結締組織生長因子在口腔鱗狀上皮細胞癌所扮演的角色探討

Abstract

口腔癌的普及率在全球常見癌症中排名第八，且發生在男性的機率特別高。結締組織生長因子CTGF是CCN (Cyr61，CTGF，NOV) 家族中一種分泌型的基質細胞蛋白，能引發許多基本生物及病理的發展。近年來，我們發現CTGF的表現與腫瘤發展與增生有一定的關係。但是，最近的研究指出CTGF在不同癌症中有相反的功能，而它在口腔癌所扮演的角色至今仍不清楚。在此我們本實驗研究CTGF在口腔癌侵入及轉移中扮演的生物學的作用與下游調控機制。內生性的CTGF表現量與有較高侵襲能力及轉移淺力的口腔癌細胞株呈現負相關。使CTGF過度表現或利用重組CTGF蛋白的做法可減少惡性度高的人類SAS細胞株侵入與移動的能力。建構small hairpin RNA (shRNA)轉染到人類TW2.6細胞株內，抑制CTGF的表現後，發現癌細胞的侵入及轉移能力有明顯增強。為了更進一步了解CTGF調控口腔癌發展的機制，我們進行microRNA的微陣列分析，結果發現，有CTGF表現的細胞株與控制組相比後，miR-504的表現量下降。且在穩定大量表現CTGF的細胞株內，使miR-504大量表現後，發現回復細胞侵入與轉移的能力。使用生物資訊分析預測miR-504下游的標靶基因，找出FOXP1為其中之一。首先利用半定量PCR方式分析FOXP1的mRNA表現量，發現跟控制組比較之後在大量表現CTGF的細胞株內上升，而在CTGF被抑制的細胞株內則表現量下降。我們進一步利用small hairpin RNA (shRNA)轉染CTGF表現及控制組細胞株，抑制FOXP1的表現後，則細胞侵入及移動的能力則大幅增加。由這些發現我們可以了解，利用CTGF抑制口腔癌細胞是有前瞻性的。本實驗證明CTGF可藉由調控miR-504及其下游的FOXP1這條訊息傳遞路徑來達到顯著抑制人類口腔鱗狀細胞侵入及轉移能力的效果，且指出CTGF可能是為口腔癌發展中的癌症抑制基因。

The prevalence of oral cancer is the eighth most common cancer worldwide and is particularly high among men. Connective tissue growth factor (CTGF), a secreted matricellular protein of CCN family engages a wide variety of basic biological and pathological processes. Recently, CTGF expression has been shown to be associated with tumor development and progression. However, recent studies demonstrated that CTGF seems to have opposite functions in different cancers and its role in oral cancer is still unknown. Here we investigated that the biological roles and underlying regulation mechanism of CTGF in oral cancer invasion and metastasis. Endogenous CTGF expression negatively correlates with invasive and metastatic potential of oral cancer cell lines. CTGF overexpression or recombinant CTGF protein treatment decreased the invasion and migration abilities in SAS cells. Transfected-small hairpin RNA (shRNA) constructs to target human CTGF in TW2.6 cells resulted in enhancement of cell invasion and migration. To further investigate the mechanism of CTGF regulated oral cancer progression, we performed microRNA microarray. The results showed that miR-504 was downregulation in CTGF transfectants compared to NEO control. Overexpression of miR-504 restored the invasion and migration abilitities of CTGF overexpression stable clones. Using bioinformatic prediction, FOXP1 was recognized as one of several miR-504 target genes. We analyzed the mRNA levels of FOXP1 by RT-PCR and found that mRNA level was increased in SAS/CTGF stable clones and decreased in TW2.6/shCTGF stable clones versus control. We further transfected-small hairpin RNA (shRNA) constructs to target human FOXP1 in CTGF-transfectants and control cells resulted in enhancement of cell invasion and migration. These findings illustrated that CTGF inhibited tumor progression in oral cancer cells. Our study demonstrated that CTGF regulated miR-504 through FOXP1 signaling significantly inhibited migration and invasion abilities in human oral squamous cells, suggesting that CTGF may be a tumor suppressor gene during oral cancer progression.