| Summary: | 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, Speckle-type POZ protein, is a highly conserved protein found
ubiquitously in human tissues. SPOP is a TRAF- and POZ-domain protein and a
member of the TD/POZ protein family. SPOP has been shown to down-regulate the
pancreatic transcription factor, PDX-1, linking SPOP to pancreas development. SPOP
also participates in protein ubiquitination and degradation. Since there are few studies
on the regulation of SPOP expression, this study aimed to investigate the expression
profiles of SPOP in normal and cancerous human tissues.
Real-time RT-PCR and western blot were performed to determine SPOP
expression in normal colon and five colorectal cancer (CRC) cell lines. The results
indicate that SPOP mRNA was down-regulated but protein expression was
up-regulated in the CRC cell lines tested. To determine possible causes of SPOP
differential expression in CRC cell lines, promoter sequence fidelity was examined
and no mutations in the SPOP promoter were found in the five CRC cell lines. We
next focused on the SPOP methylation profiles in the promoter and upstream
regulatory sequence which contained five CpG islands (CGIs). Three CGIs are
located around exon 1 and in the basal promoter; the remaining two CGIs are in the
upstream sequence overlapping with Alu transposable elements. Methylation-specific
PCR and bisulfite sequencing were performed. Promoter of SPOP was found to be
hypomethylated while upstream Alu-CGIs were hypermethylated. The promoter
activity was also tested in luciferase assays. The results show that the SPOP promoter
incorporates the three CGIs around exon 1 and the upstream CGIs. In bioinformatics
analyses, eight SPOP mRNA variants with different 5’-UTR exons were found. All
these mRNA variants contained three upstream ORFs with good Kozak consensus
sequence. It is unclear if SPOP expression is affected by these alternative mRNAs.
iv
Five microRNA (miRNA) target sites were found in the 3’-UTR which was cloned in
different lengths for luciferase assays. The results show that the hsa-548c-3p target
site in the 3’-UTR could be miRNA-regulated.
Taken together, the results show differential of SPOP expression in CRC cell
lines both at the transcriptional and translational levels which may be regulated by
miRNA. Further studies of miRNA regulation in SPOP expression and the biological
effects of differential SPOP expression in CRC cells are in progress.
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