Ming-Liang He

MiR-26a Inhibits Cell Growth and Tumorigenesis of Nasopharyngeal Carcinoma through Repression of EZH2

Several microRNAs (miRNA) have been implicated in nasopharyngeal carcinoma (NPC), a highly invasive and metastatic cancer that is widely prevalent in southern China. In this study, we report that microRNA miR-26a is commonly downregulated in NPC specimens and NPC cell lines with important functional consequences. Ectopic expression of miR-26a dramatically suppressed cell proliferation and colony formation by inducing G(1)-phase cell-cycle arrest. We found that miR-26a strongly reduced the expression of EZH2 oncogene in NPC cells. Similar to the restoring miR-26 expression, EZH2 downregulation inhibited cell growth and cell-cycle progression, whereas EZH2 overexpression rescued the suppressive effect of miR-26a. Mechanistic investigations revealed that miR-26a suppressed the expression of c-myc, the cyclin D3 and E2, and the cyclin-dependent kinase CDK4 and CDK6 while enhancing the expression of CDK inhibitors p14(ARF) and p21(CIP1) in an EZH2-dependent manner. Interestingly, cyclin D2 was regulated by miR-26a but not by EZH2, revealing cyclin D2 as another direct yet mechanistically distinct target of miR-26a. In clinical specimens, EZH2 was widely overexpressed and its mRNA levels were inversely correlated with miR-26a expression. Taken together, our results indicate that miR-26a functions as a growth-suppressive miRNA in NPC, and that its suppressive effects are mediated chiefly by repressing EZH2 expression.

microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, we identified miR-146b as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. We showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, we discovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, our findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

Lentivirus-mediated RNA interference targeting enhancer of zeste homolog 2 inhibits hepatocellular carcinoma growth through down-regulation of stathmin†

Enhancer of zeste homolog 2 (EZH2) has been shown to be overexpressed in hepatocellular (HCC). We investigated the potential role of EZH2 in HCC tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting EZH2 as a form of HCC treatment. Lentivirus‐mediated RNAi was employed to knock‐down EZH2 expression in human hepatoma cells to study the function of EZH2 in tumorigenesis and evaluate the treatment efficacy. Lentivirus‐mediated RNAi effectively reduced EZH2 expression. Suppression of EZH2 in HCC cells significantly reduced their growth rate in vitro and markedly diminished their tumorigenicity in vivo. Moreover, in a mice model of established large‐sized HCC, we showed that intratumor injection of lentiviral (Lenti)‐shRNA (short hairpin RNA) or siRNA (small interfering RNA) targeting EZH2 produced significant tumor regression. To understand its molecular mechanism of action, we employed proteomic profiling technique and found that stathmin 1 is the downstream target of EZH2, as Lenti‐shEZH2 treatment decreased stathmin protein expression, and ectopic overexpression of stathmin prevented Lenti‐shEZH2 mediated tumor growth inhibition. Conclusion: Results from our study suggested for the first time that EZH2 plays a key role in HCC tumorigenesis, and is a novel therapeutic target for HCC. (HEPATOLOGY 2007;46:200–208.)

MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells

MicroRNAs (miRNAs) are non-protein-coding RNAs that function as post-transcriptional gene regulators. Recent evidence has shown that miRNA plays a pivotal role in the development of many cancers including glioma, a lethal brain cancer. We have recently compared the miRNA expression profiles between normal brain and glioma tissues from Chinese patients by miRNA microarray and identified a panel of differentially expressed miRNAs. Here, we studied the function of one miRNA, miR-15b, in glioma carcinogenesis and elucidated its downstream targets. Over-expression of miR-15b resulted in cell cycle arrest at G0/G1 phase while suppression of miR-15b expression resulted in a decrease of cell populations in G0/G1 and a corresponding increase of cell populations in S phase. We further showed that CCNE1 (encoding cyclin E1) is one of the downstream targets of miR-15b. Taken together, our findings indicate that miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related molecules.

Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16INK4A

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 p16INK4A, 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 p16INK4A 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, p16INK4A.

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.

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.

The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat.

Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had been proposed as a powerful alternative method of cancer therapy. The present study was aimed to evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was evaluated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts using the combined treatment. These results demonstrated that the combined treatment generated additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising therapeutic strategy in treating glioma.

Ascorbic acid inhibits ROS production, NF-κB activation and prevents ethanol-induced growth retardation and microencephaly

In this study, we established an embryo model to study the effects of ethanol on fetal development. When embryos of Xenopus laevis (the African clawed frog) were exposed to ethanol, the resultant tadpoles had significantly reduced brain sizes (microencephaly) and retarded growth rates. These effects, similar to those observed in human fetal alcohol syndrome (FAS), were dose- and time-dependent. We further showed that the antioxidant ascorbic acid (vitamin C) could inhibit the ethanol-induced reactive oxygen species (ROS) production and NF-kappaB activation and protect the ethanol-treated embryos against microencephaly and growth retardation. These results suggest the involvement of NF-kappaB and oxidative stress in ethanol-mediated developmental defects, and the potential use of ascorbic acid as a new and effective protective agent for FAS.