Annexin A10與其變異物Annexin A10S在癌症細胞之角色

Abstract

Annexin 家族是真核細胞結合鈣離子沒有 EF hands 的最大一類蛋白，Annexin家族擁有一個由70個胺基酸所組成的相似結構，每個成員擁有四或八個這樣的重複結構，及一個高度變異的N’端。整個家族可參與廣泛的生理作用，包括抗凝血、胞吞作用、胞吐作用、免疫抑制、細胞分化、細胞分裂及抑制鈣離子通道、phospholipase A2和protin kinase C等。目前至少有13個Annexin成員被發現，他們對於腫瘤的形成扮演著不同的角色，可能是個致癌基因也可能是個抑癌基因。 Annexin A10 (ANXA10) 是Annexin家族的新成員，擁有幾個獨特的特徵,包括在第三重複序列的codon丟失，第四重核心重複序列第二型鈣離子結合區的不正常缺失，明顯偏離annexin結構應保存的特性，無疑在功能上會有影響。 先前，我們實驗室選殖到了一個新的ANXA10的異構物，稱之為ANXA10S。我們發現ANXA10S mRNA 在人類肝癌細胞中表現量下降與腫瘤侵犯轉移，再發性與預後不良息息相關。 在這篇論文裡我們試著去探討ANXA10S 與ANXA10 的生物功能，並且也嘗試去偵測內生性的ANXA10S蛋白。 關於ANXA10Su部分的實驗結果如下： （1）在colony formation分析中，轉染ANXA10S的Hep3B細胞株，相對於控制組細胞會形成較少的細胞群落。 （2）在動物腫瘤細胞形成實驗中，皮下控制組細胞會形成較大的腫瘤，並且擁有典型惡性腫瘤的型態，然而這些特徵是在轉染ANXA10S的腫瘤內所無法觀察到的。 （3）我們嘗試利用抗體免疫沉澱法以及蛋白質濃縮的方式，去偵測肝臟組織中內生性的ANXA10S蛋白，但不幸的，到目前為止我們始終無法成功的偵測到內生性的ANXAS蛋白。 關於ANXA10部分的實驗結果如下： （1）在colony formation分析中，轉染ANXA10的 HA22T、HCC36與Hep3B細胞株，相對於控制組細胞會形成較少的細胞群落。 （2）在MTT分析中，利用RNAi系統降低ANXA10表現的HeLa細胞株與控製組細胞在5%、7%與10%血清培養基中，細胞增殖速度並沒有顯著差異，而在低濃度的血清培養基（2%）與無血清的培養基中，降低ANXA10表現的HeLa細胞增殖速度會較快。 （3）在soft agar實驗中，降低ANXA10表現的HeLa細胞株能在缺乏依附的情況下，形成較大細胞群落。 （4）利用西方墨點法，我們發現降低ANXA10的表現量，會伴隨著p-MEK、p-ERK與c-myc表現量的增加，但不影響MEK與ERK的表現。 （5）透過microarray資料分析，我們發現一些與生長相關的基因表現量，會隨著ANXA10的表現量降低而有所改變。 綜合而言，即使到目前為止我們還是無法成功偵測到內生性的ANXA10S蛋白，但我們相信ANXA10S與ANXA10具有抑制癌細胞生長的潛力。

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.