). Other studies have revealed that miR-122 is a tumor suppressor through modulating oncogenes including CDK4, cyclin G1, and AKT3 in hepatocellular carcinoma (HCC) (Nassirpour et al., 2013). Meanwhile, another study demonstrated the overexpression of miR-122 in types of renal carcinoma (ccRCC) (Chow et al., 2010), implying miR-122 as an onco-microRNA in renal cancer (Fong et al., 2015). miR-122 physiological function in cancer tends to vary depending on the kind of cancer, and the exact mechanism through which miR-122 affects ccRCC growth is yet undetermined (Jingushi et al., 2017).

miR-483 was shown to be highly elevated in the serum of people who had HCV infection (Shwetha et al., 2013), suggesting that upregulation of miR-483-5 could be a mechanism for regulating various pathways involved in HCV pathogenesis or infection defense (Shwetha et al., 2013). In HCC cells, miR-483 promotes tumor invasion using activated leukocyte cell adhesion molecule (ALCAM) as a functional target (Tang et al., 2017). ALCAM is suggested to have a crucial role in proliferation and migration of cancer cells as a member of the immunoglobulin super family (Jiang et al., 2020). ALCAM expression and function varied among tumors; in certain tumors, it promotes malignancy, while in others, it suppresses it (Shen et al., 2013). These data suggest that the pathway of miR-483-5/ALCAM is a key regulator in HCC intrahepatic metastasis and that it can be used as a prognostic indicator and the base for tailored treatment (Lu et al., 2018). In cancer cell lines, miR-483-3p was discovered to modulate antiapoptotic oncogenes including HCT116 in colorectal carcinoma and HEPG2 in liver carcinoma. In addition, miR-483-5 was suggested to have a growth-promoting effect in an in vitro study of adrenocortical cancer. These findings suggest that miR-483-5 may have a malignant effect in carcinogenesis (Shen et al., 2013).

miR-335 promotes cancer growth by acting as an oncogene (Fouda et al., 2021). In gallbladder cancer, miR-335 suppresses the myocyte enhancer factor-2D (MEF2D) and improves cell proliferation as well as increasing the sensitivity of cancer cells to 5-fluorouracil chemotherapy (Ye et al., 2021). MEF2D is a member of the MEF2 family of transcription factors. In muscle, heart, and cancer cells, it can control cell division, differentiation, and apoptosis (McKinsey et al., 2002). Another study has demonstrated that miR-335 can promote cell proliferation and progression through targeting the cancer suppressor RAS-p21 protein activator-1 (RASA1) in CRC (Lu et al., 2016). The expression of miR-335 is aberrant in a range of malignancies (Zarfeshani et al., 2015); yet, it is still debated in some cancer types. Furthermore, the role of miR-335 in numerous cellular processes as well as the miR-335 complex regulatory networks is unknown.

The current study aimed to investigate the expression profiles of miR-122, miR-483, and miR-335 in sera from Egyptian patients with non-malignant HCV and HCV-related HCC in an attempt to use them as potential non-invasive biomarkers for HCV-related HCC diagnosis.

Gender did not differ substantially between the tested groups (p = 0.127). However, there was a male predominance of 80 percent, 73.3 percent, and 58.3 percent in HCC-related HCV, non-malignant HCV, and control groups, respectively. In addition, there was no significant difference between the studied groups in age (Table 1). Serum levels of AST, ALT, ALP, and GGT were significantly higher than the normal ranges in the HCC group. Albumin and prothrombin concentrations decreased significantly through the progression of liver disease among the HCC group (Table 2).

Table 3 shows the clinicopathological features of HCC patients. At the time of diagnosis, HCC patients had a wide range of AFP values (mean SD), with normal AFP levels (up to 10 ng/ml) present in as many as 23.3 percent of patients, 35.5 percent having AFP >20 ng/ml, and 41.2 percent having AFP>400 ng/ml. HCC patients had Child–Pugh grade B, and 20% and 80% had stage C with the mean Child–Pugh score of 6.46 ± 1.36. 66.7% of the HCC patients had mild ascites, while 6.7% had severe ascites. In BCLC staging, 46.7% had grade B, 13.3% grade D, and 26.7% had grade C. The HCC patients had a mean “Model for End-Stage Liver Disease” (MELD) score of 8.40 ± 7.89. In patients with cirrhosis, alcoholic hepatitis, acute liver failure, and acute hepatitis, the MELD score has been proven as a predictor of survival. The number of extra-hepatic organ failures is more predictive of mortality in terminally sick cirrhotic patients than the MELD score, which ranges from 6 to 40.

Cancer detection now has a low sensitivity since many cancers are not detected early enough, delaying therapy until it is too late (Fiala and Diamandis, 2018). MiRNA expression is typically dysregulated in cancer (Shaker et al., 2020), resulting in a distinct expression profile that aids in early cancer detection. MiRNAs linked to tumor growth are overexpressed, while suppressors are under-expressed (Shaker et al., 2021). As a result, tissue-specific miRNAs are becoming more widely used in cancer diagnosis (Drees and Pegtel, 2020; Zaafan and Abdelhamid, 2021). The focus of this research was to investigate the expression profiles of selected miR-122, miR-483, and miR-335 in sera from Egyptian patients with HCV-related HCC in order to be used as new non-invasive markers for HCV-related HCC diagnosis.

The expression of miRNA-122 is constrained to the liver, where it is assumed to account for 70 percent of all miRNA expression (Fu and Calin, 2018). In the present research, serum expression levels of miR-122 in the HCV and HCC groups were significantly higher than those in the normal control group (p value 0.01), although serum expression levels of miR-122 in the HCC group were significantly lower than those in the HCV group (p value 0.01). According to recent research, miRNA-122 expression levels decrease during hepatocarcinogenesis, suggesting that miRNA-122a can act as a tumor suppressor. Our findings were in line with earlier research, which found a decrease in miRNA-122 levels in HCC patients compared to HCV patients (Luo et al., 2013). The levels of miRNA-122, on the contrary, have arisen significantly in the HCV group, implying that miRNA-122 levels increase significantly during hepatocyte damage and subsequently fall dramatically once the liver enters carcinogenesis. According to a study by Jiang et al. [25], miRNA-122a levels in the blood have been found to be higher in HCC patients than in healthy people. Matching with our results (El- Garem et al., 2014), Trebicka et al. (2013) examined hepatic miR-122 expression in HCV-linked HCC in comparison with healthy liver samples and found that miR-122 was significantly upregulated in malignant liver nodules compared to healthy liver. They hypothesized that miR-122 would suppress the expression of undiscovered tumor suppressor genes, resulting in tumor development (Trebicka et al., 2013). miR-122 expression was considerably higher in ccRCC tissues with lymphatic invasion than those without lymphatic invasion, despite the fact that it was unrelated to clinical stages and grades. While more research on the exact process is required, the findings of this study are congruent with those of Lian et al., who found that miR-122 expression increased cancer cell malignancy characteristics in RCC cells (Lian et al., 2013), reliable with the preceding study in ccRCC.

Previous research has demonstrated higher expression of miR-483 in HCC patients’ serum (Gui et al., 2011; Shen et al., 2013), which is compatible with our current findings, which show considerable elevation of miRNA-483 in HCV-related HCC patients compared to HCV and healthy control groups. miR-483 was revealed to function as an anti-apoptotic oncogene with a pathogenic effect and molecular method of action in cancer cell lines (HEPG2, liver carcinoma, and HCT116, colorectal carcinoma) (Taheri et al., 2021). An in vitro investigation of adrenocortical cancer indicated that miR-483-5p promotes proliferation. These findings show that miR-483 may play a carcinogenic role in carcinogenesis (Yang et al., 2017). Several earlier investigations in Wilms tumor, colorectal cancer, malignant pheochromocytoma, and hepatocellular carcinoma tissues demonstrated positive relationships between miR-483 expression and IGF-II mRNA levels, supporting this theory (Ma et al., 2011). miR-483-5 expression was also observed to be higher in serum from hepatocellular cancer patients in a previous investigation (Shen et al., 2013), which is consistent with our existing observation.

In the current investigation, we discovered that HCV-related HCC patients had significantly lower levels of miRNA-335 expression than HCV patients and healthy controls. miR-335 has been demonstrated to behave as a tumor suppressor and inducer in a variety of cancer types in numerous investigations (Slattery et al., 2015; Wang et al., 2017). Many cancers, including breast cancer, lung cancer, colorectal cancer, and ovarian cancer (Heyn et al., 2011), have dysregulated miR-335, which serves as an oncogene or tumor suppressor in many malignant tumors. Furthermore, miR-335’s impact on the biological effect of numerous malignancies, including proliferation, apoptosis, migration, and invasion, is critical to its function in cancer (Liang et al., 2019). miR-335 has been shown to impact proliferation and apoptosis in a number of malignancies in several research studies. The serine/threonine kinase protein Rho-associated coiled-coil containing protein kinase 1 (ROCK1) promotes the growth of malignant tumors (Cui et al., 2015). miR-335 inhibits cell proliferation and cell cycle progression in NSCLC by suppressing ROCK1 expression. In HCC, miR-335 can influence cell proliferation, migration, and invasion in addition to cell proliferation. miR-335 inhibits ROCK1 expression by binding directly to its untranslated region, resulting in decreased cell proliferation and migration in cancer development (Ye et al., 2021). According to our results, the downregulation of miR-335 observed in HCC group patients increases the ROCK1 expression which facilitates the development and the progression of HCC.

The ROC curve was constructed to test the diagnostic ability of miRNA-122, producing a diagnostic accuracy (AUC = 95%) and a sensitivity of 100% in distinguishing HCC cases from controls. MicroRNA-483 had a high diagnostic accuracy (AUC = 98.6%) and 100% sensitivity for distinguishing healthy controls from HCC patients. It had a respectable capability to discriminate between HCV and HCC (AUC = 95.3%) and an 88 percent sensitivity. For distinguishing control subjects from HCC patients, microRNA-335 demonstrated a good diagnostic accuracy (AUC = 90.8 percent) and 100 percent sensitivity. It established a notable ability to differentiate HCV from HCC (AUC = 87.5% and sensitivity of 95.4%).

To summarize, early identification of HCV-related HCC is crucial for efficient disease therapy and can improve the survival rate of HCC patients. The findings of the current study highlighted the possible use of miR-122, miR-483, and miR-335 as potential diagnostic and prognostic biomarkers for clinical application. The current study elucidates that serum miRNA-335 expression is significantly under-expressed in patients with HCV-related HCC, while the expressions of miR-122 and miRNA-483 are overexpressed compared to those in the non-malignant HCV patients. The miRNA-122, miR-483, and miR-335 panel presented a great diagnostic potential as it demonstrated the highest diagnostic accuracy for discriminating non-malignant HCV patients from HCV-related HCC patients.