Adjacent Hepatocytes Research Articles

Induced collagen type-I secretion by hepatocytes of the melanoma liver metastasis is associated with a reduction in tumour-infiltrating lymphocytes.

Overall patients with melanoma liver metastasis (MLiM) have a dismal prognosis and poor responses to the standard of care treatment. Understanding the role of the tumour microenvironment (TME) is critical for discovering better strategies to overcome intrinsic therapy resistance in MLiM. The aim was to understand the crosstalk signalling pathways between hepatocytes and metastatic melanoma cells in the TME of MLiM. Hepatocytes and melanoma tumour cells of MLiM were assessed using transcriptomic NanoString GeoMx digital spatial profiling (NGDSP) assay. Functional assays were performed using normal hepatocytes and MLiM-derived cell lines. Validation was performed using multiplex immunofluorescence. In NGDSP analysis adjacent normal hepatocytes (ANH) had higher CXCR4 and COL1A1/2 levels than distant normal hepatocytes (DNH), while melanoma cells had higher TNF-α levels. In vitro, MLiM cell lines released TNF-α which upregulated CXCR4 and CXCL12 levels in ANH. CXCL12 activated CXCR4, which triggered AKT and NFκB signalling pathways. Consequently, AKT signalling induced the upregulation of collagen type I. MLiM were significantly encircled by a shield of collagen, whereas other liver metastases showed reduced levels of collagen. Of all the liver metastasis analyzed, the presence of collagen in melanoma liver metastasis was associated with a reduction in tumour-infiltrating lymphocytes. MLiM modified ANH to increase collagen production and created a physical barrier. The collagen barrier was associated with a reduction of immune cell infiltration which could potentially deter MLiM immune surveillance and treatment responses. Spatial analyses of melanoma liver metastasis show that adjacent normal hepatocytes have increased collagen-type I levels. Melanoma liver metastases tumour cells secrete enhanced levels of TNF-α to stimulate CXCR4/CXCL12 upregulation in adjacent normal hepatocytes. Activation of CXCR4 promotes AKT and NF-κB signalling pathways to promote collagen-type I secretion in adjacent normal hepatocytes. Elevated collagen levels were associated with reduced tumour-infiltrating lymphocytes.

Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography.

The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.

Assessment of large droplet fat in frozen sections of donor liver biopsies: utility and interobserver variability of the newly described Banff method compared to a simplified Average of Fields method

AimsThere is great variability in the assessment and reporting of fat in frozen sections of donor liver biopsies. The Banff Working Group has proposed a novel method and definition for...

Massive hepatic trematodosis in 5 juvenile bald eagles.

Hepatic trematodosis by opisthorchiid flukes has been reported sporadically in North American fish-eating raptors. Bald eagles (Haliaeetus leucocephalus) infected by these flukes often have various degrees of granulomatous cholangitis, pericholangitis, necrosis of adjacent hepatocytes, and subsequent hepatic fibrosis. Species identification has been complicated by the inability to dissect intact specimens from liver tissue. Between 2007 and 2018, 5 juvenile bald eagles with massive hepatic trematodosis were identified at autopsy. Histologically, flukes were non-spinous. Parasitologic identification revealed ventral suckers (80-93 µm diameter), and uteri containing golden, operculated eggs (~25.0 × 12.0 µm). An unfixed frozen liver sample of one eagle was analyzed by PCR and DNA sequencing targeting the large subunit rRNA, ITS region, and cox1 genes of the parasite. The fluke DNA sequences shared 99.6%, 98.4%, and 87.0% similarity, respectively, with Erschoviorchis anuiensis, a newly described opisthorchiid species infecting the liver and pancreas of fish-eating birds in Europe and Asia. Infection by E. anuiensis is highly pathogenic in several piscivorous bird species. The clinical significance of trematodosis in our 5 cases is uncertain because all birds had comorbidities.

Liver in guinea pig: Scanning and transmission electron microscopic studies

The liver performs many functions, which support metabolism, immunity, digestion, detoxification, vitamin storage, and so on. The liver is the storage organ for fat-soluble vitamins, iron and copper. Six adult healthy guinea pigs of 16-32 weeks of age (Irrespective of sex) were procured from the Department of Laboratory Animal Medicine, TANUVAS as per ethical committee approval. Animals were dissected as per CPCSEA norms and liver pieces were utilized for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) study. The study was performed to document the ultrastructural details of liver of guinea pigs by SEM and TEM. By SEM, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. The sinusoidal lumen was lined by two types of cells namely endothelial cells and Kupffer cells. Endothelial cells had numerous large and small cytoplasmic fenestrations. Presence of Kupffer cells along with Ito cells were observed in the sinusoids. By TEM, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus. Bile canaliculi were formed by two adjacent hepatocytes. Fenestrated endothelial cells with flat and elongated shape were found to be in the formation of hepatic sinusoidal wall. A narrow space of Disse was found between the sinusoidal endothelium and hepatocytes. Kupffer cells and pit cells were found within the sinusoidal lumen. Ito cells were found in the space of Disse. RESEARCH HIGHLIGHTS: By scanning electron microscopy, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. By transmission electron microscopy, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus.

Abstract 1580: Cancer cells promote phenotypic alterations in adjacent hepatocytes to facilitate vessel co-option in colorectal cancer liver metastasis

Abstract Vessel co-option is associated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opted lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding non-malignant tissue and hijack them to gain access to nutrient. In order to access the sinusoidal vessels, the cancer cells must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, motility, epithelial-mesenchymal transition (EMT) and autophagy pathways in hepatocyte’s displacement by cancer cells. We demonstrated that cancer cells induce the expression of the proteins that associated with upregulation of apoptosis, motility and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observed upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and Actin Related Protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cells, while we noticed lower expression levels of E-cadherin. Importantly, Runt-related transcription factor 1 (RUNX1) knockdown in the cancer cells has impaired their function towards adjacent hepatocytes in vitro and in vivo. Altogether, our data indicating that cancer cells may exploit various mechanisms to displace the hepatocytes and access to the sinusoidal vessels to establish vessel co-option. Citation Format: Miran Rada, Anthoula Lazaris, Peter Metrakos. Cancer cells promote phenotypic alterations in adjacent hepatocytes to facilitate vessel co-option in colorectal cancer liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1580.

Cancer Cells Promote Phenotypic Alterations in Hepatocytes at the Edge of Cancer Cell Nests to Facilitate Vessel Co-Option Establishment in Colorectal Cancer Liver Metastases.

Simple SummaryTumour cells in colorectal cancer liver metastases (CRCLM) obtain their blood supply via two major mechanisms: (i) sprouting angiogenesis, through the generation of new vessels; (ii) vessel co-option, where the cancer cells hijack the pre-existing vasculature. The current treatment for CRCLM targets angiogenesis; however, these treatments are ineffective on cancer cells utilizing vessel co-option to gain their blood supply. Our study suggests that cancer cells stimulate phenotypic alterations in the cells of surrounding liver tissue (hepatocytes) in vessel co-opting lesions. These modifications facilitate cancer cells to infiltrate through the liver tissue and hijack the pre-existing vasculature to obtain oxygen and nutrients.Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial–mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option.

Tetramethylpyrazine prevents liver fibrotic injury in mice by targeting hepatocyte-derived and mitochondrial DNA-enriched extracellular vesicles.

Liver fibrosis is the common consequence of almost all liver diseases and has become an urgent clinical problem without efficient therapies. Recent evidence has shown that hepatocytes-derived extracellular vesicles (EVs) play important roles in liver pathophysiology, but little is known about the role of damaged hepatocytes-derived EVs in hepatic stellate cell (HSC) activation and following fibrosis. Tetramethylpyrazine (TMP) from Ligusticum wallichii Franchat exhibits a broad spectrum of biological activities including liver protection. In this study, we investigated whether TMP exerted liver-protective action through regulating EV-dependent intercellular communication between hepatocytes and HSCs. Chronic liver injury was induced in mice by CCl4 (1.6 mg/kg, i.g.) twice a week for 8 weeks. In the last 4 weeks of CCl4 administration, mice were given TMP (40, 80, 160 mg·kg-1·d-1, i.g.). Acute liver injury was induced in mice by injection of a single dose of CCl4 (0.8 mg/kg, i.p.). After injection, mice were treated with TMP (80 mg/kg) every 24 h. We showed that TMP treatment dramatically ameliorated CCl4-induced oxidative stress and hepatic inflammation as well as acute or chronic liver fibrosis. In cultured mouse primary hepatocytes (MPHs), treatment with CCl4 or acetaminophen resulted in mitochondrial dysfunction, release of mitochondrial DNA (mtDNA) from injured hepatocytes to adjacent hepatocytes and HSCs through EVs, mediating hepatocyte damage and fibrogenic responses in activated HSCs; pretreatment of MPHs with TMP (25 μM) prevented all these pathological effects. Transplanted serum EVs from TMP-treated mice prevented both initiation and progression of liver fibrosis caused by CCl4. Taken together, this study unravels the complex mechanisms underlying the protective effects of TMP against mtDNA-containing EV-mediated hepatocyte injury and HSC activation during liver injury, and provides critical evidence inspiring the development of TMP-based innovative therapeutic agents for the treatment of liver fibrosis.

Banff consensus recommendations for steatosis assessment in donor livers.

Background and AimsNo consensus criteria or approaches exist regarding assessment of steatosis in the setting of human donor liver suitability for transplantation. The Banff Working Group on Liver Allograft Pathology undertook a study to determine the consistency with which steatosis is assessed and reported in frozen sections of potential donor livers.Approach and ResultsA panel of 59 pathologists from 16 countries completed a questionnaire covering criteria used to assess steatosis in donor liver biopsies, including droplet size and magnification used; subsequently, steatosis severity was assessed in 18 whole slide images of donor liver frozen sections (n = 59). Survey results (from 56/59) indicated a wide variation in definitions and approaches used to assess and report steatosis. Whole slide image assessment led to a broad range in the scores. Findings were discussed at a workshop held at the 15th Banff Conference on Allograft Pathology, September 2019. The aims of discussions were to (i) establish consensus criteria for defining “large droplet fat” (LDF) that predisposes to increased risk of initial poor graft function and (ii) develop an algorithmic approach to determine fat droplet size and the percentage of hepatocytes involved. LDF was defined as typically a single fat droplet that expands the involved hepatocyte and is larger than adjacent nonsteatotic hepatocytes. Estimating severity of steatosis involves (i) low magnification estimate of the approximate surface area of the biopsy occupied by fat, (ii) higher magnification determination of the percentage of hepatocytes within the fatty area with LDF, and (iii) final score calculation.ConclusionsThe proposed guidelines herein are intended to improve standardization in steatosis assessment of donor liver biopsies. The calculated percent LDF should be provided to the surgeon.

MicroRNA-375 inhibits the malignant behaviors of hepatic carcinoma cells by targeting NCAPG2.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide. Emerging evidence has revealed the vital functions of microRNAs (miRNAs) in cancer malignant progressions. miR-375 has been verified to serve as an antioncogene in tumorigenesis and a potential therapeutic target in various types of cancer. In this study, we aimed to determine the role of miR-375 in the regulation of chemoresistance and metastasis of HCC. Differentially expressed miR-375 and NCAPG2 were externally validated using expression data from The Cancer Genome Atlas (TCGA) database. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of miR-375 in HCC tissues and cell lines. miR-375 mimics and NCAPG2-overexpression were transfected into HepG2 and Huh7 cells to establish miR-375 overexpression models. Cell Counting Kit-8, Transwell, and flow cytometry experiments were conducted to monitor cell proliferation, migration, and apoptosis. The targeting relationship between miR-375 and non-SMC condensin II complex subunit G 2 (NCAPG2) was determined by qRT-PCR, western blot, and luciferase reporter gene assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using Gene Set Enrichment Analysis (GSEA). The pathway enrichment analysis was used to predict the potential pathways for further study. miR-375 was significantly downregulated in HCC tissues and cells compared to adjacent tissue and normal hepatocyte cell line respectively while NCAPG2 was upregulated. The targeting relationship was verified by luciferase reporting assay, and miR-375 could target the 3'UTR of NCAPG2 mRNA and effectively suppress NCAPG2 protein expression. Replenishing of miR-375 significantly repressed HCC cell proliferation and migration, and induced cell apoptosis. Overexpression of NCAPG2 recovered those biological abilities in miR-375 overexpressed cells. Collective data suggested that miR-375 served as a tumor suppressor via regulating NCAPG2. Replenishing of miR-375 or knockout of NCAPG2 could be therapeutically exploited for HCC.

Small nucleolar RNA 42 promotes the growth of hepatocellular carcinoma through the p53 signaling pathway

Recent studies show that small nucleolar RNAs (snoRNAs) play an important role in tumorigenesis. SNORA42 is a potential therapeutic target and prognostic biomarker for various cancers, and the aim of the present study was to investigate the function and clinical relevance of SNORA42 in hepatocellular carcinoma (HCC). We detected the expression levels of SNORA42 in HCC and normal liver tissue samples, as well as in tumor and hepatocyte-derived cell lines. SNORA42 was significantly upregulated in the HCC tissues and cells compared to the adjacent liver tissues and normal hepatocytes. Furthermore, overexpression of SNORA42 correlated with poor prognosis in the HCC patients. Knocking down SNORA42 in HCC cell lines decreased their proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. In contrast, ectopic expression of SNORA42 promoted HCC cell proliferation and inhibited apoptosis. Mechanistically, SNORA42 exerted its oncogenic effects by targeting the p53 signaling pathway and cell cycle transition. In conclusion, SNORA42 acted as an oncogene in HCC and was a potential prognostic biomarker and therapeutic target.

Rab35 controls formation of luminal projections required for bile canalicular morphogenesis.

Hepatocytes display a unique biaxial polarity with shared apical luminal connections between adjacent hepatocytes that merge into a network of bile canaliculi. Belicova et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103003) discovered that hepatocyte apical membranes generate Rab35-dependent extensions that traverse the lumen and are essential for bile canalicular formation and maintenance.

The role of different edible vegetable oils as hypolipidemic agents on experimental hyper-lipidemic rat model

Hyperlipidemia is a common disorder that includes genetic and functional disorders represented by elevated lipid levels in the body. The objective of this study was to characterize the physicochemical parameters of the edible seed oils separated from linseed, soybean, olive seed and blends from different proportion, then identify the optimum blend in controlling serum lipid profile in experimental hyperlipidemic rat model. Histopathological examinations of the liver section were carried out to support the laboratory investigation. Sixty male albino rats were used for the study. The animals were divided into ten groups, where group 1 represents negative control, while rats in group 2 received high fat diet and served as positive control. Groups from 3 to 10 were fed high-fat diet and then supplemented with different vegetable oils. At the termination of the 6 weeks trial, blood samples and organs were collected for estimation of lipid profile. Results showed that rats administered different vegetable oils significantly decrease levels of serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) cholesterol as well as increase levels of high-density lipoprotein (HDL) cholesterol in comparison with rats fed high-fat diet. Histopathological examination of liver tissue in positive control showed severe dilatation and congestion in the central vein with fatty change in the surrounding adjacent hepatocytes. While administration of different vegetable oils showed marked improvement in the histological feature of liver tissues. In conclusion, the findings suggest that balanced blend from linseed, soybean and olive oils help in controlling hyperlipidemia and restoring the lipid profile.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Anatomy of the Liver of Mizoram Local Pig (Zovawk)

Background: The present study was aimed for the promotion and advancement of the anatomical knowledge at the gross, light microscopic and electron microscopic level in Zovawk (mizo local pig). Methods: The current investigation was done at the Instructional livestock farm, Department of Veterinary Anatomy and Histology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram and Sophisticated Analytical Instrument Facility (SAIF), North Eastern Hill University (NEHU), Shillong, Meghalaya. Six liver samples were collected from six apparently healthy Zovawk animal of either sex and gross morphological observations were done directly after collection. Thereafter tissue samples were collected as such and were preserved in neutral buffer formalin (NBF) and in Karnovsky’s fixative for routine histology and transmission electron microscopic examination, respectively.Result: Zovawk liver shows four distinct borders, i.e. medial, lateral, dorsal and ventral border, two surfaces, i.e. parietal surface or diaphragmatic surface and caudal or visceral surface and six distinct lobes. The average weight of Zovawk liver was 1.402 kg. Weight of the liver was highly correlated with body weight of animal. Histologically, Zovawk liver was characterized with thick Glisson’s capsule and thick connective tissue septa emerging from it, which gives the hepatic lobules its hexagonal shape. Sinusoids of adjacent hepatocytes were lined by stellate shaped Kupffer cells. The ultra-structural examination of liver shows that, the hepatocytes were rich in mitochondria, endoplasmic reticulum and glycogen granules. Free ribosome and well developed rER and dense lysosomal granules were common in those hepatocyte.

Histological Patterns of Hepatitis and Cholangitis

Histological Patterns of Hepatitis and Cholangitis

REC8 promotes tumor migration, invasion and angiogenesis by targeting the PKA pathway in hepatocellular carcinoma.

REC8 is a member of the cohesin family, and its abnormal activation has been detected in cancer cells. This study explored the role and possible mechanism of REC8 in hepatocellular carcinoma (HCC). A total of 40 pairs of HCC and adjacent tissues were collected, and the clinical significance of REC8 expression in HCC was evaluated. REC8 expression in human HCC tissues and HCC cell lines was investigated by quantitative real-time PCR, Western blotting, immunohistochemistry and immunofluorescence staining. The biological functions of REC8 in HCC cell lines were detected by wound-healing assay, Matrigel invasion assay and tube formation assay. The proteins interacting with REC8 were identified by mass spectrometry after immunoprecipitation screening. There was a correlation between the high expression of REC8 and positive alpha-fetoprotein levels. The expression level of REC8 protein in HCC tissues was higher than that in adjacent tissues. REC8 has mainly located in the nucleus of HCC tissue cells and HCC cell lines, but it was expressed in the cytoplasm of adjacent normal tissue cells and hepatocytes. The results of wound healing, transwell invasion and tubular formation assays indicated that the overexpression of REC8 accelerated the metastasis of HCC in vitro; however, metastasis was suppressed after REC8 was silenced by small interference RNA. A total of 57 differentially expressed proteins were identified by mass spectrometry, and it was found that REC8 and PKA RII-α staining was colocalized in the nucleus. The expression levels of MMP-9 and VEGF-C were decreased after treatment with the PKA inhibitor H89. Overall, REC8 promotes the migration, invasion and angiogenesis of HCC cells through the PKA pathway.

MicroRNA miR-509-3p inhibit metastasis and epithelial-mesenchymal transition in hepatocellular carcinoma

ABSTRACT Our study seeks to obtain data which help to assess the impacts and related mechanisms of microRNA miR-509-3p in hepatocellular carcinoma (HCC). We found that the expression of miR-509-3p was down-regulated and Twist was up-regulated in HCC tissues and cell lines (HepG2, HCCLM3, Bel7402, and SMMC7721) compared with the adjacent normal tissues and normal human hepatocyte (L02). Moreover, cell proliferation, invasion, migration and epithelial–mesenchymal transition (EMT) in HepG2 and HCCLM3 cells were appeared to be markedly suppressed by overexpressed miR-509-3p. Overexpression of miR-509-3p also performed inhibition of the growth and metastasis in vivo. In addition, miR-509-3p could target and inhibit Twist expression, and it could further reverse the tumor promotion by Twist in HCC. All in all, miR-509-3p overexpression causes inhibition of the proliferation, migration, invasion and EMT of HCC cells by negatively regulating Twist, thereby suppressing HCC development and metastasis.

Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel

Drug-induced hepatocellular cholestasis leads to altered bile flow. Bile is propelled along the bile canaliculi (BC) by actomyosin contractility, triggered by increased intracellular calcium (Ca2+). However, the source of increased intracellular Ca2+ and its relationship to transporter activity remains elusive. We identify the source of the intracellular Ca2+ involved in triggering BC contractions, and we elucidate how biliary pressure regulates Ca2+ homeostasis and associated BC contractions. Primary rat hepatocytes were cultured in collagen sandwich. Intra-canalicular Ca2+ was measured with fluo-8; and intra-cellular Ca2+ was measured with GCaMP. Pharmacological modulators of canonical Ca2+-channels were used to study the Ca2+-mediated regulation of BC contraction. BC contraction correlates with cyclic transfer of Ca2+ from BC to adjacent hepatocytes, and not with endoplasmic reticulum Ca2+. A mechanosensitive Ca2+ channel (MCC), Piezo-1, is preferentially localized at BC membranes. The Piezo-1 inhibitor GsMTx-4 blocks the Ca2+ transfer, resulting in cholestatic generation of BC-derived vesicles whereas Piezo-1 hyper-activation by Yoda1 increases the frequency of Ca2+ transfer and BC contraction cycles. Yoda1 can recover normal BC contractility in drug-induced hepatocellular cholestasis, supporting that Piezo-1 regulates BC contraction cycles. Finally, we show that hyper-activating Piezo-1 can be exploited to normalize bile flow in drug-induced hepatocellular cholestasis.

High Prevalence of Human Polyomavirus 7 in Cholangiocarcinomas and Adjacent Peritumoral Hepatocytes: Preliminary Findings

Cholangiocarcinoma (CCA) is a rare biliary-duct malignancy with poor prognosis. Recently, the presence of the human polyomavirus 6 (HPyV6) has been reported in the bile of diverse hepatobiliary diseases, particularly in the bile of CCA patients. Here, we investigated the presence of novel HPyVs in CCA tissues using diverse molecular techniques to assess a possible role of HPyVs in CCA. Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 42 CCA patients were included in this study. PCR-based screening for HPyVs was conducted using degenerated and HPyV-specific primers. Following that, we performed FISH, RNA in situ hybridization (RNA-ISH), and immunohistochemistry (IHC) to assess the presence of HPyVs in selected tissues. Of all 42 CCAs, 25 (59%) were positive for one HPyV, while 10 (24%) CCAs were positive for 2 HPyVs simultaneously, and 7 (17%) were negative for HPyVs. Of the total 35 positive CCAs, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 as confirmed by sequencing. The presence of viral nucleic acids was confirmed by specific FISH, while the RNA-ISH confirmed the presence of HPyV6 on the single-cell level. In addition, expression of HPyV7, HPyV6, and MCPyV proteins were confirmed by IHC. Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect bile duct epithelium, hepatocytes, and CCA cells, which possibly suggest an indirect role of these viruses in the etiopathogenesis of CCA. Furthermore, the observed hepatotropism of these novel HPyV, in particular HPyV7, might implicate a role of these viruses in other hepatobiliary diseases.