| Summary: | 碩士 === 臺灣大學 === 病理學研究所 === 95 === The annexin (ANX) family is the largest single category of eukaryotic calcium-binding proteins without EF hands. The ANXs share a similar structure characterized by the presence of four or eight repeats of a 70-amino acid motif and a highly variable N-terminal end. The ANXs play an important role in a broad range of physiological processes, including anti-coagulation,endocytosis, exocytosis, immunosuppression, differentiation, proliferation, and inhibition of calcium channels, phospholipase A2, and protein kinase C. There are at least 13 human ANX members, play different roles in tumorigenesis, as an oncogene or a tumor suppressor gene.
ANXA10, a novel member of the ANX family, has several distinct features, including codon deletion in conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats. The loss of type II calcium-binding sites is a fundamental deviation from ANX structure conservation and undoubtedly has functional consequences.
Previously, our lab identified a novel transcript isoform of ANXA10, the ANXA10S. We found the mRNA expression level of ANXA10S gene was down-regulated in hepatocellular carcinoma, and the downregulation correlated with vascular invasion, early recurrence, and poor prognosis.
In this study we tried to determine the biological function of ANXA10S and ANXA10. Moreover we tried to prove the existence of the endogenous ANXA10S protein.
Our results about ANXA10S are as follows:
(1) On colony formation assay, the Hep3B cells transfected with ANXA10S formed fewer colonies as compared with the vector control cells.
(2) In vivo tumorigenesis assay, control cells formed larger tumor than ANXA10S overexpression cells. Besides, Tumor tissue of HuH-7 control cells showed topical morphology of cancer cells. But those characteristics did not observe in tumor tissue of ANXA10S expressed HuH-7 cells
(3) We used liver tissue protein lysates for immunoprecipitation and protein concentration to detect the endogenous ANXA10S protein. Unfortunately, we could not detect ANXA10S protein expression in liver tissue.
Our results about ANXA10 are as follows:
(1) On colony formation assay, the HA22T, HCC36 and Hep3B cells transfected with ANXA10 formed fewer colonies as compared with the vector control cells.
(2) On MTT assays, the RNAi knockdown HeLa cells, which expressed ANXA10, and control cells did not differ in cell proliferation in 5%, 7%, and 10% serum medium, but the RNAi knockdown cells showed increased cell growth in lower serum (2%) and serum free media.
(3) On the soft agar assay, the RNAi knockdown HeLa cells formed more numerous and larger colonies than the control cells.
(4) By western blot analysis, we found that the down-regulation of ANXA10 led to increased amount of p-MEK, p-ERK and c-myc but not total MEK and ERK.
(5) From microarray assay data we found several growth related gene expression would change after ANXA10 knockdown.
In conclusion, even if the endogenous ANXA10S protein was hard to detect. We still believed that ANXA10S and ANXA10 might have tumor suppressor potential.
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