Regulation of lipid droplet (LD) formation in hepatocytes via regulation of SREBP1c by non-coding RNAs.

Introduction: Increased de novo lipogenesis (DNL) is one of the key factors contributing to fat accumulation and non-alcoholic fatty liver disease (NAFLD). Among the critical transcription factors (TFs) regulating DNL is mTOR and its downstream lipogenic TF, SREBP1c. In recent years, it has been established that non-coding RNAs (ncRNAs) play role in both biological processes and disease pathogenesis. Our group has previously characterized microRNAs that can target and regulate the expression of both mTOR and SREBP1c. Accordingly, this study aimed to broaden our understanding of the role of ncRNAs in regulating the mTOR/SREBP1c axis to elucidate the role of the non-coding transcriptome in DNL and lipid droplet (LD) formation. Hence, short ncRNA, miR-615-5p, and long non-coding RNA (lncRNA), H19, were chosen as they were previously proven to target mTOR by our group and in the published literature, respectively. Methodology: Huh-7 cells were treated with 800 µM oleic acid (OA) to promote LD formation. Transfection of miR-615-5p mimics or H19 over-expression vectors was performed, followed by the measurement of their downstream targets, mTOR and SREBP, on the mRNA level by quantitative real-time PCR (qRT-PCR), and on the protein level by Western blot. To determine the functional impact of miR-615-5p and H19 on LD formation and triglyceride (TG) accumulation, post-transfection LDs were stained, imaged, and characterized, and TGs were extracted and quantified. Results: miR-615-5p was able to reduce mTOR and SREBP1c significantly on both the mRNA and protein levels compared to control cells, while H19 caused a reduction of both targets on the protein level only. Both miR-615-5p and H19 were able to significantly reduce the LD count and total area, as well as TG levels compared to control cells. Conclusion: To conclude, this study shows, for the first time, the impact of miR-615-5p and H19 on the mTOR/SREBP1c axis, and thus, their functional impact on LDs and TG accumulation. These findings might pave the way for using ncRNAs as potential therapeutic targets in the management of fatty liver.

Enhancing NK cell cytotoxicity by miR-182 in hepatocellular carcinoma.

BACKGROUND AND AIM: NK-cells are the principle defense line against different malignancies. Their activation status is determined by the balance between activating and inhibitory receptors such as NKG2D and NKG2A, respectively. MicroRNAs are crucial post-transcriptional regulators of gene expression, playing key roles in modulating NK-cell development and function. The aim of this study is to investigate the role of miRNAs in regulating the activation and cytotoxic function of NK-cells in HCC. METHODS: In silico analysis was performed to predict a potential miRNA that might target NKG2D and NKG2A mRNAs. NK-cells were isolated from HCC patients and healthy controls, after which miRNA and mRNA were quantified. Manipulating miRNA expression was performed followed by investigating downstream targets and the cytotoxic activity of NK-cells against Huh-7 cell lines. RESULTS: NK-cells of HCC patients showed miR-182 overexpression compared to controls. NKG2D and NKG2A were upregulated and downregulated, respectively, in HCC NK-cells. Upon forcing miR-182 expression in the HCC NK-cells, upregulation of both receptors was observed. Finally, miR-182 was reported to induce NK-cell cytotoxicity represented in Perforin-1 upregulation and increase in cytolytic killing of co-cultured Huh-7 cells. CONCLUSION: Our findings suggest that miR-182 may augment NK-cell cytotoxicity against liver cancer via modulating NKG2D and NKG2A expressions.

Tamoxifen downregulates MxA expression by suppressing TLR7 expression in PBMCs of males infected with HCV.

Gender discrepancies in immune response to HCV infections and during HCV therapy exist and previous findings including those from this research team indicate the female sex hormone, 17ß-estradiol (E2), to be one probable cause of such inconsistencies. Also, it was recently demonstrated that estrogen receptor modulator Tamoxifen (TAM) exerts an upmodulating/enhancing effect on the TLR7 and JAK-STAT pathways in PBMCs of premenopausal females infected with HCV. Pursuing this work, a discrepancy was noticed in the results from male patients, therefore this study aimed to determine whether the effects of TAM previously observed in the PBMCs of women would hold true in PBMCs from males infected with HCV. Isolated PBMCs were pooled and relative expression of the TLR7 was quantified using RTqPCR. Sets of PBMCs were treated with exogenous interferon alpha (IFNα) or the TLR7 ligand, Imiquimod; these stimulations were performed with and without E2 and TAM pretreatment and the relative gene expressions of TLR7 and MxA were measured. Pretreatment with E2 and IFNα downregulated TLR7 (**P = 0.0080) and TAM further decreased this expression significantly (*P = 0.0284). TAM pretreatment also caused a significant downregulation in MxA expression in Imiquimod-stimulated PBMCs (*P = 0.0218). In conclusion, TAM displays several paradoxical effects in PBMCs of males infected with HCV compared to those of females. Contrary to the previous study involving premenopausal females, in PBMCs of infected males TAM may decrease IFNα release as indicated by reduced MxA expression possibly via the suppression of TLR7 expression.