S100A9蛋白表達(dá)與宮頸鱗癌細(xì)胞侵襲、轉(zhuǎn)移的關(guān)系研究

陳苗苗 張文文 王穎 朱雪瓊

S100A9蛋白表達(dá)與宮頸鱗癌細(xì)胞侵襲、轉(zhuǎn)移的關(guān)系研究

陳苗苗 張文文 王穎 朱雪瓊

目的 探討S100A9蛋白表達(dá)與宮頸鱗癌細(xì)胞侵襲、轉(zhuǎn)移的關(guān)系。方法采用S100A9重組腺病毒轉(zhuǎn)染人宮頸鱗癌C-33A細(xì)胞，S100A9 siRNA轉(zhuǎn)染人宮頸鱗癌Caski細(xì)胞；應(yīng)用Western blot檢測轉(zhuǎn)染前后2種細(xì)胞中S100A9蛋白的表達(dá)，Transwell檢測細(xì)胞侵襲與轉(zhuǎn)移能力的變化。結(jié)果轉(zhuǎn)染S100A9重組腺病毒后C-33A細(xì)胞中S100A9蛋白的表達(dá)明顯增強(qiáng)；與未轉(zhuǎn)染組、陰性對照組相比，轉(zhuǎn)染組C-33A細(xì)胞的穿膜細(xì)胞數(shù)、穿過小室的細(xì)胞數(shù)均明顯增多（均P＜0.05）。轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞中S100A9蛋白的表達(dá)明顯降低；siRNA組Caski細(xì)胞的穿膜細(xì)胞數(shù)、穿過小室的細(xì)胞數(shù)均明顯少于陰性對照組（均P＜0.05）。結(jié)論S100A9與宮頸鱗癌細(xì)胞的侵襲、轉(zhuǎn)移相關(guān)。上調(diào)S100A9的蛋白表達(dá)，可促進(jìn)C-33A細(xì)胞侵襲與轉(zhuǎn)移的能力；下調(diào)S100A9的蛋白表達(dá)，可抑制Caski細(xì)胞侵襲與轉(zhuǎn)移的能力。

S100A9 宮頸鱗癌 侵襲 轉(zhuǎn)移

S100A9是鈣結(jié)合蛋白家族的成員之一，其在細(xì)胞的分化、增殖、凋亡和遷移中發(fā)揮著重要作用[1]。目前有相關(guān)研究結(jié)果顯示S100A9在細(xì)胞惡性轉(zhuǎn)化中亦具有重要作用，它在多種惡性腫瘤如乳腺癌[2]、卵巢癌[3]、膀胱癌[4]和未分化的甲狀腺癌[5]中表達(dá)增高，并參與腫瘤的發(fā)生、發(fā)展。我們的前期研究發(fā)現(xiàn)S100A9在正常宮頸組織、宮頸上皮內(nèi)瘤變（CIN）、宮頸癌中的表達(dá)逐漸升高[6]，但對宮頸癌細(xì)胞生物學(xué)行為的影響尚不明確。此外，本課題組還發(fā)現(xiàn)在宮頸鱗癌細(xì)胞系SiHa、C-33A、Caski和MS751中，無論是mRNA水平還是蛋白水平，S100A9在Caski細(xì)胞中的表達(dá)最高，在C-33A細(xì)胞中的表達(dá)最低（待發(fā)表）。故本研究采用轉(zhuǎn)染S100A9重組腺病毒上調(diào)宮頸鱗癌C-33A細(xì)胞S100A9的表達(dá)，利用RNA干擾技術(shù)下調(diào)宮頸癌Caski細(xì)胞中S100A9的表達(dá)，以研究S100A9蛋白表達(dá)的改變對宮頸癌細(xì)胞侵襲、轉(zhuǎn)移的影響，為宮頸鱗癌的治療提供新的思路。

1 材料和方法

1.1 材料 人宮頸鱗癌細(xì)胞系C-33A和Caski均購于中國科學(xué)院上海細(xì)胞研究所細(xì)胞庫。DMEM、RPMI-1640培養(yǎng)基購于美國Gibco公司；胎牛血清購于浙江天杭生物科技有限公司；S100A9抗體購于美國Abcam公司；S100A9 siRNA和對照siRNA購于上海吉瑪制藥技術(shù)有限公司；脂質(zhì)體Lipofectamine 2000購于美國Invitrogen公司；Matrigel購于美國BD公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng) 將Caski細(xì)胞加入RPMI-1640培養(yǎng)基，C-33A細(xì)胞加入DMEM培養(yǎng)基，置于37℃、5%CO2的培養(yǎng)箱中進(jìn)行培養(yǎng)。2～3d換1次培養(yǎng)液。

1.2.2 C-33A細(xì)胞的病毒轉(zhuǎn)染 C-33A細(xì)胞以3×105/ml接種于6孔板，分為未轉(zhuǎn)染組、陰性對照組和轉(zhuǎn)染組。培養(yǎng)24h后，細(xì)胞融合至50%～60%，轉(zhuǎn)染組加入重組腺病毒Ad-S100A9，陰性對照組加入空載體Ad-RFP，孵育12h后，換普通培養(yǎng)液繼續(xù)培養(yǎng)36h，收集細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。未轉(zhuǎn)染組未予特殊處理。

1.2.3 Caski細(xì)胞的siRNA轉(zhuǎn)染 Caski細(xì)胞以2×105/ml接種于6孔板，培養(yǎng)24h后，細(xì)胞融合至30%～50%，換成無血清的培養(yǎng)基。將稀釋好的S100A9 siRNA（5′-CCUUGAACUCUAUCGACGUCUA-3′）與脂質(zhì)體Lipofectamine 2000輕輕混勻，在室溫下放置20min。加入6孔板孵育6h后，換成完全培養(yǎng)基，轉(zhuǎn)染48h后收集細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)，以陰性序列作為陰性對照。

1.2.4 Western blot檢測細(xì)胞S100A9蛋白的表達(dá) 收集各組細(xì)胞，用裂解液裂解、超聲處理后，采用二喹啉甲酸（BCA）法測定。經(jīng)12%SDS-PAGE凝膠電泳后轉(zhuǎn)膜。4℃搖床上一抗（S100A9，1∶1 000，Tubulin，1∶2 000）孵育過夜，TBST洗膜10min×3次；在辣根過氧化物酶標(biāo)記的IgG二抗中室溫孵育2h，TBST洗膜10min×3次；均勻滴加曝光液于膜上后，采用Bio-Rad公司的圖像攝取系統(tǒng)曝光并保存圖像，測得吸光度值，即細(xì)胞S100A9蛋白的表達(dá)。

1.2.5 Transwell檢測細(xì)胞遷移能力 各組細(xì)胞用無血清培養(yǎng)基重懸后，加入Transwell上室;下室加入含10%血清的完全培養(yǎng)基。孵育24h后取出上室，輕輕擦去上層未穿透的細(xì)胞，多聚甲醛固定及結(jié)晶紫染色后拍照，計(jì)數(shù)穿過小孔的細(xì)胞數(shù)。

1.2.6 Transwell檢測細(xì)胞侵襲能力 將Matrigel膠稀釋后鋪于上室，其余步驟同遷移實(shí)驗(yàn)。

1.3 統(tǒng)計(jì)學(xué)處理 應(yīng)用SPSS17.0統(tǒng)計(jì)軟件。計(jì)量資料呈正態(tài)分布，用表示，多組間比較采用單因素方差分析，兩組比較采用獨(dú)立樣本t檢驗(yàn)。

2 結(jié)果

2.1 轉(zhuǎn)染重組腺病毒后C-33A細(xì)胞中S100A9蛋白的表達(dá) Western blot結(jié)果顯示，與未轉(zhuǎn)染組、陰性對照組相比，轉(zhuǎn)染 S100A9重組腺病毒后C-33A細(xì)胞中S100A9蛋白的表達(dá)明顯增強(qiáng)，見圖1。

圖1 轉(zhuǎn)染S100A9重組腺病毒后C-33A細(xì)胞中S100A9蛋白表達(dá)的電泳圖

2.2 轉(zhuǎn)染S100A9重組腺病毒后C-33A細(xì)胞侵襲和遷移能力的變化

2.2.1 C-33A細(xì)胞侵襲能力的變化 Transwell檢測結(jié)果顯示，未轉(zhuǎn)染組、陰性對照組和轉(zhuǎn)染組的穿膜細(xì)胞數(shù)分別為（46.40±3.65）、（46.40±2.70）和（70.40±3.85）個；與未轉(zhuǎn)染組、陰性對照組相比，轉(zhuǎn)染組穿膜細(xì)胞數(shù)明顯增多（均P＜0.05）；而未轉(zhuǎn)染組與陰性對照組比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05），見圖2。

圖2 轉(zhuǎn)染S100A9重組腺病毒后C-33A細(xì)胞侵襲能力的變化（a：未轉(zhuǎn)染組；b：陰性對照組；c：轉(zhuǎn)染組；×400）

2.2.2 C-33A細(xì)胞遷移能力的變化 Transwell檢測結(jié)果顯示，未轉(zhuǎn)染組、陰性對照組和轉(zhuǎn)染組穿過Transwell小室的細(xì)胞數(shù)分別為（44.40±3.78）、（46.60±3.21）和（70.80±3.70）個，與未轉(zhuǎn)染組、陰性對照組相比，轉(zhuǎn)染組穿過Transwell小室的細(xì)胞數(shù)明顯增多（均P＜0.05）；而未轉(zhuǎn)染組與陰性對照組比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。

2.3 轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞中S100A9蛋白的表達(dá) Western blot結(jié)果顯示，與陰性對照組相比，轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞中S100A9蛋白的表達(dá)明顯降低，見圖3。

圖3 轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞中S100A9蛋白表達(dá)的電泳圖

2.4 轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞侵襲和遷移能力的變化

2.4.1 Caski細(xì)胞侵襲能力的變化 Transwell檢測結(jié)果顯示，siRNA組的穿膜細(xì)胞數(shù)為（40.20±3.27）個，明顯低于陰性對照組的（55.80±4.81）個（P＜0.05），見圖4。

圖4 轉(zhuǎn)染S100A9 siRNA后Caski細(xì)胞侵襲能力的變化（a：陰性對照組；b：siRNA組；×400）

2.4.2 Caski細(xì)胞遷移能力的變化 Transwell檢測結(jié)果顯示，siRNA組穿過Transwell小室的細(xì)胞數(shù)為（35.60± 6.80）個，明顯低于陰性對照組的（45.60±3.57）個（P＜0.05）。

3 討論

S100A9是一類只存在于脊椎動物中的小分子酸性蛋白[7]。人S100A9位于染色體lq21，此區(qū)域穩(wěn)定性差，容易發(fā)生缺失、異位和重疊等，并參與多種惡性腫瘤的發(fā)生、發(fā)展[8-9]。有文獻(xiàn)報(bào)道S100A9可抑制胃癌細(xì)胞的侵襲與轉(zhuǎn)移[10]，但多數(shù)研究發(fā)現(xiàn)S100A9可促進(jìn)多種惡性腫瘤的侵襲與轉(zhuǎn)移。Kwon等[11]發(fā)現(xiàn)S100A9可通過激活p38 MAPK/NF-κB信號通路，進(jìn)而促進(jìn)細(xì)胞的侵襲與遷移能力；在前列腺癌[12]、肝細(xì)胞癌[13]中，外源性增加S100A9的表達(dá)，可激活p38 MAPK/NF-κB信號通路，從而促進(jìn)細(xì)胞的遷移。在宮頸癌中，Zhao等[14]采用基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜發(fā)現(xiàn)有26個蛋白在正常宮頸組織、CIN和宮頸鱗癌中逐漸升高，其中包括S100A9蛋白。本課題組前期研究亦發(fā)現(xiàn)，在正常宮頸組織、CIN、宮頸癌中S100A9的表達(dá)逐漸升高[6]。但是，目前關(guān)于S100A9對宮頸鱗癌細(xì)胞生物學(xué)行為的影響尚未明確。

本研究發(fā)現(xiàn)轉(zhuǎn)染S100A9重組腺病毒能明顯提高C-33A細(xì)胞中 S100A9蛋白的表達(dá)，轉(zhuǎn)染 S100A9 siRNA能抑制Caski細(xì)胞中S100A9蛋白的表達(dá)。本實(shí)驗(yàn)通過上述方法改變宮頸鱗癌細(xì)胞中S100A9的表達(dá)，并觀察其對細(xì)胞侵襲和轉(zhuǎn)移能力的改變。宮頸鱗癌C-33A細(xì)胞轉(zhuǎn)染S100A9重組腺病毒，可促進(jìn)細(xì)胞侵襲與轉(zhuǎn)移的能力；宮頸鱗癌Caski細(xì)胞轉(zhuǎn)染S100A9 siRNA，可抑制細(xì)胞侵襲與轉(zhuǎn)移的能力。以上結(jié)果提示S100A9與宮頸鱗癌細(xì)胞的侵襲、轉(zhuǎn)移相關(guān)，但具體機(jī)制和通路有待進(jìn)一步研究。

[1]Basso D,Bozzato D,Padoan A,et al.Inflammation and pancreatic cancer:molecularand functionalinteractions between S100A8,S100A9,NT-S100A8andTGFβ1[J].CellCommunSignal, 2014,12:20.

[2]Gunaldi M,Okuturlar Y,Gedikbasi A,et al.Diagnostic importance of S100A9 and S100A12 in breast cancer[J].Biomed Pharmacother,2015,76:52-56.

[3]Nepomuceno A I,Shao H,Jing K,et al.In-depth LC-MS/MS analysis ofthe chicken ovarian cancerproteome reveals conserved and novel differentially regulated proteins in humans [J].Anal Bioanal Chem,2015,407(22):6851-6863.

[4]Ebbing J,Mathia S,Seibert F S,et al.Urinary calprotectin:a new diagnostic marker in urothelial carcinoma of the bladder[J].World J Urol,2014,32(6):1485-1492.

[5]Reeb A N,Li W,Sewell W,et al.S100A8 is a novel therapeutic target for anaplastic thyroid carcinoma[J].J Clin Endocrinol Metab,2015,100(2):E232-242.

[6]Zhu X,Jin L,Zou S,et al.Immunohistochemical expression of RAGE and its ligand(S100A9)in cervical lesions[J].Cell Biochem Biophys,2013,66(3):843-850.

[7]Leanderson T,Liberg D,Ivars F.S100A9 as a Pharmacological target molecule in inflammation and cancer[J].Endocr Metab Immune Disord Drug Targets,2015,15(2):97-104.

[8]Srikrishna G.S100A8 and S100A9:new insights into their roles in malignancy[J].J Innate Immun,2012,4(1):31-40.

[9]Lim M Y,Thomas P S.Biomarkers in exhaled breath condensate and serum ofchronicobstructivepulmonarydiseaseandnon-small-cell lung cance[J].International Journal of Chronic Diseases,2013,2013(11suppl):578613.

[10]Choi J H,Shin N R,Moon H J,et al.Identification of S100A8 and S100A9 as negative regulators for lymph node metastasis of gastric adenocarcinoma[J].Histol Histopathol,2012,27(11): 1439-1448.

[11]Kwon C H,Moon H J,Park H J,et al.S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells[J].Mol Cells,2013,35(3):226-234.

[12]Hermani A,De Servi B,Medunjanin S,et al.S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells[J]. Exp Cell Res,2006,312(2):184-197.

[13]Wu R,Duan L,Ye L,et al.S100A9 promotes the proliferation and invasion ofHepG2 hepatocellularcarcinoma cells via the activation of the MAPK signaling pathway[J].Int J Oncol,2013, 42(3):1001-1010.

[14]Zhao Q,He Y,Wang X L,et al.Differentially expressed proteins among normal cervix,cervical intraepithelial neoplasia and cervical squamous cell carcinoma[J].Clin Transl Oncol,2015,17 (8):620-631.

Relationship between S100A9 expression and invasive/metastatic ability of squamous cervical cancer cells

ObjectiveTo investigate the relationship between S100A9 expression and invasive/metastatic ability of squamous cervical cancer cells.MethodsHuman squamous cervical cancer C-33A cells were transfected with S100A9 adenoviral vectors and Caski cells were transfected with S100A9 siRNA.The expression of S100A9 protein was measured by Western blot and the ability of cell invasion and migration was evaluated by Transwell cell assays.ResultsCompared with control groups,S100A9 protein was significantly increased in C-33A cells after transfected with S100A9 adenoviral vectors.The invasion and migration assay showed that the number of cells migrating through the Transwell membrane and chamber were significantly increased after C-33A cells were transfected with S100A9 adenoviral vectors (P＜0.05).Compared with control group,the expression of S100A9 was significantly decreased in Caski cells transfected with S100A9 siRNA.In Transwell cell assays,the number of cells migrating through the Transwell membrane and chamber were significantly lower in 100A9 siRNA-transfected Caski cells compared with control group(P＜0.05).ConclusionS100A9 may play its role in the invasion and migration of squamous cervical cancer cells.Upregulation of S100A9 promoted invasion and migration of squamous cervical cancer cells,and downregulation of S100A9 reduced invasion and migration of squamous cervical cancer cells.

S100A9 Squamous cervical cancerInvasion Migration

2016-10-05）

（本文編輯：陳丹）

國家自然科學(xué)基金資助項(xiàng)目（81372381）；浙江省醫(yī)藥衛(wèi)生平臺重點(diǎn)資助計(jì)劃（2013ZDA016）

325027 溫州醫(yī)科大學(xué)附屬第二醫(yī)院婦產(chǎn)科

朱雪瓊，E-mail：zjwzzxq@163.com