Marie C.M. Lin

Microsomal triglyceride transfer protein

Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention concerns further novel methods for lowering serum liquid levels and therapeutic agents having such activity.Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention concerns further novel methods for lowering serum liquid levels and therapeutic agents having such activity.

Analysis of MiR-195 and MiR-497 expression, regulation and role in breast cancer

Purpose: To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer. Experimental Design: The expression patterns of miR-195 and miR-497 were initially examined in breast cancer tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues. Results: MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer. Conclusions: Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets. Clin Cancer Res; 17(7); 1722–30. ©2011 AACR.

miR-200a-mediated downregulation of ZEB2 and CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth, migration and invasion.

Nasopharyngeal carcinoma (NPC), a highly metastatic and invasive malignant tumor originating from the nasopharynx, is widely prevalent in Southeast Asia, the Middle East and North Africa. Although viral, dietary and genetic factors have been implicated in NPC, the molecular basis of its pathogenesis is not well defined. Based on a recent microRNA (miRNA) microarray study showing miR-200 downregulation in NPC, we further investigated the role of miR-200a in NPC carcinogenesis. We found that the endogenous miR-200a expression level increases with the degree of differentiation in a panel of NPC cell lines, namely undifferentiated C666-1, high-differentiated CNE-1, and low-differentiated CNE-2 and HNE1 cells. By a series of gain-of-function and loss-of-function studies, we showed that over-expression of miR-200a inhibits C666-1 cell growth, migration and invasion, whereas its knock-down stimulates these processes in CNE-1 cells. In addition, we further identified ZEB2 and CTNNB1 as the functional downstream targets of miR-200a. Interestingly, knock-down of ZEB2 solely impeded NPC cell migration and invasion, whereas CTNNB1 suppression only inhibited NPC cell growth, suggesting that the inhibitory effects of miR-200a on NPC cell growth, migration and invasion are mediated by distinct targets and pathways. Our results reveal the important role of miR-200a as a regulatory factor of NPC carcinogenesis and a potential candidate for miRNA-based therapy against NPC.

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.)

Loss of Brain-enriched miR-124 MicroRNA Enhances Stem-like Traits and Invasiveness of Glioma Cells

Background: miR-124 is a brain-enriched microRNA that has been shown to be down-regulated in glioma. Results: miR-124 inhibits glioma cell invasion and tumorigenicity and reduces neurosphere formation, CD133+ cell subpopulation, and stem cell marker expression in part by targeting SNAI2. Conclusion: Loss of miR-124 enhances the stem-like traits and invasiveness of glioma cells via SNAI2 signaling. Significance: Therapeutic strategies against glioma can be developed by restoring the level of miR-124. miR-124 is a brain-enriched microRNA that plays a crucial role in neural development and has been shown to be down-regulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here, we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and in all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often up-regulated in glioma, as a direct functional target of miR-124. Because SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma cell stem-like traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133+ cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2.

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.

Arsenic trioxide induces apoptosis in human gastric cancer cells through up‐regulation of P53 and activation of caspase‐3

Arsenic trioxide (As2O3) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As2O3 in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN‐28 were treated with various concentrations (0.1 to 100 μM) of As2O3 for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21waf1/cip1, c‐myc, bcl‐2 and bax were detected by Western blotting. Effects of As2O3 on caspase‐3 protease activity, its protein concentration and cleavage of poly(ADP)‐ribose polymerase (PARP) were also studied. As2O3 inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As2O3 induced apoptosis in AGS cells in a concentration‐ and time‐dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co‐incubation with p53 anti‐sense oligo‐nucleotide suppressed As2O3‐induced intracellular p53 over‐expression and apoptosis. As2O3 increased the activity of caspase‐3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD‐fmk and the specific caspase‐3 inhibitor DEVD‐fmk partially suppressed As2O3‐induced caspase‐3 activation and apoptosis. As2O3 inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over‐expression and activation of caspase‐3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.

Revisit complexation between DNA and polyethylenimine — Effect of uncomplexed chains free in the solution mixture on gene transfection

Our revisit of the complexation between anionic DNA and cationic polyethylenimine (PEI) in both water and phosphate buffered saline (PBS) by using a combination of laser light scattering (LLS) and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~3, but the PEI/DNA polyplexes have a high in-vitro gene transfection efficiency only when N:P≥10. Putting these two facts together, we not only conclude that this extra 7 portions of PEI chains are free in the solution mixture, but also confirmed that it is these free PEI chains that substantially promote the gene transfection no matter whether they are applied hours before or after the administration of the much less effective PEI/DNA polyplexes (N:P=3). The uptake kinetics measured by flow cytometry shows that the addition of free PEI leads to a faster and more efficient cellular internalization of polyplexes, but these free PEI chains mainly contribute to the subsequent intracellular trafficking. In contrast, the bound PEI chains mainly play a role in the DNA condensation and protection, leading to a different thinking in the development of non-viral vectors.

Revisit the complexation of PEI and DNA — How to make low cytotoxic and highly efficient PEI gene transfection non-viral vectors with a controllable chain length and structure?

The commercially available branched polyethyleneimine (PEI) with a molar mass of 25 kD (PEI-25K) is an effective in vitro vector to transfer genes, but its cytotoxicity limits its applications in bio-related research. To solve such an efficiency-versus-cytotoxicity catch-22 problem, the disulfide bond has been previously used to link less toxic short PEI chains (2 kD), but previous literature results are controversial. Recently, we found that it is vitally important to remove both carbon dioxide and water in the linking reaction as well as to control the structure of the resultant chains linked by dithiobis(succinimidyl propionate) (DSP). Under a programmable mixing of PEI and DSP, we can use laser light scattering (LLS) to in-situ monitor the linking reaction kinetics in DMSO in terms of the change of the average molar mass (M(w)). Therefore, we were able to withdraw a series of linked PEI chains with different molar masses from one reaction mixture. Two such linked PEI samples (M(w) approximately 7 kD, PEI-7K-L and approximately 400 kD, PEI-400K-L) were used to illustrate the effect of the sample preparation and the chain structure on the in vitro gene transfection and cytotoxicity. Our results reveal that PEI-7K-L is less cytotoxic and more effective in the gene transfection than both PEI-25K and Lipofectamine 2000 in the in vitro gene transfection. However, PEI-400K-L has no gene transfection efficiency even though it is non-toxic.

Discovery of growth hormone-releasing hormones and receptors in nonmammalian vertebrates

In mammals, growth hormone-releasing hormone (GHRH) is the most important neuroendocrine factor that stimulates the release of growth hormone (GH) from the anterior pituitary. In nonmammalian vertebrates, however, the previously named GHRH-like peptides were unable to demonstrate robust GH-releasing activities. In this article, we provide evidence that these GHRH-like peptides are homologues of mammalian PACAP-related peptides (PRP). Instead, GHRH peptides encoded in cDNAs isolated from goldfish, zebrafish, and African clawed frog were identified. Moreover, receptors specific for these GHRHs were characterized from goldfish and zebrafish. These GHRHs and GHRH receptors (GHRH-Rs) are phylogenetically and structurally more similar to their mammalian counterparts than the previously named GHRH-like peptides and GHRH-like receptors. Information regarding their chromosomal locations and organization of neighboring genes confirmed that they share the same origins as the mammalian genes. Functionally, the goldfish GHRH dose-dependently activates cAMP production in receptor-transfected CHO cells as well as GH release from goldfish pituitary cells. Tissue distribution studies showed that the goldfish GHRH is expressed almost exclusively in the brain, whereas the goldfish GHRH-R is actively expressed in brain and pituitary. Taken together, these results provide evidence for a previously uncharacterized GHRH-GHRH-R axis in nonmammalian vertebrates. Based on these data, a comprehensive evolutionary scheme for GHRH, PRP-PACAP, and PHI-VIP genes in relation to three rounds of genome duplication early on in vertebrate evolution is proposed. These GHRHs, also found in flounder, Fugu, medaka, stickleback, Tetraodon, and rainbow trout, provide research directions regarding the neuroendocrine control of growth in vertebrates.