Abrogating the interplay between IGF2BP1, 2 and 3 and IGF1R by let-7i arrests hepatocellular carcinoma growth.

IGF2BP 1, 2 and 3 control the fate of many transcripts. Immunoprecipitation studies demonstrated the IGF2BPs to bind to IGF1R mRNA, and our laboratory has recently shown them to post-transcriptionally regulate IGF1R. This study sought to identify a microRNA regulating the IGF2BPs and consequently IGF1R. All three IGF2BPs were among the top-ranked predicted targets of let-7i. Let-7i was downregulated in HCC tissues, and transfection of HuH-7 with let-7i inhibited malignant cell behaviors and decreased IGF2BPs transcripts. Direct binding of let-7i to IGF2BP2 and IGF2BP3 3'UTRs was confirmed, and the effect of let-7i caused a decrease in the IGF2BPs' target gene, the IGF1R. IGF1R mRNA was inversely correlated with let-7i in HCC tissues and was reduced upon let-7i transfection into HuH-7. Reporter assays validated IGF1R as a target of let-7i. Therefore, let-7i may control HCC tumorigenesis by regulating IGF1R directly and indirectly by interrupting the interplay between IGF1R and the IGF2BPs.

Regulation of IGF2BP1 by miR-186 and its impact on downstream lncRNAs H19, FOXD2-AS1, and SNHG3 in HCC.

AIM: We have previously characterized oncogenic properties of IGF2BP1 in HCC, and its regulation by short noncoding RNAs (ncRNAs). Recent evidence suggests that IGF2BP1 itself may regulate long ncRNAs (lncRNAs). Therefore, this study aimed at exploring the interplay between IGF2BP1 and various upstream and downstream ncRNAs and its link to HCC pathogenesis. MATERIALS AND METHODS: Bioinformatic analysis was used to identify up- and downstream ncRNAs interacting with IGF2BP1. Huh-7 cells were transfected with siRNAs against IGF2BP1 and microRNA mimics. Relative gene expression was determined using RTqPCR and IGF2BP1 protein was quantified by western blot. Luciferase binding assay was used to explore the targeting of IGF2BP1 3'UTR. HCC tumorigenesis was measured by MTT assay, BrdU-incorporation assay, colony-forming assay, and scratch assay. KEY FINDINGS: Bioinformatic analysis identified three oncogenic lncRNAs - namely H19, FOXD2-AS1, and SNHG3 - potentially regulated by IGF2BP1. Knockdown of IGF2BP1 decreased the expression of all three oncogenic lncRNAs and inhibited malignant cell behaviors. miR-186 was revealed as a possible upstream regulator of IGF2BP1. miR-186 mimics decreased IGF2BP1 mRNA and protein levels. miR-186 was significantly lower while IGF2BP1 was elevated in cancerous tissues from ten HCC patients compared to five healthy controls. In addition, miR-186 mimics caused a downregulation of the oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 and a concomitant decrease in cell viability, proliferation, migration, and clonogenicity. SIGNIFICANCE: miR-186 may exert tumor suppressor effects in HCC by repressing oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 through its effect on IGF2BP1.

Schistosoma mansoni infection and the occurrence, characteristics, and survival of patients with hepatocellular carcinoma: an observational study over a decade.

Schistosoma mansoni infection (SMI) is suspected to be directly and indirectly involved in hepato-carcinogenesis. This study evaluated the association of a previous SMI with hepatocellular carcinoma (HCC) development, patients, tumor characteristics, treatment outcomes, and survival. This observational study included patients with HCC with and without previous SMI who presented to the multidisciplinary HCC clinic, Kasr-Alainy hospital (November 2009 to December 2019). It also included 313 patients with liver cirrhosis without HCC. Clinical and laboratory features of the patients (complete blood count, liver/renal functions , alpha-fetoprotein, and hepatitis B/C status), tumor characteristics (Triphasic CT and/or dynamic MRI), liver stiffness (transient elastography), HCC treatment outcome, and overall survival were studied. This study included 1446 patients with HCC; 688(47.6%) composed group-1, defined by patients having a history of SMI, and 758(52.4%) were in group-2 and without history of SMI. Male sex, smoking, diabetes mellitus, splenomegaly, deteriorated performance status, synthetic liver functions, and platelet count were significantly higher in group-1. The groups did not differ with regard to liver stiffness, tumor characteristics, or the occurrence of post-HCC treatment hepatic decompensation or recurrence. HCC treatment response was better in group-2. Group-1 showed lower sustained virological response to hepatitis C direct-acting antivirals (DAAs) compared with group-2 (60% versus 84.3%, respectively, P = 0.027). Prior SMI was associated with HCC (adjusted odds ratio = 1.589, 95% confidence interval = 1.187-2.127), and it was concluded that it increases the risk of HCC. In addition, it significantly affects the performance status, laboratory characteristics, response to DAAs, and overall survival.

Endothelial cell protein C receptor gene 6936A/G and 4678G/C polymorphisms as risk factors for deep venous thrombosis.

Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR) is present in plasma. Two polymorphisms in the EPCR gene (6936A/G and 4678G/C) have been reported to influence the risk of venous thromboembolism. We aimed to investigate the relation between EPCR gene polymorphisms (6936A/G and 4678C/G) and deep venous thrombosis (DVT) and their relations to sEPCR level. This study involved 90 patients with DVT and 90 age and sex-matched healthy controls. Plasma levels of sEPCR were measured in 45 cases of the primary DVT by ELISA. PCR-restriction fragment length polymorphism (RFLP) was used for detection of EPCR polymorphisms (6936A/G and 4678G/C). Regarding 6936A/G, our results demonstrated that mutant genotypes (AG, GG) were associated with an increased risk for DVT [P < 0.001, odds ratio (OR) 4.125, 95% confidence interval (95% CI) 2.198-7.740] as well as its mutant allele G (P < 0.001, OR 2.549, 95% CI 1.601-4.061). The mutant genotypes were associated with increased levels of sEPCR. Although in 4678G/C, our results demonstrated that the mutant genotype (CC) was considered as a protective factor against DVT (P = 0.014, OR 0.289, 95% CI 0.108-0.776) as well as its mutant allele C (P = 0.02, OR 0.600, 95% CI 0.388-0.927), but it had no effect on sEPCR level. Our data suggest that 6936A/G polymorphism is a risk factor for DVT and is associated with elevated plasma levels of sEPCR, while 4678G/C polymorphism plays a role in protection against DVT.

Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma.

AIM: To investigate the effect of microRNA on insulin-like growth factor binding protein-3 (IGFBP-3) and hence on insulin-like growth factor-II (IGF-II) bioavailability in hepatocellular carcinoma (HCC). METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mirVana miRNA Isolation Kit. microRNA-17-5p (miR-17-5p) expression was mimicked and antagonized in HuH-7 cell lines using HiPerFect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cDNA followed by quantification of miR-17-5p and IGFBP-3 expression using TaqMan real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-II protein was measured in transfected HuH-7 cells using IGF-II ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where miR-17-5p was extensively underexpressed in HCC tissues (P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients (P = 0.0041) compared to healthy donors. Forcing miR-17-5p expression in HuH-7 cell lines showed a significant downregulation of IGFBP-3 mRNA expression (P = 0.0267) and a significant increase in free IGF-II protein (P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of miR-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone (P = 0.0474). CONCLUSION: These data suggest that regulating IGF-II bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miRNAs.

miR-34a: Multiple Opposing Targets and One Destiny in Hepatocellular Carcinoma.

Background and Aims: The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain, including its expression pattern and relevance to tumor etiology, tumor stage and prognosis, and finally, its impact on apoptosis. Methods: miR-34a expression was assessed in hepatitis C virus (HCV)-induced non-metastatic HCC tissues by RT-Q-PCR. Huh-7 cells were transfected with miR-34a mimics and the impact of miR-34a was examined on 84 pro-apoptotic/anti-apoptotic genes using PCR array; its net effect was tested on cell viability via MTT assay. Results: miR-34a expression was up-regulated in HCC tissues. Moreover, miR-34a induced a large set of pro-apoptotic/anti-apoptotic genes, with a net result of triggering apoptosis and repressing cell viability. Conclusions: HCC-related differential expression of miR-34a could be etiology-based or stage-specific, and low expression of miR-34a may predict poor prognosis. This study's findings also emphasize the role of miR-34a in apoptosis.

A pleiotropic effect of the single clustered hepatic metastamiRs miR-96-5p and miR-182-5p on insulin-like growth factor II, insulin-like growth factor-1 receptor and insulin-like growth factor-binding protein-3 in hepatocellular carcinoma.

MicroRNAs (miRs) have a major role in the pathogenesis of hepatocellular carcinoma (HCC). As the insulin-like growth factor (IGF) axis is a highly tumorigenic pathway in HCC, the present study attempted to target it with miRs. Potential targeting of crucial members of the IGF axis by miRNAs at the 3'-untranslated region (3'-UTR) was predicted using bioinformatic tools, such as microrna.org, Diana lab and Targetscan, while 5'-UTR targeting was predicted using bibiserv software. Expression profiling of obtained miRNAs was performed using quantitative polymerase chain reaction (qPCR) in 22 non-metastatic HCC biopsy samples and 10 healthy tissues. To investigate the impact of miRNAs on their potential downstream targets, transfection of miRNAs was performed in HuH-7 cells and the targets' expression was quantified using qPCR. Transcripts of insulin-like growth factor-1 receptor (IGF-1R), insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-II were found to be potentially targeted at the 5'-UTR and 3'-UTR regions by the single clustered hepatic metastamiRs miR-96-5p and miR-182-5p. The two miRNAs showed a similar expression pattern in HCC tissues compared to those in healthy tissues. Forced expression of miR-96-5p and miR-182-5p in the HCC cell line HuH-7 had inducing effects on IGFBP-3 and IGF-II transcripts. Of note, the two miRs had differential effects on IGF-1R, where miR-96-5p induced IGF-1R mRNA expression and miR-182-5p inhibited its expression. The present study revealed the pleiotropic impact of the single clustered hepatic metastamiRs miR-96-5p and miR-182-5p on IGF-1R, and an inducing effect on IGF-II and IGFBP-3 in hepatocellular carcinoma.

miR-1275: A single microRNA that targets the three IGF2-mRNA-binding proteins hindering tumor growth in hepatocellular carcinoma.

This study aimed to identify a single miRNA or miR (microRNA) which regulates the three insulin-like growth factor-2-mRNA-binding proteins (IGF2BP1, 2 and 3). Bioinformatics predicted miR-1275 to simultaneously target the three IGF2BPs, and screening revealed miR-1275 to be underexpressed in hepatocellular carcinoma (HCC) tissues. Transfection of HuH-7 cells with miR-1275 suppressed IGF2BPs expression and all three IGF2BPs were confirmed as targets of miR-1275. Ectopic expression of miR-1275 and knockdown of IGF2BPs inhibited malignant cell behaviors, and also reduced IGF1R protein and mRNA. Finally IGF1R was validated as a direct target of miR-1275. These findings indicate that the tumor-suppressor miR-1275 can control HCC tumor growth partially through simultaneously regulating the oncogenic IGF2BPs and IGF1R.