Li-Sheng Wang

Overexpression of microRNA-29b induces apoptosis of multiple myeloma cells through down regulating Mcl-1

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids (ncRNAs), which regulate gene expression by targeting mRNAs for translational repression and degradation. Several lines of evidences have indicated that miRNAs act as tumor suppressors and oncogenes. However, the role of miRNAs in pathogenesis of multiple myeloma (MM) remains unclear. In this study, we examined the profile of miRNA expression of primary MM cells, using miRNA microarray and quantitative real-time polymerase chain reaction (qPCR) techniques. These results showed that in the bone marrow specimens analyzed, miRNA-29b was significantly downregulated. Similar results were also observed in human myeloma cell lines (HMCLs). Adenovirus-mediated overexpression of miR-29b induced apoptosis and elevated caspase-3 activation in HMCLs. Using a bioinformatics approach, we found a perfect complementarity between miRNA-29b and the 3UTR of myeloid-cell-leukemia 1(Mcl-1). It is further confirmed that miRNA-29b downregulated the level of Mcl-1 without effect on the mRNA level using both qRT-PCR assays and Western blot analyses. Moreover, we observed that enforced miR-29b expression by using a retarget miRNA-29b expression vector (Ad5F11p-miR-29b) could induce apoptosis and elevate caspase-3 activation in HMCLs. Our results also indicated that miRNA-29b-induced apoptosis acted antagonistically with IL-6 in HMCLs. These findings suggest that miRNA-29b may play an important role in MM as a tumor suppressor.

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis.

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.

Beclin1 inhibition promotes autophagy and decreases gemcitabine–induced apoptosis in Miapaca2 pancreatic cancer cells

BackgroundBeclin1 is a well-known key regulator of autophagy, which is also a haploinsufficient tumor suppressor. Current studies revealed that down-regulation or monoallelic deletions of Beclin1 were frequently found in various cancers. The purpose of this study was to investigate the effects of Beclin1 inhibition on autophagy and Gemcitabine-induced apoptosis of pancreatic cancer cells.MethodsBeclin1 expression was inhibited by siRNA transduction and gene expression was determined by Real-time PCR and Western blot. The effects of Beclin1 inhibition on autophagy and Gemcitabine-induced apoptosis of Miapaca2 cells were analyed through LC3 expression, cell viability, cell cycle and apoptosis by using Western blot.ResultsWe observed that Beclin1 silence promoted microtubule-associated protein 1 light chain 3-II (LC3-II) protein formation and increased punctate fluorescent signals in Miapaca2 cells transfected with green fluorescent protein (GFP)-tagged LC3. Beclin1 inhibition showed a greater suppressive effect on Gemcitabine-induced apoptosis of Miapaca2 cells.ConclusionOur data suggested that Beclin1 silence not only up-adjusted autophagy process, but also played an important role in the regulation of apoptosis. Beclin1 inhibition could inhibit apoptosis signaling induced by Gemcitabine in Miapaca2 cells.

Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Background Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells. Methods MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed. Results The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs. Conclusion PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs.

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn

Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion‐exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [3H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)‐based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30‐kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine‐rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Conclusion: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration. (HEPATOLOGY 2008;47:986–995.)

MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal.

MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases.

The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats

Background:u2002 Hepatopoietin Cn (HPPCn) is a member of the leucine‐rich acidic nuclear protein family (LANP), and studies of partially hepatectomized (PH) mice show that levels of HPPCn mRNA increase following liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo.

SENP1 inhibition induces apoptosis and growth arrest of multiple myeloma cells through modulation of NF-κB signaling

SUMO/sentrin specific protease 1 (Senp1) is an important regulation protease in the protein sumoylation, which affects the cell cycle, proliferation and differentiation. The role of Senp1 mediated protein desumoylation in pathophysiological progression of multiple myeloma is unknown. In this study, we demonstrated that Senp1 is overexpressed and induced by IL-6 in multiple myeloma cells. Lentivirus-mediated Senp1 knockdown triggers apoptosis and reduces viability, proliferation and colony forming ability of MM cells. The NF-κB family members including P65 and inhibitor protein IkBα play important roles in regulation of MM cell survival and proliferation. We further demonstrated that Senp1 inhibition decreased IL-6-induced P65 and IkBα phosphorylation, leading to inactivation of NF-кB signaling in MM cells. These results delineate a key role for Senp1in IL-6 induced proliferation and survival of MM cells, suggesting it may be a potential new therapeutic target in MM.

A novel rich source of human mesenchymal stem cells from the debris of bone marrow samples

The debris from human bone marrow (BM) samples is generally filtered out and discarded prior to isolation of mesenchymal stem cells (MSCs). The purpose of this study is to develop a method to harvest MSCs from the debris and investigate their biological characteristics compared with the marrow counterparts. The BM tissue fragments were digested with collagenase and this treatment yielded mononuclear cells half to those from the corresponding filtered BM. The frequencies of colony-forming unit-fibroblast in these two cell populations were not significantly different. MSCs of two origins exhibited similar morphological and phenotypic features. Fluorescent dye-dilution assay showed that they grew at comparable rates both in the primary and passaging cultures. Further, they could be induced into osteoblasts, chondroblasts and adipocytes, as revealed by histological and molecular examinations. Thus, BM tissue fragments may serve as a new source of MSCs in the settings of bench experiments and clinical trials.

SENP1 regulates hepatocyte growth factor-induced migration and epithelial-mesenchymal transition of hepatocellular carcinoma

The deregulation of HGF/c-Met signaling is implicated in epithelial-mesenchymal transition (EMT) and progress of hepatocellular carcinoma (HCC). However, the epigenetic mechanisms that HGF/c-Met regulates EMT and metastasis of HCC cells are less explored. In this study, we demonstrated that HCC cells express a high level of SUMO/sentrin-specific protease 1 (Senp1) which is induced by HGF/c-Met signals. Lentivirus-mediated small hairpin RNA (shRNA) transduction results in Senp1 silence in HCC cells. Senp1 silence reduces the HGF-induced proliferation and migration of HCC cells. Senp1 inhibition also induces HCC cell apoptosis and growth arrest. Furthermore, Senp1 knockdown inhibits epithelial-to-mesenchymal transition, with increase of E-cadherin and ZO-1 expression, decrease of fibronectin and N-cadherin expression. The EMT-related transcription factor Zeb1 was SUMO-modified and decreased in Senp1-silenced HCC cells. These results delineate that senp1 might play an important role in the regulation of HGF-induced invasion and migration of HCC cells.