MicroRNA signatures as predictive biomarkers in transarterial chemoembolization‐treated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Unfortunately, many patients are diagnosed at a late stage with a delay in the optimal timing for tumor resection. Transarterial chemoembolization (TACE) is currently considered the standard of care for patients with unresectable HCC Barcelona system class B classification. However, the treatment response of HCC patients to TACE varies widely, and there is no reliable marker for predicting a patient's response to TACE. Thus, the identification of patients who are sensitive or resistant to TACE is important for individualized therapy. Recently, our understanding of cancer cell biology has progressed enormously. Much of this progress has been driven by technological advances enabling previously unachievable studies to be performed and yield a constantly evolving picture of the regulatory role of noncoding RNAs (ncRNAs) in tumor biology. MicroRNAs (miRNAs) are a class of ncRNA molecules that regulate nearly one‐third of all protein‐coding RNAs. The existing literature indicates that the deregulation of miRNAs can contribute to tumorigenesis and metastasis in multiple cancers, including HCC, via the control of cell proliferation, apoptosis, invasion, or metastasis. Analysis and evaluation of this type of regulatory RNA could shed new light on the behavior of many cancers and provide new diagnostic and prognostic biomarkers as well as pharmacological targets for novel treatment strategies. To this end, this review highlights the expression and functional roles of miRNAs in TACE‐treated HCC and explores the potential applications of miRNAs as diagnostic markers and therapeutic targets.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide, with a high incidence in Eastern Asia and sub-Saharan Africa. 1,2 Multiple HCC treatment modalities, such as chemotherapy, irradiation, resection, and radiofrequency ablation, are available, although selective transarterial chemoembolization (TACE) using chemotherapeutic agents-doxorubicin and cisplatin-is usually performed. 3,4 According to the Barcelona clinic liver cancer (BCLC) guidelines, TACE is the standard treatment for intermediate HCC. 5 However, increasing evidence suggests that TACE may also be used to treat patients with early or advanced HCC. 6 Although TACE may result in a favorable clinical course, 7 the long-term prognosis varies substantially among patients with TACE-treated HCC because of the heterogeneous pathogenic nature of the disease and differences in disease etiology, liver function, and tumor burden. 8 Furthermore, the prognosis might remain unfavorable due to chemotherapy resistance and the high recurrence rate. 9 Thus, identifying noninvasive predictive biomarkers is necessary to improve the prognosis of this disease and evaluate the effectiveness of TACE. 10,11 Recent studies provide clear evidence that microRNAs (miRNAs) are abundant in the liver and modulate a diverse spectrum of liver functions. 12 Deregulation of miRNA expression may be a key pathogenic factor in multiple liver diseases and could be a potential predictive biomarker. 12,13 In the following sections, we will address the role of miRNAs and the differential miRNA expression signature in HCC to provide a better understanding of the predictive value of miRNAs in TACEtreated HCC.

| MicroRNAs: key players in HCC
MiRNAs are small (20-24 nucleotides) noncoding RNA (ncRNA) molecules that regulate gene expression post-transcriptionally by various mechanisms. 12 Although the most common mechanism is translational repression resulting from miRNA binding to the 3 0 UTR of an mRNA, mechanisms involving mRNA destabilization and degradation have also been described. 14 MiRNAs control cellular processes in carcinogenesis, including proliferation, cell cycle regulation, apoptosis, invasion, angiogenesis, and metastasis. 15 Several miRNAs have been reported to be involved in HCC development, progression, and theranostics. 12,[16][17][18][19] Given that they are extremely stable in bodily fluids, including serum or plasma, where they are associated with RNA-binding proteins or packaged into various microparticles, circulating miRNAs have potential applications as minimally invasive biomarkers for HCC diagnosis and prognosis. 17   A low exosomal miR-122 ratio (post-/pre-TACE levels) was shown to be an independent factor for poor prognosis in HCC patients with liver cirrhosis. 11 Also, serum miR-122 levels were shown to be negatively correlated with the Model for End-Stage Liver Disease score 31 and to be associated with poor prognosis in patients with decompensated liver disease. 32 Moreover, in HCV-induced fibrosis, a decrease in the level of circulating miR-122 reflects the development of liver fibrosis and the loss of viable liver cells. 33 Although the mechanisms of action and functions of miR-122, especially post-treatment, are not well known, some investigators have speculated that under the hypoxic conditions occurring after embolization, miR-122 may induce hypoxia-inducible factor 1α (HIF-1α) expression in noncancerous liver tissue and cancer cells. 11 HIF-1α and vimentin are miR-122 targets in hepatocytes. 34 Furthermore, changes in miR-122 levels may be associated with epithelial-mesenchymal transition (EMT), as miR-122 has been found to inhibit EMT by targeting the Snail (a zinc finger transcriptional repressor) and Wingless/Integrated (WNT)/β-cadherin signaling pathways. 35 Additionally, miR-122 plays a key role in mitochondrial metabolism by indirectly regulating mitochondrial genes, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), 36 and it can inhibit angiogenesis and intrahepatic metastasis by suppressing the expression of a disintegrin and metalloprotease 17 (ADAM17), among other genes. 37 Phosphoinositide 3-kinase and Bcl-w have also been identified as miR-122 targets; these proteins mediate the downregulationassociated escape from apoptosis through the activation of the receptor tyrosine kinase survival pathway and anti-apoptotic signaling, respectively. 38 Additionally, miR-122 was reported to regulate cell cycle checkpoints by targeting cyclin G1. 39 T A B L E 1 Summary of the literature concerned with trans-arterial chemoembolization-treated hepatocellular carcinoma and miRNAs MiR-1268a has been implicated in embryogenesis and cell differentiation. 49 The genetic variables in its seed region were found to be correlated with tumor angiogenesis and to be involved in HCC carcinogenesis. 50 Also, decreasing levels of miR-1268a expression were found to be strongly associated with poor outcome in HCC. 51 Interestingly, upon stratifying HCC patients by different levels of miR-1268a expression and investigating the effects of TACE on HCC prognosis in the differential expression status strata, Lu and colleagues revealed that TACE treatment could significantly improve the survival of HCC patients with low miR-1268a tissue expression but not those with high miR-1268a expression; this finding suggests that miR-1268a expression could modify the sensitivity of tumor cells to TACE treatment and therefore implies that miR-1268a expression is a useful marker for the selection of patients for TACE treatment. 51 1.5 | MicroRNA-1285-3p (miR-1285-3p) The expression of the differentially expressed plasma miR-1285-3p was consistently downregulated in HCC patients with a poor response to TACE than those with a good response to TACE. 52 Also, miR-1285-3p might directly target the JUN oncogene in HCC cells, 52 which has been implicated in both the extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways that impact cancer cell proliferation as well as migration. 53,54 This last finding could partially explain the observed association of low levels of miR-1285 (which could act as a potential tumor suppressor in HCC patients) with a poor response to TACE due to JUN derepression via miR-1285 and enhanced JUN pathway activation in the HCC lesions of these patients.

| MicroRNA-200 (miR-200)
The miR-200 family has been identified as a potential diagnostic marker in HCC. 55 Liu et al 56

| MicroRNA-21 (miR-21)
MiR-21 is considered a typical "oncomiR". This miRNA is associated with a wide variety of cancers, including liver cancer, 40 and is a potential diagnostic biomarker for early HCC. 41 However, few studies have evaluated the prognostic value of miRNA-21 for HCC, especially after TACE. Although exosomal levels were associated with prothrombin time and the Child-Pugh score, circulating miR-21 levels did not significantly change after TACE therapy for HCC. 11 Another study reported that higher levels of miRNA-21 were associated with shorter postoperative survival in patients with HCC. 60 In contrast, other researchers found that serum miRNA-21 levels were not significantly associated with survival after TACE. 56

| MicroRNA-26a (miR-26a)
MiRNA-26a is an emerging key regulator of carcinogenesis and tumor progression 8 and is consistently downregulated in HCC. 67 Kim  expression levels had shorter overall survival times than those with high expression levels. 67 The discrepancy between these findings might be partly due to the different cellular contexts of the tumors and different study populations. 8 Concerning the molecular mechanism by which miRNA-26a performs its regulatory function, several studies reported that miR-26a with early tumor recurrence after HCC surgery, especially in patients with BCLC stage 0/A disease. 72 Another study proposed that high serum miR-29a-3p expression levels were associated with poor overall survival and progression-free survival in patients with HCC treated with resection or local ablation. 73 In contrast, Xiong et al demonstrated downregulation of miR-29 (miR-29a-3p/b/c) expression in HCC tissue, supporting its potential tumor-suppressive role. 74 The reason for the discrepancy in the findings above is not clear, but "a deeper investigation into different miRNA- Notably, the level of miR-29a-3p alone failed to show statistically significant predictive power for early TACE refractoriness, in contrast to the panel combining miR-29a-3p, miR-21, and miR-26a, which was suggested to be a predictive factor for early TACE refractoriness, supporting the view that this multi-biomarker panel could have stronger diagnostic and prognostic power than miR-29a-3p alone. 8

| MicroRNA-335 (miR-335)
MiR-335 can act as an oncogene or tumor suppressor in several types of cancers. As a suppressor of tumor metastasis, miR-335 has been shown to act by regulating several classical signaling pathways. [76][77][78][79] Moreover, this miRNA has been found to predict sensitivity to anticancer treatment in some types of cancer, suggesting its involvement in the development of chemoresistance. 80 Cui and colleagues 81 83 Overexpression of the miR-23a-miR-27a-miR-24-2 cluster could promote hepatic metastasis by stimulating cell growth and attenuating, transforming growth factor-beta (TGF-β)-induced apoptotic cell death. 84 Also, miR-30 expression was found to be higher in HCC patients with cancer metastasis than in those without, 85 and miR-30 was found to inhibit TGF-β-mediated EMT in hepatocytes by targeting Snail1. 86 MiR-106b is involved in the TGF-β signaling pathway, and its overexpression is implicated in HCC metastasis via the activation of the EMT process. 87 Also, miR-133a could mediate TGFβ-dependent derepression of collagen synthesis in hepatic stellate cells during liver fibrosis. 88 Finally, miR-199a-3p could regulate mammalian target of rapamycin (mTOR) signaling, which is required for tumor cell motility/cancer invasion), as well as c-Met, which codes for a transmembrane tyrosine kinase receptor that binds hepatic growth factor and plays an important role in metastatic ability. 89 1.14 | Functional enrichment analysis in hallmark biological processes of cancer, such as ECM-receptor interaction (hsa04512, P = 3.57e-16), the hippo signaling pathway (hsa04390, P = 1.30e-05), adherens junctions (hsa04520, P = .008), the TGF-β signaling pathway (hsa04350, P = 1.92e-05), the p53 signaling pathway (hsa04115, P = .002), and signaling pathways regulating the pluripotency of stem cells (hsa04550, P = 3.29e-05).
Furthermore, these miRNAs were enriched in the cancer-related pathways as "pathways in cancer" (hsa05200, P = 2.39e-04), and "proteoglycans in cancer" (hsa05205, P = 6.34e-12) (Figure 1). The present review revealed that a high miRNA expression signature, especially in the plasma/serum of TACE-treated HCC patients, could predict early TACE refractoriness or response. Therefore, if there is a high probability of early TACE refractoriness, considering timely changes in the treatment modality from repetitive TACE to sorafenib or combination therapy might improve results. Additionally, the establishment of standardized miRNA quantification methods may be crucial for the stratification of treatment modalities in HCC patients.

| CONCLUSION AND FUTURE PERSPECTIVES
Furthermore, targeted treatments that increase the endogenous levels of downregulated miRNAs or decrease the levels of upregulated miRNAs implicated in the poor prognosis of TACE-treated HCC patients ( Figure 2) might be new therapeutic options. 81 However, it is appropriate to prospectively validate these suggestions in a largescale randomized controlled clinical trial to confirm these findings before immediate clinical implementation. Hence, advances in scientific research, along with overcoming the current limitations, will pave the way towards truly personalized therapies and clinical applications of miRNAs in diagnosis, prognosis, and therapy, a major step forward in cancer epigenetics.

AUTHORS' CONTRIBUTION
Performing the data search and preparing the primary draft conducted by Manal S Fawzy and Eman A Toraih. Manal S Fawzy edited the manuscript. All authors reviewed, commented, and approved the final draft.

CONFLICT OF INTEREST
The authors declare no competing interests.

ETHICS STATEMENT
Ethics approval and consent to participate: Not applicable.
Consent for publication: Not applicable.