Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16(INK4A).

zMicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs that regulate approximately one-third of human genes at post-transcription level. Previous studies have shown that miRNAs were implicated in many cellular processes and participated in the progress of various tumors including hepatocellular carcinoma (HCC). Among all miRNAs, the let-7 family is well recognized to play pivotal roles in tumorigenesis by functioning as potential growth suppressor. In the present study, we aimed to investigate the role of let-7 family, particularly the hsa-let-7g, in the molecular pathogenesis of HCC. By use of MTT, qPCR, Western blotting and 2-dimensional electrophoresis (2-DE), over-expression of hsa-let-7g was found to inhibit the proliferation of HCC cell line via negative and positive regulations of c-Myc and p16(INK4A) , respectively. The expression of hsa-let-7g was noted to be markedly lowered in the HepG2, Hep3B and Huh7 cells, yet higher in the Bel-7404 HCC cell line. Proliferation of HCC cell line was significantly inhibited after the transfection of hsa-let-7g mimics, while hsa-let-7g inhibitor transfection exerted an opposite effect. Concurrently, the mRNA and protein levels of c-Myc were found significantly decreased in HepG2 cells after transfection of hsa-let-7g mimics, but obviously increased in Bel-7404 cells after transfection of hsa-let-7g inhibitor. As revealed by 2-DE, a significant upregulation of p16(INK4A) was revealed after the gain-of-function study using hsa-let-7g. Therefore, we suggest that hsa-let-7g may act as a tumor suppressor gene that inhibits HCC cell proliferation by downregulating the oncogene, c-Myc, and upregulating the tumor suppressor gene, p16(INK4A) .

Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus.

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta1 (IFN-beta1). In this connection, the IFN-beta1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-beta1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, under defined conditions, the migration capacity of BM- and P-MSC was found to be 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Interestingly, the expression levels of those proteins reflect perfectly the migration capacity of corresponding MSC, which is also proved by in vitro overexpression and silencing techniques. Our study indicates that a bunch of migration-related proteins are pivotal in governing the migration capacity of MSC.

Ethanol extract of Fructus Ligustri Lucidi promotes osteogenesis of mesenchymal stem cells.

Fructus Ligustri Lucidi (FLL) has been used in traditional Chinese medicine for over 1000 years. The ethanol extract of FLL (EFLL) has been shown to be a potential candidate in the prevention and treatment of osteoporosis. The present study aimed to determine whether EFLL carries out the effect by promoting osteogenesis in mesenchymal stem cells (MSCs). The osteogenic differentiation of MSCs was evaluated by their alkaline phosphatase (ALP) activities and mineralization. Expression of genes was detected by RT-PCR. We found that EFLL significantly stimulated the ALP activities and shortened the time needed for the mineralization of MSCs during osteogenic differentiation. The expression of several osteoblast differentiation regulators was also upregulated by EFLL during this process. Our study demonstrated that the EFLL is capable of enhancing osteogenic differentiation of MSCs. It might be useful for treating diseases with inadequate bone formation, including osteoporosis.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM- and P-MSC was found 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC-MSC when compared with those in BM- and P-MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor-1 (PAI-1) and manganese superoxide dismutase, higher expression was found in the UC-MSC. We also showed that the overexpression of the PAI-1 impaired the migration capacity of BM- and P-MSC while silencing of PAI-1 enhanced the migration capacity of UC-MSC. Our study indicates that PAI-1 and other migration-related proteins are pivotal in governing the migration capacity of MSC.

Proteomic identification of molecular targets of gambogic acid: role of stathmin in hepatocellular carcinoma.

Gamboge has been developed as an injectable drug for cancer treatment in China. In this study, the inhibition ratio and their IC(50) values of two derivatives from Gamboge in hepatocellular carcinoma (HCC) were determined. Proteomic approach was employed to reveal the target proteins of these two derivatives, gambogic acid (GA), and gambogenic acid (GEA). HCC cells were cultured under varied conditions with the addition of either GA or GEA. Twenty differentially expressed proteins were identified and the four most distinctly expressed proteins were further validated by Western blotting. GA and GEA revealed inhibitory effects on HCC cell proliferation. The expression of cyclin-dependent kinase 4 inhibitor A and guanine nucleotide-binding protein beta subunit 1 were upregulated by both xanthones, whilst the expression of 14-3-3 protein sigma and stathmin 1 (STMN1) were downregulated. Furthermore, overexpression of STMN1 in HCC cells decreased their sensitivity, whilst small interfering RNAs targeting STMN1 enhanced their sensitivity to GA and GEA. In conclusion, our study suggested for the first time that STMN1 might be a major target for GA and GEA in combating HCC. Further investigation may lead to a new generation of anticancer drugs exerting synergistic effect with conventional therapy, thus to promote treatment efficacy.

The four-and-a-half-LIM protein 2 (FHL2) is overexpressed in gliomas and associated with oncogenic activities.

Four-and-a-half-LIM protein 2 (FHL2) is a member of FHL protein family, which plays a crucial role in regulating gene expression, cell survival, and migration. Although its function in oncogenesis appears to be tumor type-specific, its roles in glioma formation and development are yet to be elucidated. In the present study, we demonstrated that the mRNA level of FHL2 was elevated in both low- and high-grade glioma samples. Overexpression of FHL2 stimulated the proliferation, anchorage-independent growth, and migration of human glioblastoma cells. Conversely, FHL2 knockdown by short hairpin RNA (shRNA-FHL2) inhibited glioblastoma cell proliferation and migration. Overexpression of FHL2 increased the tumorigenicity of glioblastoma cells in nude mice and decreased the mRNA levels of p53 and its downstream proapoptotic genes, including p21, Bcl2-associated protein X (Bax), and p53-upregulated modulator of apoptosis. It also enhanced the promoter activities of activator protein-1 (AP-1), human telomerase reverse transcriptase, and survivin genes. Together, these results provide the first evidence that FHL2 contributes to glioma carcinogenesis.

Two-dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma.

Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN-induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a two-dimensional differential gel electrophoresis (2D-DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN-induced HCC and healthy liver cells. Western blotting and semiquantitative RT-PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN-induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione S-transferase super family, aldo-keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN-induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis.

Inhibition of HBV gene expression and replication by stably expressed interferon-alpha1 via adeno-associated viral vectors.

BACKGROUND: Interferon-alpha2 (IFNalpha2) is routinely used for anti-hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half-life, relatively low local concentration and strong side-effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector-delivered IFNalpha1 was tested for its anti-HBV effects. METHODS: Adeno-associated viral vector (AAV-IFNalpha1) was generated to deliver the IFNalpha1 gene into hepatocytes. IFNalpha1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme-linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real-time polymerase chain reaction. RESULTS: AAV-IFNalpha1 effectively transduced HBV-producing cells (HepAD38) and mouse hepatocytes, where IFNalpha1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV-producing mice, the concentration of IFNalpha1 in the liver was eight-fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten-fold from day 1-5, and dropped to an undetectable level on day 9 in the AAV-IFNalpha1 group. Concurrently, the level of viral DNA decreased over 30-fold for several weeks. CONCLUSIONS: A single dose administration of AAV-IFNalpha1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV-IFNalpha1 might be a potential alternative strategy for anti-HBV therapy.

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery.

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.

RNA interference and antiviral therapy.

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

The mechanism of aqueous humour formation.

Aqueous humour (AH) is an important intraocular fluid responsible for the supply of nutrients to and removal of metabolic wastes from the avascular tissues of the eye. It is also indispensable for the maintenance of the optical properties of the eye. The fluid dynamics of AH are frequently associated with the potentially blinding disease called glaucoma. Pharmacological treatment of glaucoma generally aims to lower the intraocular pressure by reducing AH formation. However, the mechanism underlying the formation of AH is still not well understood. Understanding the mechanism of AH formation and its regulation is paramount to develop rational and target specific drugs for the treatment of glaucoma. It is now generally believed that AH is formed mostly by active transport of ions and solutes across the ciliary epithelium. Many studies have been carried out in the past half a century to understand these transport processes. In the past several years, new information has emerged and a comprehensive review of these new developments is necessary. This review covers the ion transports in the ciliary epithelium, including the possible roles of sodium, chloride and bicarbonate ions as the driving forces. It also examines the current ionic models for AH formation and its regulation from a cellular transport perspective.

Transactivation of the TIEG1 confers growth inhibition of transforming growth factor-ß-susceptible hepatocellular carcinoma cells.

AIM: To investigate the role of transforming growth factor (TGF)-ß-inducible early gene 1 (TIEG1) in TGF-ß-induced growth inhibition in hepatocellular carcinoma (HCC) cells. METHODS: Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-ß1 were tested by methylthiazoletetrazolium (MTT) assay. The expression changes of Smad2, Smad3, Smad4, Smad7, TIEG1 and TIEG2 gene following treatment with TGF-ß1 in a TGF-ß-sensitive hepatocyte cell line (MIHA), a TGF-ß-sensitive hepatoma cell line (Hep3B) and two TGF-ß-insensitive hepatoma cell lines (HepG2 and Bel7404) were examined. SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-ß1 was examined. Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-ß1-resistant hepatoma cell lines (Bel7404 and HepG2). MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis, respectively. The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis, and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system. RESULTS: TIEG1 was significantly upregulated by TGF-ß1 in the TGF-ß1-sensitive HCC cell line, Hep3B, but not in the resistant cell lines. The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-ß1, whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-ß1-resistant HCC cell lines, which resembled those of TGF-ß1-sensitive HCC cells treated with TGF-ß1. Our data further suggested that stathmin was a direct target of TIEG1, as stathmin was significantly downregulated by TIEG1 overexpression, and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner. CONCLUSION: Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-ß-susceptible human HCC cells.

Proteomic analysis of human mesenchymal stem cells.

Umbilical cord (UC) and placenta (P) are generally believed to be potential alternatives to bone marrow (BM), as sources of mesenchymal stem cells (MSC) for cell therapy. They possess immunophenotypic and functional characteristics which are similar to that of BM-MSC, yet one of the crucial factors in determining the tissue regeneration process--the migration capacity--is still unclear. In our previous study, the migration capacity of BM- and P-MSC was found 5.9- and 3.2-fold higher than that of UC-MSC, respectively. By using 2-dimensional gel electrophoresis (2-DE) and combined MS and MS/MS analysis, six proteins were identified as differentially expressed among these MSC samples. Five out of the six proteins were known to be involved in cell migration as migration inhibiting or enhancing proteins.

Dynamic transcriptional changes of TIEG1 and TIEG2 during mouse tissue development.

TGF-beta-inducible early-response gene (TIEG) is a family of primary response genes induced by TGF-beta, which are well recognized in regulating cellular proliferation and apoptosis. However, their expression profile has never been investigated during embryogenesis in different organs. In this study, we aimed to investigate the transcriptional level of both TIEG1 and TIEG2 during development in various mice organs, including the brain cortex, cerebellum and stem, brain striatum, muscle, heart, liver, kidney, and lung. Quantitative real-time PCR was used to profile the change of transcriptional level of the two TIEG members in the mice tissues at six developmental stages. Taken together, the expression of TIEG1 and TIEG2 was specific in different organs yet varied with different developmental time points. Their dynamic changes were moderately consistent in most organs including the brain cortex, striatum, liver, kidney, and lung. However, their mRNA expression in both the heart and muscle was significantly different at all developmental stages, which might propose a compensation of functions in the TIEG family. Nevertheless, our data indicate that both the TIEG genes are essential in regulating the normal organ development and functioning in murine model, as their expressions were ubiquitous and tissue specific at various developmental stages.

Microarray profile of brain aging-related genes in the frontal cortex of SAMP8.

This study examined the protein expression profile changes in the brain of senescence-accelerated mice/prone 8 (SAMP8) model. Two approaches, namely microarray and RT-PCR, were used in the study. Four genes, which are orthologous to human, were found to differentially express in the aging brain of mice. In this study, we examined the differentially expressed genes in the frontal cortex of the SAMP8 mice of two different ages (4 and 12 month old). Four orthologous genes (i.e., guanine nucleotide binding protein-alpha q polypeptide, kinesin family member 1B, sortilin 1, and somatostatin) showed significant changes in expression with aging. This study may provide important information on the mechanism of aging or aging-related diseases such as Alzheimer's diseases.

RNAi targeting EZH2 inhibits tumor growth and liver metastasis of pancreatic cancer in vivo.

The function of EZH2 in tumorigenesis and liver metastasis of pancreatic cancer has never been elucidated in vivo. EZH2 was overexpressed in pancreatic carcinomas and its overexpression was associated with tumor differentiation and pT status. Suppression of EZH2 caused a significant growth inhibition of pancreatic cancer cells in vitro and markedly diminished their tumorigenicity in vivo. Knock-down of EZH2 inhibited liver metastasis of pancreatic cancer in vivo. EZH2 has a crucial role in tumor growth and liver metastasis of pancreatic cancer.

Flavonoids of Herba Epimedii regulate osteogenesis of human mesenchymal stem cells through BMP and Wnt/beta-catenin signaling pathway.

Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, beta-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/beta-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/beta-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.

Total flavonoids of Herba Epimedii improves osteogenesis and inhibits osteoclastogenesis of human mesenchymal stem cells.

INTRODUCTION: In China Herba Epimedii is one of the most common herbs that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD). The present study was aimed at investigating the effect of flavonoids of Herba Epimedii (HEF) on osteogenesis in human MSCs. METHODS: The human bone marrow-derived MSCs (BM-MSCs) were isolated and their osteogenic differentiation was evaluated by their alkaline phosphatase (ALP) activities and level of mineralization. After treating with total flavonoids during osteogenic differentiation process, differential mRNA expression was examined by RT-PCR. RESULTS: The total time needed for osteogenic differentiation of BM-MSCs was significantly shortened by adding HEF. Up-regulation of mRNA expression by HEF was observed for several marker genes and osteogenic regulators. HEF was also found to inhibit osteoclastogenesis of MSCs by enhancing the ratio OPG/RANKL. CONCLUSIONS: Our study demonstrated that the HEF could improve osteogenic differentiation and inhibit the osteoclast differentiation of BM-MSCs concurrently.