MicroRNA-150 (miR-150) is frequently dysregulated in cancer and is involved in carcinogenesis and cancer progression. In this study, we found that miR-150 was significantly downregulated in hepatocellular carcinoma (HCC) tissues compared to adjacent noncancerous tissues. Low levels of miR-150 were significantly associated with worse clinicopathological characteristics and a poor prognosis for patients with HCC. miR-150 overexpression inhibited cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo. Further experiments indicated that Grb2-associated binding protein 1 (GAB1) was a direct target of miR-150 in HCC cells. In addition, GAB1 expression was increased in HCC tissues and inversely correlated with miR-150 levels. Knockdown of GAB1 mimicked the tumor-suppressive effects of miR-150 overexpression on HCC cells, whereas restoration of GAB1 expression partially abolished the inhibitory effects. Moreover, miR-150 overexpression decreased GAB1 expression, subsequently downregulated phospho-ERK1/2 and suppressed epithelial-mesenchymal-transition (EMT). These effects caused by miR-150 overexpression were alleviated by exogenous GAB1 expression. Taken together, this study demonstrates that miR-150 may be useful as a prognostic marker and that the identified miR-150-GAB1-ERK axis is a potential therapeutic target for HCC.
Objective To investigate the potential of directed differentiation of fetal liver stem cells (FLSCs) in mice with sponataneous green fluorescence in vitro and in vivo.Methods FLSCs were taken from green fluorescent protein transgenic mice at embryonic day 13.After mechanical dissection and enzyme digestion with collagenase,FLSCs were further cultured.The expression of stem cell related markers and the self-renewal ability were identified by flow cytometry.The expression of alpha fetoprotein (AFP) was detected by RT-PCR and Western-blot.FLSCs were induced to differentiation by hepatic growth factor (HGF) and transforming growth factor β (TGF-β),respectively,and the ability of dual-direction (hepatocyte and cholangiocyte) differentiation was measured by exploring the expression alteration of Albumin (Alb),cytokeratin (CK)-7,8,19 by using RT-PCR and the expression alteration of CK-7,8,19 and Alb by using Western blot.Suspended FLSCs were injected to wild type C57BL/6J mouce hepatectomy model via caudal vein,and the ability of hepatic tissue reconstruction was observed.All data were analyzed using the analysis of variance or LSD-t test.Results FLSCs cells were successfully cloned.Stem cell related markers including epithelial cell adhesion molecule (EpCAM),CD133 and CD49f were detected by flow cytometry.The positive expressions of EpCAM,CD133 and CD49f were 78.6％ ± 3.3％,31.7％ ± 2.5％ and 47.6％ ± 1.8％,respectively.The relative mRNA and protein expression levels of AFP in FLSCs and mature hepatocytes were 0.640 ± 0.115,0.053 ± 0.012 and 1.383 ± 0.265,0.243 ± 0.227,there were significant differences in AFP expression between FLSCs and mature hepatocytes (t =8.772,5.354,P ＜ 0.05).FLSCs and mature hepatocytes were both cultured in ultra-low attachment dishes for 2 weeks.There was a significant difference in the clone formation rate beteween the FLSCs and mature hepatocytes,which were 234.0 ± 57.0 and 5.0 ± 2.0,respectively (t =12.711,P ＜ 0.05).After induced differentiation by HGF in vitro,the expression level of Alb and CK-8 in fetal liver stem cells peaked at the 8th day and the 10th day,which were 0.851 ± 0.030 and 0.771 ± 0.031,respectively.The protein expression levels of Alb and CK-8 in FLSCs peaked at the 10th day,which were 4.573 ±0.015 and 1.562 ±0.029,respectively.After the induction of differentiation by TGF-3 in vitro,the mRNA expression levels of CK-7 and CK-19 in FLSCs peaked at the 12th day,which were 15.454 ±2.152 and 10.675 ± 1.822,respectively.The mRNA expression levels of CK-7 and CK-19 in FLSCs peaked at the 14th day,which were 3.423 ± 0.105 and 1.892 ± 0.081,respectively.EGFP could be detected in both hepatoeyte and cholangiocyte after transplanting FLSCs to mouse hepatectomy model.Conclusions The FLSCs isolated from C57BL/6JEGFP mice can stably express the green fluorescence protein.These cells possess the traits of hepatic stem cells and have the potential ability of dual-direction differention into both hepatocytes and cholangiocytes in vitro and in vivo.Furthermore,FLSCs derived from green fluorecent protein transgenic mice have a good tracing effect in vivo.
Key words:
Green fluorescent protein transgenic mice; Fetal liver stem cell; Isolation; Identification ; Tracing
This review aims to compile recent advances regarding the significance of Notch signaling in different types of intrahepatic cells during liver injury and repair. The functions of Notch signaling in regulating cell development, fate decisions, and organ homeostasis have been widely acknowledged. Notch is also expressed and activated in hepatocytes, macrophages, liver sinusoidal endothelial cells, endothelial progenitor cells, and hepatic progenitor cells during the process of development, injury, inflammation, fibrosis, and carcinoma. During acute/chronic liver injury, Notch interacts with many signaling pathways that are involved in liver repair. Recent research, including ours, has confirmed the crucial role of Notch signaling in modulating the function of diverse intrahepatic cells during liver injury and reconstruction. Thus, Notch signaling may serve as a potential therapeutic target for liver diseases.
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies.MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy.In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines.The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line.We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a.The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis.We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC.Its expression was inversely correlated with miR-135a expression in PDAC.Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3'-untranslated region (3'-UTR) of Bmi1.Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor.miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.
Due to high incidence of invasion and intrahepatic metastasis, hepatocellular carcinoma (HCC) is one of the most aggressive tumors in the world, which is also associated with the acquisition of epithelial-mesenchymal transition (EMT). Increasing evidence suggests that cancer cells with EMT traits share many biological characteristics with cancer stem cells. And miR-200a has been known as a powerful regulator of EMT. Here, we sought to investigate the role of miR-200a in regulation of EMT phenotype of liver cancer stem cells (LCSCs). We used side population (SP) sorting to obtain cancer stem-like cells from HCC cell lines and identified that the SP fraction could be enriched with LCSCs. Then, we detected the expression of miR-200a and EMT makers in SP and non-SP cells. Our results suggested that miR-200a was down-regulated in SP cells, along with relatively low epithelial marker and high mesenchymal marker. In order to find the role of miR-200a in the manipulation of EMT, we transfected miR-200a mimic into LCSCs and found that overexpression of miR-200a resulted in down-regulation of N-cadherin, ZEB2, and vimentin, but up-regulation of E-cadherin. Moreover, overexpression of miR-200a resulted in decreased migration and invasion ability in LCSCs. In conclusion, our study revealed that miR-200a played an important role in linking the characteristics of cancer stem cells with EMT phenotype in HCC, and targeting miR-200a might be an effective strategy to weaken the invasive behavior of LCSCs.
Abstract Aging leads to systemic metabolic disorders including nonalcoholic steatohepatitis (NASH). Here, we showed that aging-induced liver sinusoidal endothelial cell (LSEC) senescence accelerated liver sinusoid capillarization and promoted steatohepatitis by reprogramming liver endothelial zonation and inactivating pericentral endothelium-derived C-kit. Abrogation of endothelial C-kit triggered cellular senescence which disturbed LSEC homeostasis. During diet-induced NASH development, C-kit deletion aggravated hepatic steatosis and exacerbated NASH-associated fibrosis and inflammation. Mechanistically, CXCR4/SDF-1 signaling was inhibited by C-kit. Blocking CXCR4/SDF-1 signaling by AMD3100 abolished LSEC-macrophage crosstalk and recovered the aggravated NASH in C-kit deficient mice. For therapeutic purpose, C-kit + LSECs were implanted into NASH or aged mice, which counteracted LSEC senescence and improved diet or aging-induced NASH by restoring the homeostasis of pericentral liver endothelium.
Abstract Hidden hearing loss (HHL) is characterized by normal audiometric thresholds but impaired auditory function, particularly in noisy environments. In vivo, we employed auditory brainstem response (ABR) testing and ribbon synapses counting to assess changes in mouse hearing function, and observed the morphology of hair cells through scanning electron microscopy. SRT1720 was administered to the cochlea via round window injection. In vitro, western blot analysis and RT‐qPCR were used, and Lenti‐shNrf2 was used to knockdown Nrf2 expression. In addition, various oxidative stress indicators were detected by immunofluorescence, kit‐based assays, and flow cytometry. ABR measurement of HHL mouse showed a significant increase in hearing threshold, as well as a decrease and delay in the I wave amplitude and latency on the first day after noise exposure. Histological observation showed a significant loss of ribbon synapses and stereocilia lodging. HHL mice exhibited oxidative stress, which was reduced by pretreatment with SRT1720. Additionally, SRT1720 could reduce hydrogen peroxide‐induced oxidative stress in HEI‐OC1 cells through activating the SIRT1/Nrf2 pathway. Subsequent experiments with Nrf2 knockdown confirmed the importance of this pathway. findings highlight oxidative stress as the primary contributor to HHL, with the SIRT1/Nrf2 signaling pathway emerging as a promising therapeutic target for alleviating HHL.
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