Cloning, Expression And Preliminary Function Study Of DRR1 Gene

Xenopus laevis was recently determined as a novel study platform ofgene function in recent years. In this study, we cloned a cDNA, which ishomologous to human down-regulated in renal carcinoma (DRR1) gene, byRT-PCR from the total RNA of frog oocytes. The cDNA product was named asxDRR1. Sequence analysis of the DRR1 protein indicated that the xDRR1 shared74ï¼…identity human DRR1 and 66ï¼…with murine DRR1. The xDRR1 cDNAwasused to construct expression plasmids pEGFP-N1/xDRR1 andpcDNA3.1(+)/xDRR1. RT-PCR revealed that xDRR1 gene was expressed in allstages of the early development embryo and all kinds of tissues. Cellular locationanalysis of the xDRR1 protein revealed that the expression of the xDRR1 genewas in nuclei by transfecting A549 cells with plasmid pEGFP-N1/xDRR1 usingliposome. As a coiled-domain-containing nuclear protein, the DRR1 protein mayinteract with other proteins or DNA to regulate gene transcription and signaltransduction and thus be involved in development regulation. Study in lungcancer A549 cells, which loses the DRR1 expression, showed that expression ofthe xDRR1 could suppress the cell growth by transfecting A549 cells withplasmid pcDNA3.1(+)/xDRR1. Further investigation remains needed.Human down-regulated in renal cell carcinoma 1 is widely expressed innormal tissues but dramatically reduced or even undetectable in a number ofdifferent cancer cell lines and primary tumor types. In this work, human DRR1cDNA was cloned into the pQE30 vector for fusion protein expression with sixhistidine residues in Escherichia coli strain BL21(DE3). A soluble protein with a molecular mass of approximately 19 kDa on SDS-PAGE that matches theexpected rDRR1 molecular weight was obtained. The soluble and insolubleexpression of recombinant protein DRR1(rDRR1)was temperature-dependent.The expression rDRR1 was in soluble and insoluble forms at 37℃and about80ï¼…of total rDRR1 was soluble at 37℃, while rDRR1 was almost exclusivelyexpressing in soluble form at 20℃. The expressed rDRR1 at 20℃wasaffinity-purified by Ni²⁺-charged resin under native conditions. The purifiedprotein was further identified by electrospray ionization-mass spectrometry(ESI-MS). The purified recombinant protein rDRR1 was further used to raiseanti-human DRR1 polyclonal antibodies, which were suitable to detect both therecombinant exogenous DRR1 and the endogenous DRR1 from tissues and cellsby immunoblotting and immunohistochemistry. The purified rDRR1 and ourprepared anti-human DRR1 polyclonal antibodies may provide useful tools forfuture biological function studies for DRR1. RNA interference (RNAi) technology is emerging as a very potent toolto obtain a cellular knochdown of a desired gene. Polo-like kinase 1(PLK1) actsas a key cell cycle regulator in different stages of mitosis and modulates thespindle checkpoint at metaphase-anaphase transition. Overexpression of PLK1 isobserved in virous human tumors. PLK1 is one of the negative prognostic factorsin patients suffering diverse cancers.In this work we used vector-based RNAi to inhibit PLK1 expression in lungcancer in vitro and in vivo. A549 lung cancer cells were transfected with threeplasmids carrying U6 promotor-driven shRNAs against human PLK1 mRNA(pGPU6/PLK1-184, pGPU6/PLKI-1412 and pGPU6/PLKl-1424) or negativecontrol plasmid (pGPU6/NC). Semi-quantitative RT-PCR and western blotanalysis were used to measure PLK1 mRNA and protein expression, respectively,in transfected cultured cells. We also used a xenograf mouse model to determinewether an RNAi-based strategy that used short short haipin RNAs (shRNAs) tosuppress PLK1 eapression could inhibit tumor growth in vivo. Nude mice bearingxenograf tumor were injected with shRNA plasmids into tail vein. Plasmids wereencapsulated in liposome to prolong its half life in murine blood. The xenograftumor growth was assessed. Immunohistochemical staining was used to measurePLK1 protein expression and in xenograf tumors. We further used one of thethree PLK1 shRNAs for treatment of xenograf tumor in combination withgemcitabine. We demonstrated that transduction with these PLK1-shRNA plasmids,dramatically suppressed the expression of PLK1 gene in human lung cancer cellline A549. As a consequence, proliferation of A549 cells transfected PLK1shRNA was lower than that of cells transfected with negative control shRNA (NCshRNA) in vitro. Most importantly, these vector-based PLK1 shRNAs result ininhibition of tumor growth rate, suppression of PLK1 protein expression, and anincreased rate of apoptaosis. In mice with human xenograf tumor, RNAi targetingposition 1424 of PLK1, namely PLK1-1424 shRNA enchances lung cancerchemosensitivity to gemcitabine in vivo.In conclusion, shRNA-based gene silencing may be an effective gene therapystrategy of treating PLK1-overexpressed cancers, including non-small-cell lungcancer. The application of shRNA-mediated gene therapy approach can enhancethe efficacy of anti-tumor nucleoside analog, suggesting that vector-based RNAiwill be a feasible therapeutic means for cancer combinant therapy in future.