異常的PDGFR-α基因表現對誘發甲狀腺濾泡癌癌化之探討

Abstract

甲狀腺腫瘤約佔所有腫瘤的1%。腫瘤會發生在所有的年齡層，而且會隨著年齡的增加發生率也會增加。女性與男性罹患的比率約3:1。目前，確定可行的診斷方式，是藉著組織學的觀察。但臨床上發現，有些早期的吸引性細胞學檢查，有時是不可信賴的，特別是針對濾泡型的甲狀腺腫瘤。通常濾泡型腫瘤或較少數的乳突型腫瘤，病理學家無法僅以吸引性細胞學檢查的結果，做出正確的診斷。 為了發展可以早期偵測的分子標的，我們首先藉著描繪出在不同的甲狀腺腫瘤組織與細胞株的表現具有差異的基因，從其中尋找具有潛力的腫瘤標的基因。cDNA表現基因陣列(cDNA expression array)的技術，在本研究中用來描繪出在甲狀腺濾泡癌中，差別表現的基因，以顯現適合的腫瘤標誌。針對在濾泡型的甲狀腺腫瘤，表現具有差異的基因，進行進一步的研究，將有助於瞭解，這些基因改變在甲狀腺腫瘤癌化過程中，所扮演的角色。 在利用了cDNA表現基因陣列的技術分析差別表現基因過後，我們發現PDGF-A 與PDGFR-α的mRNA 相較於良性的結節性增生細胞(nodular hyperplasia cells)而言，在甲狀腺濾泡癌細胞株中，具有高度表現。因此，這些結果引起我們的動機，想瞭解是否具有PDGF相關的自分泌活化(autocrine activation)的現象，存在甲狀腺腫瘤細胞中且在癌化過程中，扮演重要角色。PDGFR本身具有激脢活性(kinase activity)，當PDGF 配位蛋白(ligand)結合上受體蛋白(receptor)時，可活化位於細胞內激脢活性。已知PDGFR活化與一些細胞的癌化，具有相關性，包含星狀細胞瘤(astrocytomas),寡樹突神經膠質瘤(oligodendrogliomas),及神經膠質胚胎細胞瘤(glioblastoma)。在cDNA表現基因陣列的分析，我們發現在濾泡癌細胞中，PDGF-A 與PDGFR-α之mRNA，皆會增加表現。之後，利用西方墨點法分析，確認在濾泡癌細胞中，PDGF-A 與PDGFR-α蛋白，也會提升表現。更進一步，觀察PDGFR-α的磷酸化現象，發現利用針對第720位置的酪胺酸磷酸化專一性抗體，在濾泡癌細胞中，可偵測到磷酸化現象。而PDGFR-α所具有的酪胺酸激脢活性，可以被抑制劑tyrphostin AG1295所抑制，而且顯現隨著抑制劑劑量的增加，而提升抑制腫瘤細胞增生的作用。另一方面，在免疫組織呈色實驗(immunohistochemistry)中，結果顯示PDGFR-α蛋白的表現，主要是呈現在甲狀腺濾泡周邊(thyroid follicle )且是與惡性的腫瘤階段(tumor stage)，呈現統計上的相關性。這些發現暗示，藉著PDGFR-α所進行的自分泌活化現象，在甲狀腺細胞的癌化過程中，扮演重要的角色。 進一步為了確認是否能藉著抑制這不正常的訊息傳遞，能夠達到解除癌化的可能性。我們檢驗PDGFR-α siRNA 與 tyrphostin AG1295，這兩種方式，來觀察對抑制CGTH W-1 細胞增生的功效。目前，已知有數種酪胺酸激脢抑制劑，可以阻擋具酪胺酸激脢活性的受體之訊息傳遞，從而抑制不正常的訊息傳遞。這裡我們設計小而具有干擾特性的RNA (short interfering RNAs; siRNA)專門針對PDGFR-α基因，且達到壓制濾泡型的甲狀腺腫瘤細胞(CGTH W-1)增生的效果。即時定量聚合脢連鎖反應(real-time quantitative PCR)、流式細胞儀(flow cytometry)測定、免疫螢光細胞染色(immunofluorescence cell staining)及MTT 測定後，結果顯示送入PDGFR-α siRNA的CGTH W-1細胞，可達到減少細胞中PDGFR-α mRNA 的表現程度。之後，可以達到減少CGTH W-1細胞表面受體的分子數目及最後可以達到抑制細胞增生的效果。而利用non-silencing siRNA所進行的對照組實驗，則不管在PDGFR-α基因的表現或者是細胞增生，皆無顯著影響。 最後，我們比較利用PDGFR-α siRNA 與 tyrphostin AG1295，在抑制CGTH W-1細胞增生的影響，發現利用PDGFR-α siRNA 時，功效的呈現，相對於tyrphostin AG1295，需要多24小時的時間，才能達到顯著的效果;但是它的作用可以達到240小時(第十天)之久。這些發現指示了PDGFR-α siRNA可以是一個有潛力的工具，來抑制不正常的PDGFR-α基因表現。另外也進一步的證明，在甲狀腺細胞癌化過程中，PDGFR-α基因的表現，扮演重要的角色。

Thyroid cancer accounts for about 1% of all cancers. Thyroid cancer occurs at all ages and increases frequently in each decade. The female:male ratio is about 3:1. Definite diagnosis is possible only by histological examination. Fine needle aspiration cytology (FNAC) reports are sometime unreliable, especially for follicular thyroid tumors. Frequently, the pathologist cannot diagnose follicular carcinoma on FNAC or (to a lesser extent) papillary carcinoma. To develop an early detection method by using biological markers, we have first searched the potent tumor markers by outline the differentially gene expressions in various thyroid tissues or thyroid cell lines. The cDNA expression array technology is utilized herein to profile differentially expressed genes from human follicular thyroid carcinoma and reveals new tumor markers. An expression profile of genes that are associated with malignant process of follicular thyroid cancer was further discussed. Further investigation is required to understand the precise relationship between the altered expression of these genes and the malignant process of follicular thyroid cancer. After analysis of differentially gene expression by a cDNA microarray technique, we found that mRNA of PDGF-A and PDGFR-α were highly expressed in thyroid carcinomas but not in nodular hyperplasia cells. These results cause the motive to understand whether PDGF autocrine activation exists in thyroid cells and play a crucial role in carcinogenesis. Platelet-derived growth factor receptor (PDGFR) possesses a kinase activity and can be activated through binding with PDGF. The activation of PDGFR is associated with the carcinogenesis of some cell types, including astrocytomas, oligodendrogliomas, and glioblastoma. In a cDNA microarray analysis, we discovered the over-expressed mRNA of both PDGF-A and PDGF α-receptor in thyroid carcinoma cells. And the elevated protein expressions of PDGF-A and PDGF α-receptor in thyroid carcinoma cells were confirmed by a western blot analysis. Furthermore, the phosphorylation of PDGF α-receptor measured by an antibody against Tyr 720-phosphate was found in thyroid carcinoma cells. The tyrosine kinase activity of PDGF α-receptor was inhibited by tyrphostin AG1295 and showed a dose-dependent inhibition for cell proliferation. In an immunohistochemistry study, data showed that the expression of PDGF α-receptor was primarily localized around the follicle and significantly correlated with malignant tumor stage. These findings imply that autocrine activation of PDGF-α receptor plays a crucial role in the carcinogenesis of thyroid cells. To identify whether inhibition of the aberrant signal transduction could have the potential to dismiss the possibility of carcinogenesis, we examined the efficacy of PDGFR-α siRNA and tyrphostin AG1295 on repressing cell proliferation of follicular thyroid carcinoma cell line (CGTH W-1). Some tyrosine kinase inhibitors have shown to block the tyrosine-like receptors and achieve the inhibition of some aberrant signal transduction. Here we designed short interfering RNAs (siRNA) specific for PDGFR-α to repress cell proliferation in CGTH W-1. Real-time quantitative PCR, flow cytometry, immunofluorescence cell staining, and MTT assay results demonstrated that the transfected CGTH W-1 cells reduce the cellular PDGFR-α mRNA level, reduce the PDGF α-receptor in cell membrane, and repress cell proliferation. While control studies of non-silencing siRNA showed no significant effects in PDGFR-α expression and cell proliferation. Finally, we compared the effects of repressing cell proliferation of CGTH W-1 cells by PDGFR-α siRNA and a tyrosine kinase inhibitor, tyrphostin AG1295. PDGFR-α siRNA required 24 hours more than tyrphostin AG1295 to show significant inhibition of cell proliferation, but the effects last up to 240 hours. The findings indicate that the PDGFR-α siRNA could be a potential tool to suppress aberrant PDGFR-α gene expression and furthermore show that PDGF-α receptor plays a crucial role in the carcinogenesis of thyroid cells.