Fibrotic liver extracellular matrix induces cancerous phenotype in biomimetic micro-tissues of hepatocellular carcinoma model.

BACKGROUND: Despite considerable advancements in identifying factors contributing to the development of hepatocellular carcinoma (HCC), the pathogenesis of HCC remains unclear. In many cases, HCC is a consequence of prolonged liver fibrosis, resulting in the formation of an intricate premalignant microenvironment. The accumulation of extracellular matrix (ECM) is a hallmark of premalignant microenvironment. Given the critical role of different matrix components in regulating cell phenotype and function, this study aimed to elucidate the interplay between the fibrotic matrix and malignant features in HCC. METHODS: Liver tissues from both control (normal) and carbon tetrachloride (CCl4)-induced fibrotic rats were decellularized using sodium dodecyl sulfate (SDS) and Triton X-100. The resulting hydrogel from decellularized ECM was processed into micro-particles via the water-in-oil emulsion method. Micro-particles were subsequently incorporated into three-dimensional liver biomimetic micro-tissues (MTs) comprising Huh-7 cells, human umbilical vein endothelial cells (HUVECs), and LX-2 cells. The MTs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at day 11, immunofluorescence staining, immunoblotting, and spheroid migration assay at day 14 after co-culture. RESULTS: Fibrotic matrix from CCl4-treated rat livers significantly enhanced the growth rate of the MTs and their expression of CCND1 as compared to the normal one. Fibrotic matrix, also induced the expression of epithelial-to-mesenchymal transition (EMT)-associated genes such as TWIST1, ACTA2, MMP9, CDH2, and VIMENTIN in the MTs as compared to the normal matrix. Conversely, the expression of CDH1 and hepatic maturation genes HNF4A, ALB, CYP3A4 was decreased in the MTs when the fibrotic matrix was used. Furthermore, the fibrotic matrix increased the migration of the MTs and their secretion of alpha-fetoprotein. CONCLUSIONS: Our findings suggest a regulatory role for the fibrotic matrix in promoting cancerous phenotype, which could potentially accelerate the progression of malignancy in the liver.

MiR-29a-laden extracellular vesicles efficiently induced apoptosis through autophagy blockage in HCC cells.

BACKGROUND: In spite of significant advancements in theraputic modalities for hepatocellular carcinoma (HCC), there is still a high annual mortality rate with a rising incidence. Major challenges in the HCC clinical managment are related to the development of therapy resistance, and evasion of tumor cells apoptosis which leading unsatisfactory outcomes in HCC patients. Previous investigations have shown that autophagy plays crucial role in contributing to drug resistance development in HCC. Although, miR-29a is known to counteract authophagy, increasing evidence revealed a down-regulation of miR-29a in HCC patients which correlates with poor prognosis. Beside, evidences showed that miR-29a serves as a negative regulator of autophagy in other cancers. In the current study, we aim to investigate the impact of miR-29a on the autophagy and apoptosis in HCC cells using extracellular vesicles (EVs) as a natural delivery system given their potential in the miRNA delivery both in vitro and in vivo. METHOD: Human Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles were lately isolated through 20,000 or 110,000 × g centrifugation (EV20K or EV110K, respectively), characterized by western blot (WB), scanning electron microscopy (SEM), and dynamic light scattering (DLS). miR-29a was subsequently loaded into these EVs and its loading efficiency was evaluated via RT-qPCR. Comprehensive in vitro and in vivo assessments were then performed on Huh-7 and HepG2 cell lines. RESULTS: EV20K-miR-29a treatment significantly induces cell apoptosis and reduces both cell proliferation and colony formation in Huh-7 and HepG2 cell lines. In addition, LC3-II/LC3-I ratio was increased while the expression of key autophagy regulators TFEB and ATG9A were downregulated by this treatment. These findings suggest an effective blockade of autophagy by EV20K-miR-29a leading to apoptosis in the HCC cell lines through concomitant targeting of critical mediators within each pathway.

Proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Impact of isolation methods on protein cargo.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with EVs isolated through different methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cell-cell interactions and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Autophagy orchestrates resistance in hepatocellular carcinoma cells.

Treatment resistance is one of the major barriers for therapeutic strategies in hepatocellular carcinoma (HCC). Many studies have indicated that chemotherapy and radiotherapy induce autophagy machinery (cell protective autophagy) in HCC cells. In addition, many experiments report a remarkable crosstalk between treatment resistance and autophagy pathways. Thus, autophagy could be one of the key factors enabling tumor cells to hinder induced cell death after medical interventions. Therefore, extensive research on the molecular pathways involved in resistance induction and autophagy have been conducted to achieve the desired therapeutic response. The key molecular pathways related to the therapy resistance are TGF-ß, MAPK, NRF2, NF-κB, and non-coding RNAs. In addition, EMT, drug transports, apoptosis evasion, DNA repair, cancer stem cells, and hypoxia could have considerable impact on the hepatoma cell's response to therapies. These mechanisms protect tumor cells against various treatments and many studies have shown that each of them is connected to the molecular pathways of autophagy induction in HCC. Hence, autophagy inhibition may be an effective strategy to improve therapeutic outcome in HCC patients. In this review, we further highlight how autophagy leads to poor response during treatment through a complex molecular network and how it enhances resistance in primary liver cancer. We propose that combinational regimens of approved HCC therapeutic protocols plus autophagy inhibitors may overcome drug resistance in HCC therapy.

Regulatory Non-Coding RNAs in Familial Hypercholesterolemia, Theranostic Applications.

Familial hypercholesterolemia (FH) is a common monogenic disease which is associated with high serum levels of low-density lipoprotein cholesterol (LDL-C) and leads to atherosclerosis and cardiovascular disease (CVD). Early diagnosis and effective treatment strategy can significantly improve prognosis. Recently, non-coding RNAs (ncRNAs) have emerged as novel biomarkers for the diagnosis and innovative targets for therapeutics. Non-coding RNAs have essential roles in the regulation of LDL-C homeostasis, suggesting that manipulation and regulating ncRNAs could be a promising theranostic approach to ameliorate clinical complications of FH, particularly cardiovascular disease. In this review, we briefly discussed the mechanisms and pathophysiology of FH and novel therapeutic strategies for the treatment of FH. Moreover, the theranostic effects of different non-coding RNAs for the treatment and diagnosis of FH were highlighted. Finally, the advantages and disadvantages of ncRNA-based therapies vs. conventional therapies were discussed.

Bioinformatics analysis of regulated MicroRNAs by placental growth factor signaling in cancer stem cells.

AIM OF STUDY: Since the effect of placental growth factor (PlGF) on MicroRNAs (miRNAs) at molecular level was remained unknown, the aim was to predict the transcription factors (TFs) and their regulated miRNAs that activated by PlGF and analysis the function, biological processes, and cancer stem cells (CSCs)-related signaling pathways of miRNAs that regulated in PlGF signaling pathway. SUBJECTS AND METHODS: The aim of this study is to find the TFs that activated by PlGF, we used three online software programs PCViz, PubAngioGen, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Then, the regulatory miRNAs downstream of the TFs were identified by four software TMHD, chipbase, circuits, and transmir databases. Target genes of miRNAs were predicted by three online software program TargetScan, Pictar, and miRanda algorithms. Moreover, Mirwalk database was used to find the validated miRNAs in angiogenesis process. Furthermore, Gene ontology (GO) biological process, GO molecular function, KEGG pathway, BIOCARTA pathway, Panther pathway, and Reactome pathway in Database for Annotation and Visualization and Integrated Discovery tools were used to find the functions and signaling pathways of target genes. RESULTS: Many target genes of miRNAs in PlGF pathway were involved in CSCs-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, epidermal growth factor EGF, and transforming growth factor-beta signaling pathways. Regulatory miRNAs in PlGF signaling pathway probably promote cell proliferation, migration, tubulogenesis, and metastasize in CSCs. CONCLUSIONS: Bioinformatic analysis revealed that regulatory miRNAs and their target genes in PlGF pathway played important roles in the progression of CSCs-related signaling pathways.