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The Effect of Haxl Gene and Its Specific Small Interference RNAs on the Apoptosis of 293 Cells

Author: LongLin
Tutor: LiuLi
School: Beijing Union Medical College
Course: Pathogen Biology
Keywords: Haxl siRNA 293 cells Apoptosis
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 14
Quote: 0
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Abstract


The Hax1 (HS-1-associated protein X-1, Hax1) initially in 1997 by Suzuki et al. Using the yeast two-hybrid technology found with HS1 (hematopoietic lineage cell-specific protein 1, HS1), a protein interaction. The current study proved Hax1 is a multifunctional protein able found Hax1 the presence in human tissue, and its innovative features, including anti-apoptotic cell transfer and endocytosis and mRNA 3'-UTR binding, and endoplasmic reticulum and nuclear membrane compared to the higher the amount of its expression in the mitochondria. Because Hax1 and a variety of cellular or viral protein interactions, then participate in different signaling pathways and cellular processes, so it is with the occurrence of the disease in humans has a close relationship. Hax1 gene deletion or overexpression of some genetic diseases or systemic disease caused by the body's changes, such as cancer, psoriasis, systemic lupus (SLE), red and white, polycystic kidney disease, congenital neutropenia psychosis However, the mechanism of action is still not very clear, therefore further explore Hax1 function of popular concern. Of this study by RNAi construct Hax1 gene-specific small interfering RNA (sihax), to detection sihax on Hax1 gene expression inhibition, apoptosis research Hax1 Haxl siRNAs in 293 cells, preliminary study Hax1 proapoptotic function. The research topics include: 1) amplification Hax1 gene coding sequence of full-length, constructed the eukaryotic expression vector pEGFP-C1-Hax1. The 2) Select the Hax1 gene siRNA two target sites, to build Hax1 siRNA the expression vector pBS/U6-siHaxA and pBS/U6-siHaxB. 3) detection pBS/U6-siHaxA and pBS/U6-siHaxB on Hax1 gene expression. 4) the impact of gene expression. The detection Hax1 gene specific siRNAs to the EGFP-Hax1 fusion 5) the detection Hax1 its siRNA 293 apoptosis. This experiment, as a template pCMV-Myc-Hax1 the PCR amplified Hax1 gene coding sequence of full-length, was cloned into the eukaryotic expression vector pEGFP-C1; choose the human source Hax1 gene siRNA target sites (300-320 and 860 -880), according to published siRNA target sequence design principles, as well as the NCBI Hax1 the cDNA sequence, BLAST analysis and synthesis of two specific target sequences, cloned into pBS/U6 to build PBS/U6-siHaxA and pBS/U6 the-siHaxB expression vector; pCMV-Myc-Hax pEGFP-C 1-Hax 1 pBS/U6-siHaxA and pBS/U6-siHaxB after different grouping transfected 293 cells, EGFP-Hax1 fusion was observed under an inverted fluorescence microscope the level of gene expression to determine Hax1 in 293 cell positioning, and observe the impact the gene specificity siRNAs for Hax1 of the EGFP-Hax1 fusion gene; flow cytometry Annexin V-EGFP/PI double staining the proportion of 293 apoptosis; through the detection Hax1 mRNA expression levels in 293 cells conclusion by RT-PCR and Western-blot method: In this experiment, was successfully constructed the eukaryotic expression vector pEGFP-C1-Hax1 293 cells overexpressing EGFP-Hax1 fusion The protein was mainly distributed in the cytoplasm. By inverted fluorescence microscopy and flow cytometry to detect the expression of green fluorescent protein, and RT-PCR and Western-blot to detect the expression of endogenous Hax1 display successfully build two Hax1 of siRNA eukaryotic expression vector (pBS/U6-siHaxA and pBS/U6-siHaxB) inhibitory effect of significantly stronger than siHaxA, siHaxB inhibition of target genes. And Haxl siRNA inhibition of Expression of endogenous Hax1 in can significantly increase the proportion of late apoptosis, and significant inhibition of early apoptotic cells process Based on the above results, we believe that different Hax1 play a role in the different stages of apoptosis, the complexity of its function sexual performance in two completely opposite role in the same tissue. Inhibition of the target gene target sequence conserved Hax1 siRNA correlation the Hax1 genes involved in apoptosis in cells related to a variety of regulatory process, this study to further investigate the Hax1 laid the foundation.

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