Matriptase表達(dá)下調(diào)對(duì)胰腺癌細(xì)胞株SW1990侵襲的影響

王楠 楊劍峰 賈振宇 何楊 許春芳

·論著·

Matriptase表達(dá)下調(diào)對(duì)胰腺癌細(xì)胞株SW1990侵襲的影響

王楠 楊劍峰 賈振宇 何楊 許春芳

目的探討matriptase基因沉默對(duì)胰腺癌細(xì)胞株SW1990侵襲力的影響。方法采用脂質(zhì)體法將不同濃度的靶向matriptase的siRNA(Ma-siRNA)轉(zhuǎn)染人胰腺癌SW1990細(xì)胞株，以轉(zhuǎn)染陰性對(duì)照的siRNA(NC-siRNA)作為對(duì)照組。采用熒光定量PCR法和蛋白質(zhì)印跡法檢測(cè)各組細(xì)胞matriptase mRNA和蛋白的表達(dá)水平，采用Transwell小室檢測(cè)細(xì)胞侵襲能力，明膠酶譜法檢測(cè)細(xì)胞matriptase及MMP-9的活性。結(jié)果NC-siRNA組及12.5、25、50 nmol/L Ma-siRNA組SW1990細(xì)胞matriptase mRNA相對(duì)表達(dá)量分別為1.000、0.417±0.006、0.233±0.068、0.221±0.092，蛋白表達(dá)量分別為0.736±0.066、0.498±0.036、0.341±0.118、0.239±0.050，Ma-siRNA各組細(xì)胞matriptase mRNA及蛋白表達(dá)量均顯著低于NC-siRNA組，差異具有統(tǒng)計(jì)學(xué)意義(P值均<0.01)；matriptase活性分別為1.501±0.165、1.211±0.265、0.645±0.165、0.620±0.003，MMP-9活性分別為0.929±0.260、0.484±0.364、0.352±0.113、0.346±0.121，其中25、50 nmol/L Ma-siRNA組細(xì)胞的matriptase及MMP-9活性較NC-siRNA組顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P值<0.05或<0.01)。NC-siRNA組的穿膜細(xì)胞數(shù)為(256±1)個(gè)/200倍視野，25 nmol/L Ma-siRNA組為(109±3)個(gè)/200倍視野，25 nmol/L Ma-siRNA組細(xì)胞侵襲力較對(duì)照組下降(57.4±5.4)%。結(jié)論抑制matriptase基因表達(dá)可降低胰腺癌SW1990細(xì)胞的侵襲能力，其機(jī)制可能與matriptase及MMP-9酶活性下降有關(guān)。

胰腺腫瘤； 腫瘤侵襲； 絲氨酸蛋白酶類(lèi)； 基質(zhì)金屬蛋白酶9

Matriptase是Ⅱ型跨膜絲氨酸蛋白酶家族成員，主要表達(dá)于各種組織的上皮細(xì)胞，免疫細(xì)胞中也有微量表達(dá)[1]。Lee等[2]報(bào)道，matriptase的表達(dá)量與宮頸癌的臨床分期和病理學(xué)分級(jí)呈正相關(guān)。Uhland等[3]發(fā)現(xiàn)，matriptase參與胰腺導(dǎo)管癌的病理進(jìn)程，抑制matriptase活性可以抑制疾病的進(jìn)展。本研究通過(guò)特異性沉默matriptase基因表達(dá)，觀察其對(duì)胰腺癌細(xì)胞株SW1990侵襲能力的影響。

材料和方法

一、細(xì)胞株及實(shí)驗(yàn)材料

白血病細(xì)胞株HL60、Namalwa，胰腺癌細(xì)胞株SW1990、PaTu-8988、BxPC-3、AsPC-1系唐仲英血液研究中心贈(zèng)予。HL60、Namalwa在含10%胎牛血清的RIPM-1640培養(yǎng)液中培養(yǎng)、傳代，其他各細(xì)胞株均在含10%胎牛血清的DMEM培養(yǎng)液中培養(yǎng)、傳代。靶向matriptase的siRNA(Ma-siRNA)、陰性對(duì)照siRNA(NC-siRNA)及熒光標(biāo)記的siRNA(NC FAM-siRNA)均購(gòu)自蘇州吉瑪公司(GenePharma)。Ma-siRNA的序列為5′-UGCCAGUCAACAACGUCAATT-3′。鼠抗人matriptase單克隆抗體購(gòu)自Santa Cruz公司，羊抗鼠HRP標(biāo)記的二抗購(gòu)自美國(guó)Bioworld公司。Trizol試劑購(gòu)自Sigma公司，反轉(zhuǎn)錄試劑盒購(gòu)自Thermo公司。SYBR green PCR Master Mix購(gòu)自美國(guó)ABI 公司，Transwell小室購(gòu)自Millipore公司。

二、RT-PCR法檢測(cè)細(xì)胞株matriptase mRNA表達(dá)

收集各對(duì)數(shù)生長(zhǎng)期細(xì)胞，用Trizol提取細(xì)胞總RNA。采用RT-PCR方法檢測(cè)細(xì)胞matriptase mRNA表達(dá)。根據(jù)Genbank中的基因序列設(shè)計(jì)引物，matriptase上游引物序列為5′-GGCACGAGAAAGTGAATGGC-3′，下游引物序列為5′-GAAGACCTTCTGGACACGCA-3′，產(chǎn)物大小197 bp；內(nèi)參β-actin上游引物序列為5′-AGCGAGCATCCCCCAAAGTT-3′，下游引物序列為5′-GGGCACGAAGGCTCATCATT-3′，產(chǎn)物大小285 bp。引物均由蘇州金唯智生物科技有限公司合成。先用反轉(zhuǎn)錄試劑盒將RNA反轉(zhuǎn)錄為cDNA。PCR反應(yīng)條件： 94℃ 4 min，94℃ 20 s、60℃ 20 s、72℃ 30 s，29個(gè)循環(huán)。PCR擴(kuò)增產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳分離，BIO-RAD/Gel DocXR成像系統(tǒng)觀察拍照。

三、蛋白質(zhì)印跡法檢測(cè)細(xì)胞株matriptase蛋白的表達(dá)

收集各對(duì)數(shù)生長(zhǎng)期細(xì)胞，細(xì)胞裂解液裂解細(xì)胞獲取蛋白勻漿，BCA比色法定量蛋白后常規(guī)行蛋白質(zhì)印跡法檢測(cè)matriptase蛋白表達(dá)。最后應(yīng)用圖像分析儀掃描，以目的條帶與內(nèi)參GAPDH條帶的灰度值比表示蛋白的相對(duì)表達(dá)量。實(shí)驗(yàn)重復(fù)3次，取均值。

四、Matriptase基因沉默的胰腺癌SW1990細(xì)胞建立及鑒定

選取高表達(dá)matriptase mRNA的SW1990細(xì)胞株對(duì)數(shù)生長(zhǎng)期細(xì)胞，以5×105/孔密度接種于6孔板，37℃培養(yǎng)箱中孵育過(guò)夜，待細(xì)胞融合至70%～90%時(shí)，在200 μl無(wú)血清DMEM中加入終濃度為12.5、25、50 nmol/L的Ma-siRNA及5 μl lipofectamine 2000混勻，以加入NC-siRNA作為對(duì)照。繼續(xù)培養(yǎng)24、48 h后收集各組細(xì)胞。采用實(shí)時(shí)PCR及蛋白質(zhì)印跡法檢測(cè)細(xì)胞的matriptase mRNA及蛋白表達(dá)。實(shí)時(shí)PCR的反應(yīng)條件同上，由儀器自帶軟件獲取Ct值，以公式2-ΔΔCt計(jì)算mRNA相對(duì)表達(dá)量，ΔΔCt=(Ma-siRNA組Ct值-β-actin組Ct值)-(NC-siRNA組Ct值-β-actin組Ct值)。實(shí)驗(yàn)重復(fù)3次，取均值。

五、明膠酶譜檢測(cè)細(xì)胞matriptase及MMP-9活性

取上述各組培養(yǎng)4 h后的SW1990細(xì)胞，換無(wú)血清的DMEM繼續(xù)培養(yǎng)24 h，收集各組細(xì)胞培養(yǎng)上清液。將培養(yǎng)上清液與非還原性上樣緩沖液1∶1混合，置4℃行SDS-PAGE電泳。電泳結(jié)束后用2.5%洗脫液洗脫凝膠2次，每次20 min，然后用ddH2O漂洗2次，每次30 min，置孵育液中洗滌30 min，換新鮮孵育液后置37℃孵育42 h。孵育結(jié)束后用考馬斯藍(lán)染色30 min，脫色2 h，掃描儀掃描獲取各條帶灰度值。實(shí)驗(yàn)重復(fù)3次，取均值。

六、Transwell小室檢測(cè)細(xì)胞侵襲能力

用Matrigel涂布Transwell小室的Polycarbonate膜，凝固后在上室加入200 μl用無(wú)血清DMEM重懸的25 nmol/L Ma-siRNA轉(zhuǎn)染24 h的SW1990細(xì)胞(1×105/室)，在下室中加入500 μl含10% FBS的DMEM，置37℃培養(yǎng)箱中培養(yǎng)36 h，用棉簽輕輕擦去上室中未穿膜的細(xì)胞，將膜用甲醇固定15 min，結(jié)晶紫(碧云天公司)染色10 min。用智能圖像導(dǎo)航儀(奧林巴斯公司FSX100)拍照，200倍光鏡下取5個(gè)視野，計(jì)數(shù)穿膜細(xì)胞數(shù)。以轉(zhuǎn)染NC-siRNA的SW1990細(xì)胞作為對(duì)照。實(shí)驗(yàn)重復(fù)3次，取均值。

七、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、胰腺癌細(xì)胞株matriptase mRNA及蛋白表達(dá)

胰腺癌細(xì)胞SW1990及AsPC-1均有matriptase mRNA及蛋白表達(dá)，以SW1990細(xì)胞的表達(dá)量高，而PaTu-8988細(xì)胞不表達(dá)；陰性對(duì)照的HL60細(xì)胞不表達(dá)，陽(yáng)性對(duì)照的Namalwa有表達(dá)(圖1)。

圖1 HL60(1)、Namalwa(2)、AsPC-1(3)、SW1990(4)、PaTu-8988(5)細(xì)胞matriptase mRNA(1A)及蛋白(1B)表達(dá)

二、胰腺癌細(xì)胞株SW1990的matriptase基因沉默效果

NC-siRNA組及12.5、25、50 nmol/L Ma-siRNA組SW1990細(xì)胞的matriptase mRNA表達(dá)量分別為1.000、0.417±0.006、0.233±0.068，0.221±0.092；蛋白表達(dá)量分別為0.736±0.066、0.498±0.036、0.341±0.118、0.239±0.050(圖2)。Ma-siRNA各組細(xì)胞matriptase mRNA及蛋白表達(dá)量均顯著低于NC-siRNA組，差異具有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。

圖2 NC-siRNA組(1)及12.5(2)、25(3)、50 nmol/L(4)Ma-siRNA組SW1990細(xì)胞 matriptase蛋白表達(dá)

三、SW1990細(xì)胞matriptase及MMP-9活性的變化

NC-siRNA組及12.5、25、50 nmol/L Ma-siRNA組SW1990細(xì)胞的matriptase活性分別為1.501±0.165、1.211±0.265、0.645±0.165、0.620±0.003，其中25、50 nmol/L Ma-siRNA組細(xì)胞的matriptase活性較NC-siRNA組顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.01，圖3)。

NC-siRNA組及12.5、25、50 nmol/L Ma-siRNA組SW1990細(xì)胞的MMP-9活性分別為0.929±0.260、0.484±0.364、0.352±0.113、0.346±0.121，其中25、50 nmol/L Ma-siRNA組細(xì)胞的MMP-9活性較NC-siRNA組顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P值均<0.05，圖3)。

圖3 NC-siRNA組(1)及12.5(2)、25(3)、50 nmol/L(4)Ma-siRNA組SW1990細(xì)胞matriptase、MMP-9活性(明膠酶譜)

四、Matriptase基因沉默對(duì)SW1990細(xì)胞侵襲力的影響

NC-siRNA組和25nmol/L Ma-siRNA組的穿膜細(xì)胞數(shù)分別為(256±1)、(109±3)個(gè)/200倍視野，沉默后SW1990細(xì)胞的侵襲力下降(57.4±5.4)%(圖4)。

圖4 NC-siRNA組(4A)和25 nmol/L Ma-siRNA組(4B)的穿膜細(xì)胞(×200)

討 論

Matriptase最早于1993年在體外培養(yǎng)的乳腺癌細(xì)胞培養(yǎng)基中被鑒定出來(lái)[4]。編碼matriptase的基因ST14定位于人染色體11q24-25，全長(zhǎng)50 900 bp，mRNA全長(zhǎng)為3 310 bp，編碼一條855個(gè)氨基酸的多肽。研究發(fā)現(xiàn)，matriptase與上皮來(lái)源的實(shí)體腫瘤密切相關(guān)，在前列腺[5]、結(jié)腸[6]、腎臟[7]等部位的腫瘤均能檢測(cè)到matriptase的過(guò)表達(dá)；部分腫瘤的matriptase高表達(dá)與臨床預(yù)后不良相關(guān)。編碼matriptase蛋白的ST14基因突變或缺失后可導(dǎo)致炎癥性腸病[8]、瑟頓綜合征[9]等疾病。但matriptase在胰腺癌中的作用尚不清楚。

研究發(fā)現(xiàn)，matriptase可直接降解細(xì)胞外基質(zhì)或通過(guò)激活肝細(xì)胞生長(zhǎng)因子前體(pro-HGF)[10]和尿激酶型纖溶酶原激活物前體(pro-uPA)[11]降解細(xì)胞外基質(zhì)并促進(jìn)腫瘤細(xì)胞的侵襲及轉(zhuǎn)移。此外，matriptase增加HGF和c-Met[12]的表達(dá)，它們相互作用能增加STAT3磷酸化水平，從而通過(guò)PI3K/NF-kB/AKT[13]等信號(hào)通路延長(zhǎng)腫瘤的存活時(shí)間。

本研究結(jié)果顯示，胰腺癌細(xì)胞株SW1990、AsPC-1表達(dá)matriptase，但胰腺癌細(xì)胞株P(guān)aTu-8988不表達(dá)，提示matriptase在胰腺癌細(xì)胞中存在異質(zhì)性。通過(guò)siRNA技術(shù)特異性沉默SW1990細(xì)胞matriptase表達(dá)后細(xì)胞的matriptase、MMP-9活性降低，侵襲能力明顯下降，提示抑制matriptase基因表達(dá)既可抑制matriptase活性，也可抑制MMP-9活性，從而降低胰腺癌細(xì)胞株SW1990的侵襲力，表明matriptase基因參與SW1990細(xì)胞的浸潤(rùn)及轉(zhuǎn)移。

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(本文編輯：呂芳萍)

Effects of matriptase down regulation on invasion of human pancreatic cancer cells SW1990

WangNan,YangJianfeng,JiaZhenyu,HeYang,XuChunfang.

DepartmentofGastroenterology,FirstAffiliatedHospital,SoochowUniversity,Suzhou215006,China

Correspondinganthor:XuChunfang,Email:xcf601@163.com

Objective To investigate the effect of the down regulation of matriptase expression on invasion of human pancreatic cancers cells SW1990. Methods Small interfering RNA targeting at matriptase (Ma-siRNA) was transfected into human pancreatic cancers SW1990 cells, and nonsense siRNA (NC-siRNA) group was used as control. Real time PCR assay and Western blot were used to detect the expression of matriptase mRNA and protein. Transwell assay was used to examine the invasion ability of cancer cells. The enzymatic activity of matriptase and MMP-9 was determined by gelatin zymography assay. Results The expression level of matriptase mRNA in NC-siRNA group, 12.5, 25, 50 nmol/L Ma-siRNA group were 1.000, 0.417±0.006, 0.233±0.068, 0.221±0.092; and the protein expression of matriptase were 0.736±0.066, 0.498±0.036, 0.341±0.118, 0.239±0.050, respectively. The matriptase mRNA and protein expression in Ma-siRNA groups was significantly lower than those in NC-siRNA group, and the difference between the two groups was statistically significant (P<0.05). The enzymatic activity of matriptase were 1.501±0.165, 1.211±0.265, 0.645±0.165, 0.620±0.003, and the enzymatic activity of MMP-9 were 0.929±0.260, 0.484±0.364, 0.352±0.113, 0.346±0.121, and the enzymatic activity of matriptase and MMP-9 in 25, 50 nmol/L Ma-siRNA groups was significantly lower than that in NC-siRNA group, and the difference was statistically significant (P<0.05 or <0.01). The number of transmembrane cell was (256±1)/per 200 power field, and it was (109±3)/per 200 power field in 25 nmol/L Ma-siRNA group, and the invasion ability of the cells in 25 nmol/L Ma-siRNA group was decreased by (57.4±5.4)% when compared with that of control group. Conclusions Down-regulation of matriptase inhibits invasion ability of pancreatic cancer SW1990 cells, and this result may be due to the down regulated enzymatic activity of matriptase and MMP-9.

Pancreatic neoplasms; Neoplasm invasiveness; Serine proteases; Matrix metalloproteinase 9

10.3760/cma.j.issn.1674-1935.2015.04.004

215006 蘇州，蘇州大學(xué)附屬第一醫(yī)院消化內(nèi)科(王楠、賈振宇、許春芳)；蘇州大學(xué)唐仲英血液學(xué)研究中心，教育部血液與血管疾病診療藥物技術(shù)工程中心(王楠、楊劍峰、何楊)

許春芳，Email: xcf601@163.com

2014-12-08)