We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepati... more We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validat...
We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepati... more We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validat...
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology, 2009
To profile the protein expression in activated rat hepatic stellate cells (HSCs). Primary rat HSC... more To profile the protein expression in activated rat hepatic stellate cells (HSCs). Primary rat HSCs were isolated and cultured in vitro. After 10 days in vitro culture, the HSCs were activated. Total protein extracted from these activated HSCs were digested, and the obtained peptides were analyzed by using online 2D nanoLC-MS/MS. The identified proteins were classified according to their distributions and functions. 1014 proteins were identified from 50 microg HSCs protein extract, the molecular weights of these proteins ranged from 7832 Da to 588,364 Da. Most of these proteins resided in nucleus, cytoskeleton, mitochondrion and endoplasmic reticulum. And these proteins were mainly involved in nucleic acid metabolism, organelle organization, signal transduction and energy generation. Among these proteins, alpha-smooth muscle actin, vimentin and desmin were specifically expressed in activated HSCs. To the best of our knowledge, this is the most comprehensive protein expression profile...
Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essen... more Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essential step toward understanding the biological properties of HSC and clarifying the potential roles of HSCs in liver fibrosis and other liver diseases, including hepatocellular carcinoma. Highthroughput comparative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS) were performed on an in vitro HSC activation model to obtain a comprehensive view of the protein ensembles associated with HSC activation. In total, 2,417 proteins were confidently identified (false discovery rate <1%), of which 2,322 proteins were quantified. Compared with quiescent HSCs, 519 proteins showed significant differences in activated HSCs (!3.0-fold). Bioinformatics analyses using Ingenuity Pathway Analysis revealed that the 319 up-regulated proteins represented multiple cellular functions closely associated with HSC activation, such as extracellular matrix synthesis and proliferation. In addition to the well-known markers for HSC activation, such as a-smooth muscle actin and collagen types 1 and 3, some novel proteins potentially associated with HSC activation were identified, while the 200 down-regulated proteins were primarily related to immune response and lipid metabolism. Most intriguingly, the top biological function, top network, and top canonical pathway of down-regulated proteins were all involved in immune responses. The expression and/or biological function of a set of proteins were properly validated, especially Bcl2-associated athanogene 2, BAG3, and B7H3. Conclusion: The present study provided the most comprehensive proteome profile of rat HSCs and some novel insights into HSC activation, especially the suppressed immune response. (HEPATOLOGY 2012;332-349) Abbreviations: 2D nano-LC-MS/MS, two-dimensional nanoscale liquid chromatography and tandem mass spectrometry; a-SMA, a-smooth muscle actin; B2M, b2microglobulin; BAG2, Bcl2-associated athanogene 2; CCl 4 , carbon tetrachloride; COL, collagen; CRBP1, cellular retinol-binding protein-1; ECM, extracellular matrix; EdU, 5ethynyl-2 0 -deoxyuridine; ERK1/2, extracellular-regulated kinase 1/2; FDR, false discovery rate; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; Hsp, heat shock protein 70; IPA, Ingenuity Pathway Analysis; iTRAQ, isobaric tag for relative and absolute quantitation; LTA4H, leukotriene A4 hydrolase; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T cells; NTC, nontargeting control; RE, retinyl ester; siRNA, small interfering RNA; TGF-b, transforming growth factor-b.
FOXQ1 (forkhead box Q1) is a forkhead transcription factor, and FOX genes play significant roles ... more FOXQ1 (forkhead box Q1) is a forkhead transcription factor, and FOX genes play significant roles in a series of biological processes. The aim of our study was to verify the importance of FOXQ1 as a prognostic indicator in HCC patients. One-step quantitative real-time PCR was performed to identify the expression of FOXQ1 mRNA in HCC and corresponding non-cancerous tissues. FOXQ1 expression was then evaluated by immunohistochemistry (IHC) using tissue microarray. Finally, we analyzed FOXQ1 expression and clinicopathological factors in 114 HCC patients using SPSS 20.0 software. FOXQ1 expression at the transcriptional level in HCC cells was much higher than in the noncancerous cells (P=0.012, respectively). Comparison of clinicopathological characteristics and FOXQ1 expression in HCC by IHC and χ(2) tests showed that high FOXQ1 expression was linked to large tumor diameter, high serum α-fetoprotein levels and later stage grouping with tumor node metastasis classification. Kaplan-Meier a...
Aim: To investigate whether fenretinide, a clinically proved apoptosis-inducing chemopreventive a... more Aim: To investigate whether fenretinide, a clinically proved apoptosis-inducing chemopreventive agent in tumor cells, can induce apoptosis in hepatic stellate cells (HSCs) and resolve hepatic fibrosis.
Liver fibrosis a b s t r a c t Hepatic stellate cells (HSCs) activation is an initial event in li... more Liver fibrosis a b s t r a c t Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down-regulation of two over-expressed miRNAs, miR-27a and 27b allowed culture-activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor a was confirmed to be the target of miR-27a and 27b. These results indicated a new role and mechanism of miR-27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.
Objectives: Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and ... more Objectives: Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Recent studies suggested that deregulation of Spy1 expression plays a key role in oncogenesis. To investigate the potential roles of Spy1 in hepatocellular carcinoma (HCC), expression of Spy1 was examined in human HCC samples. Methods: Immunohistochemistry and Western blot analysis was performed for Spy1 in 61 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. Results: Spy1 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Spy1 was associated with histological grade and the level of alpha fetal protein (AFP) (P = 0.009 and 0.003, respectively), and Spy1 was positively correlated with proliferation marker Ki-67 (P < 0.001). Univariate analysis showed that Spy1 expression was associated with poor prognosis (P = 0.03). Multivariate analysis indicated that Spy1 and Ki-67 protein expression was an independent prognostic marker for HCC (P = 0.001 and 0.012, respectively). While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Spy1 was upregulated. Conclusions: Our results suggested that Spy1 overexpression is involved in the pathogenesis of hepatocellular carcinoma, it may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.
Biochemical and Biophysical Research Communications, 2009
Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our pre... more Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-a (C/EBP-a) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-a gene (Ad-C/EBP-a) could induce HSCs apoptosis in a dose-and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-a resulted in the up-regulation of peroxisome proliferator-activated receptor-c (PPAR-c) and P53, while P53 expression was regulated by PPAR-c. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-c and p53 in the process of apoptosis triggered by C/EBP-a in HSCs.
Hepatocellular carcinoma (HCC) patients suffer from a poor survival rate and high incidence of po... more Hepatocellular carcinoma (HCC) patients suffer from a poor survival rate and high incidence of post-operative recurrence. The hepatic microenvironment plays a significant role in the initiation, progression and recurrence of HCC; however the causal mechanisms of these phenomena are unclear. Given the predominant underlying fibrotic and cirrhotic conditions of the liver prone to HCC and its recurrence, alterations of components of the inflammatory milieu have been suggested as factors that promote HCC development. In particular, activated hepatic stellate cells (A-HSC) that play a key role in liver fibrosis and cirrhosis, have been suggested as contributors to the HCC-prone microenvironment. Here, we have identified and validated an A-HSC-specific gene expression signature among non-tumor tissues of 319 HCC patients that is significantly and independently associated with HCC recurrence and survival. Peritumoral, rather than tumor tissue-related A-HSC-specific gene expression is assoc...
We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepati... more We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validat...
We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepati... more We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validat...
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology, 2009
To profile the protein expression in activated rat hepatic stellate cells (HSCs). Primary rat HSC... more To profile the protein expression in activated rat hepatic stellate cells (HSCs). Primary rat HSCs were isolated and cultured in vitro. After 10 days in vitro culture, the HSCs were activated. Total protein extracted from these activated HSCs were digested, and the obtained peptides were analyzed by using online 2D nanoLC-MS/MS. The identified proteins were classified according to their distributions and functions. 1014 proteins were identified from 50 microg HSCs protein extract, the molecular weights of these proteins ranged from 7832 Da to 588,364 Da. Most of these proteins resided in nucleus, cytoskeleton, mitochondrion and endoplasmic reticulum. And these proteins were mainly involved in nucleic acid metabolism, organelle organization, signal transduction and energy generation. Among these proteins, alpha-smooth muscle actin, vimentin and desmin were specifically expressed in activated HSCs. To the best of our knowledge, this is the most comprehensive protein expression profile...
Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essen... more Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essential step toward understanding the biological properties of HSC and clarifying the potential roles of HSCs in liver fibrosis and other liver diseases, including hepatocellular carcinoma. Highthroughput comparative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS) were performed on an in vitro HSC activation model to obtain a comprehensive view of the protein ensembles associated with HSC activation. In total, 2,417 proteins were confidently identified (false discovery rate <1%), of which 2,322 proteins were quantified. Compared with quiescent HSCs, 519 proteins showed significant differences in activated HSCs (!3.0-fold). Bioinformatics analyses using Ingenuity Pathway Analysis revealed that the 319 up-regulated proteins represented multiple cellular functions closely associated with HSC activation, such as extracellular matrix synthesis and proliferation. In addition to the well-known markers for HSC activation, such as a-smooth muscle actin and collagen types 1 and 3, some novel proteins potentially associated with HSC activation were identified, while the 200 down-regulated proteins were primarily related to immune response and lipid metabolism. Most intriguingly, the top biological function, top network, and top canonical pathway of down-regulated proteins were all involved in immune responses. The expression and/or biological function of a set of proteins were properly validated, especially Bcl2-associated athanogene 2, BAG3, and B7H3. Conclusion: The present study provided the most comprehensive proteome profile of rat HSCs and some novel insights into HSC activation, especially the suppressed immune response. (HEPATOLOGY 2012;332-349) Abbreviations: 2D nano-LC-MS/MS, two-dimensional nanoscale liquid chromatography and tandem mass spectrometry; a-SMA, a-smooth muscle actin; B2M, b2microglobulin; BAG2, Bcl2-associated athanogene 2; CCl 4 , carbon tetrachloride; COL, collagen; CRBP1, cellular retinol-binding protein-1; ECM, extracellular matrix; EdU, 5ethynyl-2 0 -deoxyuridine; ERK1/2, extracellular-regulated kinase 1/2; FDR, false discovery rate; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; Hsp, heat shock protein 70; IPA, Ingenuity Pathway Analysis; iTRAQ, isobaric tag for relative and absolute quantitation; LTA4H, leukotriene A4 hydrolase; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T cells; NTC, nontargeting control; RE, retinyl ester; siRNA, small interfering RNA; TGF-b, transforming growth factor-b.
FOXQ1 (forkhead box Q1) is a forkhead transcription factor, and FOX genes play significant roles ... more FOXQ1 (forkhead box Q1) is a forkhead transcription factor, and FOX genes play significant roles in a series of biological processes. The aim of our study was to verify the importance of FOXQ1 as a prognostic indicator in HCC patients. One-step quantitative real-time PCR was performed to identify the expression of FOXQ1 mRNA in HCC and corresponding non-cancerous tissues. FOXQ1 expression was then evaluated by immunohistochemistry (IHC) using tissue microarray. Finally, we analyzed FOXQ1 expression and clinicopathological factors in 114 HCC patients using SPSS 20.0 software. FOXQ1 expression at the transcriptional level in HCC cells was much higher than in the noncancerous cells (P=0.012, respectively). Comparison of clinicopathological characteristics and FOXQ1 expression in HCC by IHC and χ(2) tests showed that high FOXQ1 expression was linked to large tumor diameter, high serum α-fetoprotein levels and later stage grouping with tumor node metastasis classification. Kaplan-Meier a...
Aim: To investigate whether fenretinide, a clinically proved apoptosis-inducing chemopreventive a... more Aim: To investigate whether fenretinide, a clinically proved apoptosis-inducing chemopreventive agent in tumor cells, can induce apoptosis in hepatic stellate cells (HSCs) and resolve hepatic fibrosis.
Liver fibrosis a b s t r a c t Hepatic stellate cells (HSCs) activation is an initial event in li... more Liver fibrosis a b s t r a c t Hepatic stellate cells (HSCs) activation is an initial event in liver fibrosis. MicroRNAs (miRNAs) have been found to play essential roles in cell differentiation, proliferation, and fat metabolism. In this study, we showed that down-regulation of two over-expressed miRNAs, miR-27a and 27b allowed culture-activated rat HSCs to switch to a more quiescent HSC phenotype, with restored cytoplasmic lipid droplets and decreased cell proliferation. Mechanistically, retinoid X receptor a was confirmed to be the target of miR-27a and 27b. These results indicated a new role and mechanism of miR-27a and 27b in regulating fat metabolism and cell proliferation during HSCs activation.
Objectives: Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and ... more Objectives: Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Recent studies suggested that deregulation of Spy1 expression plays a key role in oncogenesis. To investigate the potential roles of Spy1 in hepatocellular carcinoma (HCC), expression of Spy1 was examined in human HCC samples. Methods: Immunohistochemistry and Western blot analysis was performed for Spy1 in 61 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. Results: Spy1 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Spy1 was associated with histological grade and the level of alpha fetal protein (AFP) (P = 0.009 and 0.003, respectively), and Spy1 was positively correlated with proliferation marker Ki-67 (P < 0.001). Univariate analysis showed that Spy1 expression was associated with poor prognosis (P = 0.03). Multivariate analysis indicated that Spy1 and Ki-67 protein expression was an independent prognostic marker for HCC (P = 0.001 and 0.012, respectively). While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Spy1 was upregulated. Conclusions: Our results suggested that Spy1 overexpression is involved in the pathogenesis of hepatocellular carcinoma, it may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.
Biochemical and Biophysical Research Communications, 2009
Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our pre... more Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-a (C/EBP-a) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-a gene (Ad-C/EBP-a) could induce HSCs apoptosis in a dose-and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-a resulted in the up-regulation of peroxisome proliferator-activated receptor-c (PPAR-c) and P53, while P53 expression was regulated by PPAR-c. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-c and p53 in the process of apoptosis triggered by C/EBP-a in HSCs.
Hepatocellular carcinoma (HCC) patients suffer from a poor survival rate and high incidence of po... more Hepatocellular carcinoma (HCC) patients suffer from a poor survival rate and high incidence of post-operative recurrence. The hepatic microenvironment plays a significant role in the initiation, progression and recurrence of HCC; however the causal mechanisms of these phenomena are unclear. Given the predominant underlying fibrotic and cirrhotic conditions of the liver prone to HCC and its recurrence, alterations of components of the inflammatory milieu have been suggested as factors that promote HCC development. In particular, activated hepatic stellate cells (A-HSC) that play a key role in liver fibrosis and cirrhosis, have been suggested as contributors to the HCC-prone microenvironment. Here, we have identified and validated an A-HSC-specific gene expression signature among non-tumor tissues of 319 HCC patients that is significantly and independently associated with HCC recurrence and survival. Peritumoral, rather than tumor tissue-related A-HSC-specific gene expression is assoc...
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