MicroRNAs as Modulators of Tumor Metabolism, Microenvironment, and Immune Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. Molecular heterogeneity and absence of biomarkers helping patient allocation to the best therapeutic option contribute to poor prognosis in advanced stages. MicroRNAs' (miRNAs) deregulated expression contributes to tumor development and progression and influences drug resistance in HCC. Accordingly, miRNAs have been extensively investigated as both biomarkers and therapeutic targets. The diagnostic and prognostic roles of circulating miRNAs have been ascertained, though with some inconsistencies across studies. From a therapeutic perspective, miRNA-based approaches demonstrated safety profiles and antitumor efficacy in HCC animal models. Nevertheless, caution should be used when transferring preclinical findings to the clinic, due to possible molecular inconsistency between animal models and the heterogeneous patterns of human diseases. Awealth of information is offered by preclinical studies exploring the mechanisms driving miRNAs' aberrant expression, the molecular cascades triggered by miRNAs and the corresponding phenotypic changes. Ex-vivo analyses confirmed these results, further shedding light on the intricacy of the human disease often overcoming pre-clinical models. This complexity seems to be ascribed to the intrinsic heterogeneity of HCC, to different risk factors driving its development, as well as to changes across stages and previous treatments. Preliminary findings suggest that miRNAs associated with specific risk factors might be more informative in defined patients' subgroups. The first issue to be considered when trying to envisage a possible translational perspective is the molecular context that often drives different miRNA functions, as clearly evidenced by "dual" miRNAs. Concerning the possible roles of miRNAs as biomarkers and therapeutic targets, we will focus on miRNAs' involvement in metabolic pathways and in the modulation of tumor microenvironment, to support their exploitation in defined contexts

Animal models of hepatocellular carcinoma prevention

Hepatocellular carcinoma (HCC) is a deadly disease and therapeutic efficacy in advanced HCC is limited. Since progression of chronic liver disease to HCC involves a long latency period of a few decades, a significant window of therapeutic opportunities exists for prevention of HCC and improve patient prognosis. Nonetheless, there has been no clinical advancement in instituting HCC chemopreventive strategies. Some of the major challenges are heterogenous genetic aberrations of HCC, significant modulation of tumor microenvironment and incomplete understanding of HCC tumorigenesis. To this end, animal models of HCC are valuable tools to evaluate biology of tumor initiation and progression with specific insight into molecular and genetic mechanisms involved. In this review, we describe various animal models of HCC that facilitate effective ways to study therapeutic prevention strategies that have translational potential to be evaluated in a clinical context © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell popula-tions displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas

ER Stress Negatively Modulates the Expression of the miR-199a/214 Cluster to Regulates Tumor Survival and Progression in Human Hepatocellular Cancer

Background: Recent studies have emphasized causative links between microRNAs (miRNAs) deregulation and tumor development. In hepatocellular carcinoma (HCC), more and more miRNAs were identified as diagnostic and prognostic cancer biomarkers, as well as additional therapeutic tools. This study aimed to investigate the functional significance and regulatory mechanism of the miR-199a2/214 cluster in HCC progression. Methods and Findings: In this study, we showed that miR-214, as well as miR-199a-3p and miR-199a-5p levels were significantly reduced in the majority of examined 23 HCC tissues and HepG2 and SMMC-7721 cell lines, compared with their nontumor counterparts. To further explore the role of miR-214 in hepatocarcinogenesis, we disclosed that the ER stressinduced pro-survival factor XBP-1 is a target of miR-214 by using western blot assay and luciferase reporter assay. Reexpression of miR-214 in HCC cell lines (HepG2 and SMMC-7721) inhibited proliferation and induced apoptosis. Furthermore, ectopic expression of miR-214 dramatically suppressed the ability of HCC cells to form colonies in vitro and to develop tumors in a subcutaneous xenotransplantation model of the BALB/c athymic nude mice. Moreover, reintroduction of XBP-1s attenuated miR-214-mediated suppression of HCC cells proliferation, colony and tumor formation. To further understand the mechanism of the miR-199a/214 cluster down-expression in HCC, we found that thapsigargin (TG) and tunicamycin (TM) or hypoxia-induced unfolded protein response (UPR) suppresses the expression of the miR-199a/21

MicroRNA-122 Modulates the Rhythmic Expression Profile of the Circadian Deadenylase Nocturnin in Mouse Liver

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3′-untranslated region (3′-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3′-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer

BACKGROUND: MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker. METHODS: The 5-aza-2'-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a. RESULTS: The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561. CONCLUSIONS: These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker

The functional significance of microRNA-145 in prostate cancer

BackgroundMicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer.Methods and resultsWe investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis.ConclusionOne of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer

Serum MicroRNAs as Biomarkers for Hepatocellular Carcinoma in Chinese Patients with Chronic Hepatitis B Virus Infection

BACKGROUND: MicroRNAs (miRNAs) have been shown to anticipate great cancer diagnostic potential. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: This study was divided into four phases: (I) Ten candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 10 HCC patients with chronic hepatitis B virus (HBV) infection and 10 age- and sex-matched healthy subjects. (II) Marker validation by real-time RT-PCR on HBV patients with (n = 48) or without HCC (n = 48), and healthy subjects (n = 24). (III) Marker detection by real-time RT-PCR in sera from another 14 HCC patients before and 1 month after surgical resection. (IV) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of HCC patients. Although miR-222, miR-223 or miR-21 were significantly up- or down-regulated between HCC patients and healthy controls, no significant difference was observed in the levels of these miRNAs between HBV patients without and with HCC. MiR-122 in serum was significantly higher in HCC patients than healthy controls (p<0.001). More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples. Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line (p = 0.043). CONCLUSIONS/SIGNIFICANCE: Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for detection of HCC in healthy subjects, moreover, it might serve as a novel biomarker for liver injury but not specifically for detection of HCC in chronic HBV infection patients