TAZ與胰腺癌細(xì)胞增殖和抗凋亡能力的關(guān)系研究

占 婷，羅和生，田 霞，黃曉東

1.武漢大學(xué)附屬同仁醫(yī)院(武漢市第三醫(yī)院)消化內(nèi)科，湖北 武漢430060； 2.武漢大學(xué)人民醫(yī)院消化內(nèi)科

TAZ與胰腺癌細(xì)胞增殖和抗凋亡能力的關(guān)系研究

占 婷1,2，羅和生2，田 霞1，黃曉東1

1.武漢大學(xué)附屬同仁醫(yī)院(武漢市第三醫(yī)院)消化內(nèi)科，湖北 武漢430060； 2.武漢大學(xué)人民醫(yī)院消化內(nèi)科

目的 探討轉(zhuǎn)錄共激活因子TAZ對(duì)胰腺癌細(xì)胞增殖與抗凋亡能力的影響及其作用機(jī)制，為胰腺癌尋找新的診斷與治療靶點(diǎn)提供思路。方法 用空載病毒(對(duì)照組)、TAZ過(guò)表達(dá)病毒(Lv-TAZ)和TAZ干擾病毒(siTAZ)轉(zhuǎn)染胰腺癌細(xì)胞后用CCK8法檢測(cè)各組細(xì)胞的增殖情況；流式細(xì)胞儀檢測(cè)各組細(xì)胞的凋亡率；Western blotting檢測(cè)各組細(xì)胞的PTEN、Akt、p-Akt的表達(dá)情況。結(jié)果 與對(duì)照組相比，Lv-TAZ組細(xì)胞增殖率升高，siTAZ組細(xì)胞增殖率下降。與對(duì)照組相比，Lv-TAZ組細(xì)胞凋亡率下降，siTAZ組細(xì)胞凋亡率升高。Western blotting結(jié)果顯示，Lv-TAZ組細(xì)胞PTEN表達(dá)水平下調(diào)，p-Akt上調(diào)；siTAZ組細(xì)胞PTEN表達(dá)水平上調(diào)，p-Akt下調(diào)。結(jié)論 TAZ在胰腺癌細(xì)胞中可能通過(guò)調(diào)控PTEN/Akt信號(hào)通路發(fā)揮其促增殖和抗凋亡作用。

胰腺癌；TAZ；PTEN/Akt信號(hào)通路

近年來(lái)胰腺癌的發(fā)病率和死亡率不斷升高，使其成為消化系統(tǒng)惡性程度最高的腫瘤之一[1]。目前，手術(shù)切除是胰腺癌唯一根治的方法，但是由于侵襲性強(qiáng)，大部分胰腺癌患者就診時(shí)已經(jīng)發(fā)生局部或遠(yuǎn)處轉(zhuǎn)移，失去根治性手術(shù)的機(jī)會(huì)[2]。因此，我們迫切需要對(duì)胰腺癌發(fā)病機(jī)制進(jìn)行深入研究，以尋找新的治療策略。

轉(zhuǎn)錄共激活因子TAZ (transcriptional co-activator with PDZ-binding motif) 于2000年作為14-3-3蛋白的結(jié)合蛋白而被發(fā)現(xiàn)，參與哺乳動(dòng)物多種組織的生長(zhǎng)[3]。作為一種轉(zhuǎn)錄共激活因子，TAZ通過(guò)與多種轉(zhuǎn)錄因子如TEA結(jié)構(gòu)域家族轉(zhuǎn)錄因子、Runt功能域轉(zhuǎn)錄因子等結(jié)合而發(fā)揮不同的作用[4-5]。越來(lái)越多研究已經(jīng)證實(shí)，TAZ參與腫瘤形成、細(xì)胞增殖與遷移、上皮細(xì)胞間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition,EMT)等一系列過(guò)程[6-8]。研究[9]發(fā)現(xiàn)，TAZ在胰腺癌組織和細(xì)胞中高表達(dá)，且能夠促進(jìn)胰腺癌細(xì)胞的遷移、侵襲、EMT。然而TAZ對(duì)胰腺癌細(xì)胞增殖與抗凋亡能力的影響仍未見(jiàn)報(bào)道。因此，本研究著重討論TAZ對(duì)胰腺癌細(xì)胞的增殖與凋亡能力的影響及其可能機(jī)制。

1 材料與方法

1.1 細(xì)胞系及細(xì)胞培養(yǎng) 人胰腺癌細(xì)胞PANC1購(gòu)自中國(guó)科學(xué)院上海細(xì)胞庫(kù)。細(xì)胞在37 ℃、體積分?jǐn)?shù)為5%的CO2培養(yǎng)箱中培養(yǎng)，培養(yǎng)細(xì)胞的完全培養(yǎng)基為含1%雙抗(Thermo Fisher Scientific,美國(guó))、100 g/L胎牛血清(NQBB, USA)的高糖培養(yǎng)基DMEM (Gibco, Carlsbad,美國(guó))。

1.2 細(xì)胞轉(zhuǎn)染 TAZ的靶向siRNA慢病毒(siTAZ)以及過(guò)表達(dá)病毒(Lv-TAZ)由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司合成。將細(xì)胞接種于6孔板，于第2天按照操作說(shuō)明書加入適量慢病毒、Ploybrene和Enhance培養(yǎng)12 h后換成完全培養(yǎng)基后繼續(xù)培養(yǎng)。

1.3 Western blotting檢測(cè) 轉(zhuǎn)染后的細(xì)胞用RIPA裂解液(北京普利萊基因技術(shù)有限公司)裂解提蛋白后，用BCA蛋白濃度測(cè)定盒(碧云天生物技術(shù)研究所)測(cè)蛋白濃度。將蛋白與loading buffer混合后100 ℃變性10 min,再用100 g/L SDS-PAGE膠分離蛋白，然后轉(zhuǎn)到NC膜上,隨后用兔抗人TAZ(CST,美國(guó))、PTEN(CST,美國(guó))、Akt(CST,美國(guó))、p-Akt(CST,美國(guó))、GAPDH(Santa Cruz,美國(guó))多克隆抗體于4 ℃中孵育過(guò)夜。最后用羊抗兔二抗(Lincoln，美國(guó))在室溫下孵育1 h，蛋白表達(dá)水平用ECL化學(xué)發(fā)光顯色試劑盒檢測(cè)。

1.4 CCK-8檢測(cè) 將轉(zhuǎn)染后的細(xì)胞接種于96孔板中，然后用CCK8試劑(日本同仁化學(xué)研究所)分別檢測(cè)各種細(xì)胞在24 h、48 h、72 h、96 h時(shí)450 nm的OD值以反映細(xì)胞的增殖情況。

1.5 流式細(xì)胞檢測(cè) 將轉(zhuǎn)染后的細(xì)胞于6孔中培養(yǎng)3 d后，根據(jù)Annexin V-PE凋亡試劑盒(BD Bioscience,美國(guó))的說(shuō)明書處理細(xì)胞后用流式細(xì)胞儀檢測(cè)細(xì)胞的凋亡情況。

1.6 統(tǒng)計(jì)學(xué)處理 采用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，說(shuō)明數(shù)據(jù)的圖用GraphPad Prism 5制作，采用t檢驗(yàn)和單因素方差分析進(jìn)行統(tǒng)計(jì)學(xué)分析，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 TAZ對(duì)胰腺癌細(xì)胞增殖與抗凋亡能力的影響 首先用空載對(duì)照病毒(對(duì)照組)、TAZ干擾病毒(siTAZ組)和過(guò)表達(dá)病毒(Lv-TAZ 組)轉(zhuǎn)染胰腺癌細(xì)胞PANC1，轉(zhuǎn)染效率見(jiàn)圖1。CCK-8檢測(cè)結(jié)果顯示，與對(duì)照組比較，Lv-TAZ 組細(xì)胞增殖率升高，siTAZ 組細(xì)胞增殖率下降(見(jiàn)圖2)。將轉(zhuǎn)染后的胰腺癌細(xì)胞用流式細(xì)胞儀檢測(cè)其凋亡情況，結(jié)果顯示，與對(duì)照組相比，Lv-TAZ 組細(xì)胞凋亡率下降，siTAZ 組細(xì)胞凋亡率升高(見(jiàn)圖3)。

圖1 轉(zhuǎn)染病毒后各組細(xì)胞TAZ蛋白的表達(dá)情況；圖2 轉(zhuǎn)染病毒后各組細(xì)胞的增殖情況

Fig 1 The expression of TAZ protein after transfection of the virus in each group; Fig 2 The proliferation of cells after transfection of the virus in each group

2.2 TAZ在胰腺癌細(xì)胞中對(duì)PTEN/Akt信號(hào)通路的作用 將PANC1細(xì)胞用空載病毒、TAZ干擾病毒和過(guò)表達(dá)病毒轉(zhuǎn)染后提取蛋白，然后用Western blotting檢測(cè)PTEN、Akt、p-Akt蛋白的表達(dá)情況。結(jié)果顯示，與對(duì)照組相比，Lv-TAZ組蛋白PTEN表達(dá)下調(diào)，siTAZ組蛋白PTEN表達(dá)上調(diào)(見(jiàn)圖4)。而與對(duì)照組相比，Lv-TAZ組蛋白p-Akt表達(dá)上調(diào)，siTAZ組蛋白p-Akt表達(dá)下調(diào)，而Akt表達(dá)量無(wú)明顯改變(見(jiàn)圖5)。

3 討論

近幾年來(lái)雖然胰腺癌的手術(shù)治療已經(jīng)有了很大的提升，但大多數(shù)患者在確診的時(shí)候已經(jīng)在中晚期，失去了切除機(jī)會(huì)[10]，因此，我們迫切需要研究早期胰腺癌的檢測(cè)和治療靶點(diǎn)。最近研究[11-12]顯示，胰腺癌本質(zhì)上是一種由于基因和表觀遺傳學(xué)的改變而導(dǎo)致的基因性疾病。因此，研究能夠促進(jìn)胰腺癌發(fā)生、發(fā)展的關(guān)鍵基因能夠?yàn)橐认侔┰缙跈z測(cè)及治療提供新的思路。

TAZ是一種含有WW結(jié)構(gòu)域的轉(zhuǎn)錄共激活因子，是Hippo信號(hào)通路重要的下游效應(yīng)因子。該信號(hào)通路在器官大小、干細(xì)胞功能維持、腫瘤發(fā)生調(diào)節(jié)上都發(fā)揮著重要作用[13]。當(dāng)Hippo信號(hào)通路激活時(shí)，該通路中的絲/蘇氨酸蛋白激酶MST1/2作用于腫瘤抑制基因LATS1/2激酶使其磷酸化，導(dǎo)致其下游的效應(yīng)因子TAZ失活[14]。近年來(lái)，TAZ被發(fā)現(xiàn)在多種惡性腫瘤中表達(dá)升高，如肝癌[15]、胃癌[16]、乳腺癌[17]等，提示TAZ在腫瘤的發(fā)生、發(fā)展中發(fā)揮著重要作用。

研究[9]發(fā)現(xiàn)，TAZ在胰腺癌組織和細(xì)胞中高表達(dá)，并能夠促進(jìn)胰腺癌細(xì)胞的遷移、侵襲、EMT。

圖3 轉(zhuǎn)染病毒后各組細(xì)胞的凋亡情況Fig 3 Apoptosis after transfection of the virus in each group

注：與對(duì)照組比較，*P<0.05，**P<0.001。

圖4 轉(zhuǎn)染病毒后各組細(xì)胞PTEN蛋白的表達(dá)情況

Fig 4 The expression of PTEN protein after transfection of the virus in each group

注：與對(duì)照組比較，*P<0.05。

圖5 轉(zhuǎn)染病毒后各組細(xì)胞p-Akt及Akt蛋白的表達(dá)情況

Fig 5 The expressions of p-Akt protein and Akt protein after transfection of the virus in each group

然而TAZ對(duì)胰腺癌細(xì)胞增殖與抗凋亡能力的影響及作用的可能機(jī)制仍未見(jiàn)報(bào)道。而在我們的實(shí)驗(yàn)中，通過(guò)過(guò)表達(dá)TAZ的表達(dá)和干擾TAZ的表達(dá)，發(fā)現(xiàn)TAZ的表達(dá)水平與胰腺癌細(xì)胞的增殖率呈正相關(guān)，與胰腺癌細(xì)胞的凋亡率呈負(fù)相關(guān)。這些結(jié)果證實(shí)了TAZ能夠增強(qiáng)胰腺癌的增殖與抗凋亡能力。PTEN 是公認(rèn)的腫瘤抑制因子，與多種腫瘤的發(fā)生、發(fā)展密切相關(guān)[18]，而我們?cè)趯?shí)驗(yàn)中發(fā)現(xiàn)，TAZ的表達(dá)水平與PTEN蛋白的表達(dá)水平呈負(fù)相關(guān)。PTEN是PI3K-Akt通路上的一個(gè)重要的抑制因子，它可以使PIP3去磷酸化成為PIP2從而抑制 Akt的磷酸化使其失活[19]。P13K/Akt信號(hào)通路過(guò)度激活常見(jiàn)于多種腫瘤，與人類多種腫瘤的發(fā)生、發(fā)展密切相關(guān)[20]?；罨腁kt是腫瘤發(fā)生和發(fā)展的一個(gè)重要因素,它能夠使PIP2促進(jìn)細(xì)胞增殖，減少細(xì)胞凋亡，降低細(xì)胞對(duì)藥物的敏感性[21-22]。 為了探討TAZ與活化的Akt之間的潛在關(guān)系，我們檢測(cè)p-Akt與Akt在轉(zhuǎn)染后的胰腺癌細(xì)胞中的表達(dá)水平，結(jié)果發(fā)現(xiàn)在TAZ過(guò)表達(dá)的胰腺癌細(xì)胞中p-Akt表達(dá)升高，在干擾了TAZ表達(dá)的胰腺癌細(xì)胞中，p-Akt表達(dá)下降，而與Akt表達(dá)水平基本一致。這些結(jié)果提示，TAZ在胰腺癌細(xì)胞中可能通過(guò)調(diào)控PTEN的表達(dá)進(jìn)而激活A(yù)kt信號(hào)通路。

總之，我們的研究表明，TAZ在胰腺癌細(xì)胞中可能通過(guò)調(diào)控PTEN/Akt信號(hào)通路來(lái)發(fā)揮其促增殖和抗凋亡作用。因此，我們可以認(rèn)為TAZ是胰腺癌早期診斷和治療的潛在靶點(diǎn)。

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(責(zé)任編輯：李 健)

Relationship of TAZ with proliferation and anti apoptotic ability of pancreatic cancer

ZHAN Ting1,2, LUO Hesheng2, TIAN Xia1, HUANG Xiaodong1

1.Department of Gastroenterology, Wuhan Third Hospital, Tongren Hospital of Wuhan University, Wuhan 430060; 2.Department of Gastroenterology, People’s Hospital of Wuhan University, China

Objective To investigate the effect and functionary mechanism of TAZ on the proliferation and anti apoptotic ability of pancreatic cancer cells, and to provide a new way for the diagnosis and treatment of pancreatic cancer.Methods Pancreatic cancer cells were transfected with lentiviral vector expressing TAZ (Lv-TAZ) and TAZ siRNA lentiviral vector (siTAZ), and then CCK8 was used to detect the proliferation. Flow cytometry was used to detect the apoptosis. Western blotting was used to detect the expressions of PTEN, Akt, p-Akt.Results Compared with the control group, the proliferation rate of cells transfected with Lv-TAZ was increased, and the cell proliferation rate of cells transfected with siTAZ was decreased. Compared with control group, the apoptosis rate of cells transfected with Lv-TAZ was decreased, and the apoptosis rate of cells transfected with siTAZ was increased. Western blotting results showed that the expression of PTEN in cells transfected with Lv-TAZ was downregulated, and the expression of p-Akt was upregulated. On the contrary, the expression of PTEN in cells transfected with siTAZ was upregulated, and the expression of p-Akt was downregulated.Conclusion TAZ may play a role in promoting the proliferation and anti apoptosis by regulating PTEN/Akt signaling pathway in pancreatic cancer cells.

Pancreatic cancer; TAZ; PTEN/Akt signaling pathway

湖北省科技支撐計(jì)劃項(xiàng)目(2015BHE022)

占婷,在讀碩士研究生，研究方向：胰腺癌。E-mail：472308046@qq.com

黃曉東，博士，主任醫(yī)師，研究方向：消化道動(dòng)力障礙性疾病。 E-mail：13886190549@163.com

10.3969/j.issn.1006-5709.2017.07.006

R735.9

A

1006-5709(2017)07-0741-03

2016-12-17