miR-328透過靶向CRK抑制侵襲並伴隨GBM的有利生存

Abstract

多年來，癌症一直佔據國人十大死因的榜首，對國人健康產生很大的威脅。根據衛生署癌症登記統計，台灣每年大約有六百位原發性惡性腦瘤的新生病例，其中最常見的腦瘤就是多形性膠質母細胞瘤(GBM，Glioblastoma Multiforme)，約佔所有惡性腦瘤的50-60%。GBM同時也是預後最差的原發腦瘤，平均存活率只有15-18個月。一般而言，惡性腦瘤標準的治療是全部或者局部的切除腫瘤之後，進行放射治療以及施予化療藥物。然而治療效果相當有限，因此了解腦瘤發生原因進而研發新的治療方式是勢在必行。 微核醣核酸是一種長約20-23 核苷酸的內生性核醣核酸，它可以透過轉錄後機制抑制數以百計下游基因的蛋白質表現。微核醣核酸的異常表現與許多種癌症的某些特徵有關連性。我們研究結果發現微核醣核酸hsa-miR-328與病人存活率有顯著相關。同時發現hsa-miR-328會抑制U251和SNB19 GBM細胞株的侵襲和轉移能力。運用生物資訊工具發現CRK是微核醣核酸hsa-miR-328的潛在標的基因，並利用報導基因分析及西方墨點法證明其間的調控關係，證實微核醣核酸hsa-miR-328可以減少U251和SNB19 CRK的表現量。藉由減少CRK的表現，hsa-miR-328可以抑制神經膠瘤細胞的侵襲和轉移能力。在hsa-miR-328的啟動子(promoter)，我們發現有三個STAT1的可能結合位點，並且利用報導基因分析證明STAT1可以利用其中一個位點抑制hsa-miR-328的表現量。血小板衍生生長因子(Platelet-derived growth factor，PDGF) 可以調控細胞的生長和分化，常常在GBM患者腦瘤部位發現有PDGF的過量表現。我們同時進一步發現PDGF可以藉由刺激STAT1的磷酸化，活化STAT1進而抑制hsa-miR-328的表現量，使得hsa-miR-328的標的基因CRK表現量上升，促進了GBM細胞的侵襲和轉移能力。

Glioblastomas multiforme (GBM) is the most malignant brain tumor because of the high invasive property and poor prognosis of patients. Treatments of patients diagnosed with GBM have been largely ineffective which consist of surgical resection followed by radiation and/or chemotherapy. However, the survival of patients with malignant glioma remains limited. Our present study of the microRNA expression signature in GBM revealed that miR-328 is significantly downregulated in cancer tissues. Kaplan-Meier survival curves showed that low expression of miR-328 predicted a short duration of progression to GBM. Functional studies including trans-well migration and matri-gel invasion were analyzed using two GBM cell lines, U251 and SNB19. Restoration of miR-328 in cancer cells revealed that miR-328 significantly inhibited cancer cell migration and invasion. CRK was directly regulated by the miR-328 in U251 and SNB19 cells. Our data also demonstrated that STAT1 could inhibit miR-328 expression through its promoter binding and PDGF-BB could inhibit miR-328 expression by STAT1 stimulation.