Expression profiling and biochemical analysis of the human SPOP and SPOPL genes

• Main Authors: Sheng-Wen Chen, 陳生文
• Other Authors: Kong-Bung Choo
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/44780720543688114705

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 98 === SPOP and SPOPL are highly conserved proteins ubiquitously expressed in mammals and belong to the TD/POZ gene family. SPOP has been shown to participate in protein ubiquitination while the functions of SPOPL are unknown. In order to further understand SPOP and SPOPL, we aimed to investigate the expression profiles of SPOP and SPOPL in major human tissues and to perform biochemical analysis for the SPOP and SPOPL proteins. Based on NCBI UniGene Expressed Sequence Tag (EST) data, SPOP and SPOPL are ubiquitously expressed in major human tissues and the expression of SPOP is generally higher than SPOPL. On differential expression of SPOP and SPOPL in normal and tumor tissues, however, SPOP is up-regulated in pancreatic tumor for 6.33-fold and down-regulated in uterus tumor for 6.33-fold. On the other hand, SPOPL is up-regulated in ovarian tumor while SPOPL had not detected in normal pancreas and silenced in adrenal gland and bladder tumors. The results show that expression of SPOP and SPOPL are generally down-regulated in tumor tissues. To experimentally confirm the expression profiles, real-time RT-PCR and western blot analysis were performed to determine SPOP and SPOPL mRNA and protein expression levels in four cancer types in eight different cancer cell lines. The mRNA levels of SPOP and SPOPL were largely different in the eight cell lines and the protein levels also showed difference indicating post-transcriptional regulation. In fluorescence examination of SPOP-EGFP or SPOPL-EGFP fusion proteins in transfected cells, SPOP was located in the nucleus as speckles while the SPOPL was found to be diffused in the nucleus. Over-expression SPOP-EGFP or SPOPL-EGFP expression plasmid induced morphological changes in HEK293T and HeLa cells. Taken together, the results show differential expression of SPOP and SPOPL in normal and cancer cells suggesting post-transcriptional regulation. Over-expression of SPOP or SPOPL induces the morphological changes in HEK293T and HeLa cells suggesting that SPOP and SPOPL may affect cell physiology of cells. Due to high identity of SPOP and SPOPL, we predict that SPOP and SPOPL share complementary biological functions.