Biomedical subjects

Demin Zhou

Publications and source records attributed to Demin Zhou.

4 recordsLinked to original sources

PubMed2006

A new inducible RNAi xenograft model for assessing the staged tumor response to mTOR silencing.

Human xenograft tumor models are widely used for efficacy evaluation of potential cancer targets. siRNA is usually stably introduced into tumor cells prior to transplantation. However, silencing of the cancer therapeutic target usually results in reduced cell growth/survival in vitro and/or failure to establish tumors in vivo, thus hindering tumor response-based efficacy evaluation. The present study explored a new tumor response model based on regulated RNAi, which is more relevant from a clinical standpoint. As a proof of principle, an inducible lentiviral RNAi vector was used to silence the known cancer therapeutic target mTOR upon induction with Doxycycline (DOX). The responses to DOX-induced mTOR silencing were tested both in vitro and in vivo for prostate cancer PC3 models. Significant reduction in cancer cell survival was observed due to cell cycle arrest and apoptosis when mTOR silencing was induced in vitro. mTOR silencing also caused tumor regression for the early-staged PC3 tumors (100% tumor regressed and 45% became tumor-free). The advanced-staged tumors also demonstrated significant responses (100% regressed). Therefore, our results demonstrate the powerful utility of this new inducible xenograft tumor model for efficacy evaluation of cancer targets, and it provides a direct in vivo efficacy validation of mTOR as a cancer therapeutic target.

Animals↗

PubMed2006

RNA interference and potential applications.

RNA interference (RNAi) is the process of using specific sequences of double-stranded RNA (dsRNA) to knock down the expression level of sequence-homologous genes. Such ability of small interfering RNA (siRNA) in mammalian cells will undoubtedly revolutionize the study of functional genomics, the discovery of drug targets and even the treatment of human diseases. In this review we briefly describe the history of RNAi discovery, the RNAi mechanism and the general guideline for siRNA design as well as various methods for siRNA production and delivery. We also introduce the potential applications of siRNA, inducible siRNA and siRNA library in speeding up basic biomedical research and in acting as potential therapeutic agents for treatment of numerous human diseases.

Animals↗

PubMed2004

Expression, purification, and characterization of human malonyl-CoA decarboxylase.

The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.

Amino Acid Sequence↗

PubMed2003

An approach to genomewide screens of expressed small interfering RNAs in mammalian cells.

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3' terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-kappaB signaling pathway.

Animals↗