硫酸乙酰肝素蛋白多糖与涎腺腺样囊性癌肺转移关系的研究
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摘要
目的:
涎腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）是最常见的涎腺恶性肿瘤，约占涎腺恶性肿瘤的22%。此肿瘤无包膜，侵袭性很强。嗜神经生长和远处转移是涎腺腺样囊性癌的两大恶性生物学特性，其中肺脏是涎腺腺样囊性癌远处转移最常发生的部位，也是临床上涎腺腺样囊性癌患者死亡的主要原因之一。
涎腺腺样囊性癌由肿瘤性肌上皮细胞（neoplastic myoepithelial cells,NMCs）和肿瘤性腺上皮细胞构成。肿瘤性肌上皮细胞具有双向分化的特征，它不仅具有正常肌上皮细胞的收缩功能，还具有平滑肌分泌蛋白多糖（proteoglycans, PGs）的功能。蛋白多糖分布于涎腺腺样囊性癌肿瘤细胞团块中，形成大小不等的囊样腔隙，呈筛孔状分布于肿瘤实质区，形成了涎腺腺样囊性癌特征性的组织学结构。蛋白多糖为涎腺腺样囊性癌的生长提供必要的营养来源。
硫酸乙酰肝素蛋白多糖（heparan sulfate proteoglycans, HSPGs）是一类由核心蛋白和（乙酰）硫酸肝素（heparan sulfate, HS）多糖链组成的蛋白多糖的总称。根据核心蛋白结构的不同，HSPGs主要包括位于细胞膜表面的磷脂酰肌醇蛋白多糖家族（glypicans, GPCs）和多配体蛋白多糖家族（syndecans, SDCs），以及位于细胞外基质中的基膜蛋白多糖（perlecan/HSPG2）、XVIII型胶原和集聚蛋白多糖（agrin）。GPCs家族包括六个成员：GPC1、GPC2、GPC3、GPC4、GPC5、GPC6；SDCs家族包括四个成员：SDC1、SDC2、SDC3、SDC4。以往的研究表明，HSPGs广泛分布于涎腺腺样囊性癌肿瘤细胞及细胞外基质。并可作为受体、共受体参与细胞增殖、细胞粘附、基质聚集、凝血等正常生理过程，也参与伤口愈合、炎症、肿瘤细胞的迁移及侵袭等过程。
本研究拟检测HSPGs，包括细胞膜HSPGs（SDCs家族和GPCs家族）与基膜HSPG（perlecan），在涎腺腺样囊性癌高转移细胞株SACC-M、低转移细胞株SACC-2、涎腺腺样囊性癌细胞株SACC-83及伴或不伴有肺转移涎腺腺样囊性癌组织中的表达，并分析探讨HSPGs异常表达，在涎腺腺样囊性癌肺转移过程中的作用。
方法：
第一部分硫酸乙酰肝素蛋白多糖在涎腺腺样囊性癌中的表达
1HSPGs在涎腺腺样囊性癌细胞株中的表达
本部分研究选择以下细胞株：
涎腺腺样囊性癌高转移细胞株SACC-M；
涎腺腺样囊性癌低转移细胞株SACC-2；
涎腺腺样囊性癌细胞株SACC-83。
1.1涎腺腺样囊性癌SACC-M、SACC-2、SACC-83的细胞培养将SACC-M、SACC-2、SACC-83细胞培养于含15%胎牛血清的RPMI-1640培养液（100U/ml青霉素，100U/ml链霉素，pH值为7.2）中，于37C、5%CO_2饱和湿度培养箱中培养。
1.2细胞总RNA提取
Trizol法分别提取SACC-M、SACC-2、SACC-83细胞总RNA；
分光光度计260nm及280nm波长检测RNA纯度及浓度；
1%琼脂糖凝胶电泳鉴定其完整性。
1.3反转录合成第一链cDNA
应用反转录试剂盒RevertAid First Strand cDNA Synthesis Kit合成第一链cDNA。
1.4Real-Time PCR检测
本研究采用Real-Time PCR（CT相对定量法）检测主要HSPGs，两大细胞膜HSPGs（SDCs家族和GPCs家族）与基膜HSPG（perlecan），在涎腺腺样囊性癌不同细胞株（SACC-M、SACC-2、SACC-83）的相对表达水平。
2检测GPC5蛋白在涎腺腺样囊性癌细胞株中的表达
本部分研究根据前述实验结果，选择高表达的硫酸乙酰肝素蛋白多糖——GPC5作为研究目的基因，检测其在不同转移潜能的涎腺腺样囊性癌细胞株中的表达。
2.1免疫荧光细胞化学检测
采用兔抗人GPC5单克隆抗体，通过免疫荧光细胞化学法检测GPC5蛋白在涎腺腺样囊性癌细胞株SACC-M、SACC-2和SACC-83细胞中的表达与分布。
2.2Western blot检测
采用鼠抗人GPC5单克隆抗体，通过Western blot检测GPC5蛋白在SACC-M、SACC-2、SACC-83细胞中的相对表达量。
3检测GPC5蛋白在伴或不伴有肺转移涎腺腺样囊性癌中的表达
收集临床涎腺腺样囊性癌16例，其中肺转移5例，无肺转移11例。免疫组织化学法检测GPC5蛋白在涎腺腺样囊性癌肿瘤组织中的表达，观察分析伴或不伴有肺转移涎腺腺样囊性癌肿瘤中GPC5蛋白的表达差异。
第二部分靶向沉默人GPC5基因miRNA干扰质粒的构建
1miRNA干扰载体的设计及构建
针对靶基因（human GPC5, Gene ID:2262）设计干扰序列并合成一套miRNA干扰载体。一套miRNA干扰载体包含4个miRNA oligos，以及阴性对照序列Oligo，退火后序列亚克隆到pcDNA6.2-GW/EmGFP-miR载体。
2测序验证
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序，以验证重组克隆中插入片段序列是否与设计的序列一致。
3质粒中量抽提
4质粒靶向沉默效果的鉴定
脂质体LipofectamineTM2000介导质粒转染SACC-M细胞，Real-TimePCR（CT相对定量法）检测4个构建质粒的沉默效率。
第三部分沉默人GPC5基因表达对涎腺腺样囊性癌肺转移作用的研究
1试验分组
基因沉默组：GPC5-silenced，GPC5干扰质粒转染SACC-M细胞组；
阴性对照组：GPC5-NC，阴性对照质粒NC转染SACC-M细胞组；
空白对照组：SACC-M，未转染质粒的SACC-M细胞组。
2建立稳定沉默人GPC5基因细胞株
2.1SACC-M细胞培养，确定杀稻瘟菌素对SACC-M细胞的最小致死浓度及筛选浓度。
2.2通过脂质体LipofectamineTM2000介导靶向沉默GPC5基因的miRNA质粒转染SACC-M细胞，并利用药物（杀稻瘟菌素）筛选单克隆，建立稳定沉默人GPC5基因表达的细胞株（GPC5-silenced细胞），同时转染阴性对照质粒（NC质粒）进入SACC-M细胞株，建立阴性对照细胞株（GPC5-NC细胞）。
2.3质粒靶向沉默效率的鉴定
mRNA水平鉴定：Real-Time PCR检测GPC5基因在GPC5-silenced、GPC5-NC、SACC-M细胞的表达；
蛋白水平鉴定：Western blot检测GPC5蛋白在GPC5-silenced、GPC5-NC、SACC-M细胞的表达。
3涎腺腺样囊性癌肺转移体内抑制试验
选择18只BLAB/C nu裸鼠，4周龄，雄性，随机分为3组，分别进行尾静脉注射GPC5-silenced细胞、GPC5-NC细胞和SACC-M细胞，2×107/ml×0.2ml/只。SPF条件下饲养，第4周麻醉处死，称重新鲜肺组织湿重。10%福尔马林固定，HE染色，光镜下观察，进行统计学分析。
结果：
第一部分硫酸乙酰肝素蛋白多糖在涎腺腺样囊性癌中的表达
1HSPGs在涎腺腺样囊性癌细胞株中的表达
Real-Time PCR相对定量（CT相对定量法）分析结果显示，与SACC-2细胞相比，11种主要HSPGs在SACC-M细胞中的表达，其中SDC2、GPC3、GPC5表达分别上调1.50、2.58、3.24倍；与SACC-83细胞相比，11种主要HSPGs在SACC-M细胞中的表达，其中SDC2和GPC5表达分别上调15.32倍和815.69倍。
GPC5在涎腺腺样囊性癌肺转移细胞株SACC-M细胞中的表达明显上调3倍以上。
2GPC5蛋白在SACC-M、SACC-2、SACC-83中的表达
免疫荧光细胞化学法检测结果显示，GPC5蛋白在涎腺腺样囊性癌细胞株SACC-M、SACC-2、SACC-83中均有表达，其中在涎腺腺样囊性癌肺转移株SACC-M中表达最高，SACC-2表达次之，在SACC-83细胞中呈弱表达。另外，GPC5不仅在细胞膜表面有表达，而且在细胞浆部位亦有表达。
Western blot检测结果显示，GPC5蛋白在涎腺腺样囊性癌肺转移细胞株SACC-M中的表达是SACC-2的3.41倍，是SACC-83表达的675.00倍。
3GPC5蛋白在伴或不伴有肺转移涎腺腺样囊性癌原发肿瘤中的表达
免疫组织化学检测结果显示，GPC5蛋白在涎腺腺样囊性癌肿瘤组织中表达，主要沉积在肿瘤细胞的细胞膜、细胞浆。另外，在部分假性囊腔和腺腔内细胞外基质中也有沉积。且GPC5蛋白在伴有肺转移的涎腺腺样囊性癌组织表达明显高于不伴有肺转移的肿瘤，差别具有显著性（P <0.05）。
第二部分靶向沉默人GPC5基因miRNA干扰质粒的构建
1构建靶向沉默GPC5基因的miRNA干扰质粒
成功构建靶向沉默GPC5基因的miRNA干扰质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2及阴性对照质粒NC，并进行测序验证；
2质粒靶向沉默效率的鉴定
脂质体介导质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2及NC转染SACC-M细胞。质粒转染48小时后，均可见有绿色荧光蛋白表达，分别收集细胞并提取细胞总RNA，Real-Time PCR检测结果显示，质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2对靶基因GPC5的沉默效率分别达到80.5%、78.1%、80.3%、89.7%。
第三部分沉默人GPC5基因表达对涎腺腺样囊性癌肺转移作用的研究
1建立稳定沉默GPC5基因的细胞株
通过脂质体介导miRNA质粒成功转染SACC-M细胞株，在基因转染48小时，加入杀稻瘟菌素，进行药物筛选，建立靶向沉默GPC5基因的稳定细胞株GPC5-silenced，以及阴性对照细胞株GPC5-NC。
mRNA水平检测结果显示沉默效率达到89.7%；
蛋白水平检测结果显示沉默效率达到96.5%。
2涎腺腺样囊性癌肺转移体内抑制试验
裸鼠肺转移试验结果显示，GPC5基因沉默组（GPC5-silenced）裸鼠肺转移2例（转移率33.33%），阴性对照组（GPC5-NC组）裸鼠发生肺转移6例（100%），空白对照组SACC-M组裸鼠肺转移6例（100%），GPC5-silenced组与GPC5-NC组、SACC-M组相比，差异具有显著性（P <0.05）；GPC5-NC组与SACC-M组比较，差异没有统计学意义（P>0.05）。
新鲜肺组织湿重，GPC5基因沉默组（GPC5-silenced组）肺湿重（0.507±0.223g）明显低于阴性对照GPC5-NC组（1.177±0.342g）和空白对照SACC-M组（1.045±0.539g），差异具有显著性（P <0.05）；GPC5-NC组与SACC-M组比较，差别无显著性（P>0.05）。
形态学观察发现，SACC-M组和GPC5-NC组中正常肺组织结构消失，双肺内充满肿瘤细胞转移灶，肿瘤灶呈圆形或椭圆形。肿瘤细胞排列成致密的实性团块。肿瘤细胞圆形或多边性，排列紧密，胞浆嗜伊红染色，着色较浅，细胞胞核呈圆形，核分裂像多见，细胞核异型性明显。另外，肺组织内可见大量毛细血管扩张、充血。GPC5-silenced组2个肺转移标本中，正常肺组织结构清晰，偶见散在分布的肿瘤转移灶，且体积较SACC-M组及GPC5-NC组转移灶小，包膜不完整包膜。其余4个肺组织未见转移，肺组织结构正常。
结论：
硫酸乙酰肝素蛋白多糖GPC5基因高表达，能够增强涎腺腺样囊性癌肺转移的能力。
Objectives:
Salivary adenoid cystic carcinoma (SACC) is one of the most commonsalivary malignant tumors without envelope and strongly aggressive. Itaccounts for about22%of the salivary gland malignanies. SACC has a highpropensity of perineural invasion and distant metastasis, the most common siteof distant metastases is the lung which is also the chief cause of clinical death.
SACC is composed of duct epithelial cells and neoplastic myoepithelialcells (NMCs) which are the chief proliferative cells of the tumor. Because ofthe bilateral differentiation, NMCs hold the function of constractile andsecreting proteoglycans which is different from the normal myoepithelial cells.Abundant proteoglycans produced by NMCs make SACC typical cribriformstructures full with proteoglycans and provide nutrition and microenvironmentfor the biological behavior of SACC.
Heparan sulfate proteoglycans (HSPGs), consist of a protein core towhich heparan sulphate glycosaminoglycan chains are attached are abundantcell-surface and extra cellular matrix (ECM). Secreted extra cellular matrixHSPGs include perlecan (HSPG2), agrin and type XVIII collagen. Syndecansand glypicans represent the two major membrane HSPGs, in mammals fourseparate syndecans (SDC1, SDC2, SDC3, and SDC4) and six separateglypicans (GPC1, GPC2, GPC3, GPC4, GPC5, and GPC6) have beenidentified. The syndecans have a transmembrane and cytoplasmic domain,whereas the glypicans are anchored to the extracytoplasmic face of the plasmamembrane via glycosylphosphatidylinositol (GPI).
HSPGs have been shown to act as receptors, co-receptors and playcentral role in many biological process such as cell proliferation, celladhesion/anti-adhesion, inflammation, wound healing, coagulation, matrix assembly, embryo development, tumor metastasis.
In present study, the two major membrane HSPGs and perlecanexpression were analyzed in SACC with and without lung metastasis toexplore the role of HSPGs in the lung metastasis of SACC.
Methods:
Part I HSPGs expression in SACC
1HSPGs expression in SACC cell lines
In this study, the following cell lines with different metastatic abilitywere used:
Highly metastatic SACC cell line: SACC-M cell line;
Poorly metastatic SACC cell lines: SACC-2;
SACC cell lines: SACC-83cell lines.
1.1Cell culture
SACC-M, SACC-2, SACC-83cells were routinely cultured inRPMI-1640medium containing15%fetal bovine serum, penicillin (100U/ml),and streptomycin (100U/ml) at37C in humidified air containing5%CO2.
1.2Total RNA extraction
Total RNA was extracted from the tumor cells by using TRIzol reagent(Invitrogen, USA) following the manufacturer’s instructions. RNAconcentration was obtained through spectrophotometric readings at260and280nm, and quality was evaluated by1%agarose gel electrophoresis.
1.3Reverse transcription
Three microgram of total RNA from the sample were used in reversetranscribed with RevertAid First Strand cDNA Synthesis Kit.1.4Real-Time PCR
In this study, Real-Time PCR (CTRelative quantification) was used totest the expression of HSPGs, the two major membrane HSPGs and perlecan,in SACC cell lines with highly and poorly metastatic ability.
2GPC5protein expression in SACC cell lines
In this part, we choose the high expression HSPG (GPC5) as the studygene, and investigated the GPC5expression in SACC cell lines.
2.1Immunolocalization
GPC5expression and distribution in SACC-M, SACC-2, SACC-83cellswas tested by Immunofluorescence staining with rabbit anti-GPC5monoclonal antibody.
2.2Western blot analysis
The relative quantification of GPC5expression level in SACC-M,SACC-2, SACC-83cells was tested by Western blot with Mouse Anti-GPC5Monoclonal Antibody.
3GPC5expression in SACC with and without lung metastasis
Sixteen cases of salivary adenoid cystic carcinoma (SACC) werecollected from Department of Oral Pathology and collected from Departmentof Oral and Maxillofacial Surgery, College&Hospital of Stomatology, andthe Fourth Hospital, Hebei Medical University. Five out of16cases with lungmetastasis, the other11cases without lung metastasis. Immunohistochemistrystaining was used to investigate the GPC5expression in SACC with andwithout lung metastasis.
Part II construction of GPC5miRNA expression vector
1Construction of GPC5miRNA expression vector
The human gene GPC5(glypican5, Gene ID:2262) was selected fromGenBank. Go to the BLOCK-iT RNAi Designer and input the accessionnumber (NM-004466), the Designer will automatically generate highprobability DNA duplexes miRNA oligos. Hybridize the oligos to form a60-bp duplex with4-nt5′overhangs. Cohesively ligate the duplex into theBLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP.
2Sequence validation
Three clonies from each plate were picked and sequenced to verify therecombinational cloning insertion sequence and oligo sequence alignmentdesign.
3Plasmid midi-preparation
4Evaluation of silencing efficiency
Plasmids were transfected into SACC-M cells through LipofectamineTM2000, and the silencing efficiency was tested by Real-TimePCR.
Part III The effect of GPC5down-regulation on the lung metastasis ofSACC in vivo
1Group of experiment
Gene silenced group: Group GPC5-silenced, SACC-M cells transfectedwith miRNA vector targeting GPC5;
Negative control group: Group GPC5-NC, SACC-M cells transfectedwith negative control miRNA vector;
Black control group: Group SACC-M, SACC-M cells withouttransfection.
2Establishment of stable cell lines with silenced GPC5
2.1SACC-M cell cultrue and definition of the minimal lethal concentration
2.2SACC-M cells were transfected with miRNA plasmid targeting GPC5according to the manufacturer’s of LipofectamineTM2000, transfected with NCmiRNA plasmid as negative control. Three weeks after transfection, stable cellclones (GPC5-silenced and GPC5-NC cell lines) were aquired by usingBlasticidin S HCl.
2.3Evaluation of silencing efficiency
mRNA level: the GPC5mRNA expression was evaluted by Real-TimePCR.
Protein level: the GPC5protein expression was evaluted by Western blot.4The inhibition of lung metastasis of SACC in vivo
Eighteen four-week-old,14~15g, male BALB/C nude mice were dividedinto3groups of6mice each randomly. GPC5-silenced cells, GPC5-NC cellsand SACC-M cells were collected respectively,0.2ml cell suspensioncontaining1×107cells per milliliter was injected into the vena caudalis. Themice were anesthetized and killed at the4th week after injection. The freshlung samples were harvested and weighed. Formalin fixed/paraffin-embeddedspecimens were prepared by ordinary procedures,2.5μm thick sections werestained with hematoxylin and eosin (HE), and then examined by the microscope to evaluate the morphological changes.
Results:
Part I HSPGs expression in SACC
1HSPGs expression in highly and poorly metastatic SACC cell lines
By relative-quantitive Real-Time PCR, HSPGs were expressed atdifferent levels in the three kinds of SACC cell lines, with no expression ofGPC4. Compared with SACC-2, the mRNA expression of SDC2, GPC3andGPC5in SACC-M cell line increased1.50-flod,2.58-fold and3.24-foldrespectively. And two HSPGs (SDC2and GPC5) expression increased(15.32-fold and815.69-fold) in SACC-M compared with SACC-83, onlyGPC5increased significantly (more than3-fold), while others decreased inSACC-M compared with both SACC-2and SACC-83cells.
2GPC5protein expression in highly and poorly metastatic SACC cell lines
2.1Immunolocalization
By Immunofluorescence staining, GPC5expressed not only on cellmembrane, it also be found in cytoplasm of SACC cells. GPC5expressed atdifferent levels in the SACC-M, SACC-2and SACC-83cell lines, weaklypositive in SACC-83cells, and strong positive in SACC-M cells.
2.2Western blot analysis
Western blot analysis showed that the expression level of GPC5proteinin SACC-M was3.41-flod as SACC-2and675.00-flod as SACC-83cells.This is consistent with the results of Real-Time PCR and immunofluorescencestaining.
3GPC5expression in SACC with and without lung metastasis
In16clinical cases of SACC which were confirmed by pathologicaldiagnosis as SACC with description as cribriform, tumular and solid type, theexpression of GPC5showed strong positive in4cases out of5, and1caseshowed positive in SACC with lung metastasis, whereas negative in3out of11cases,7cases with weakly positive and1case with positive in SACCwithout lung metastasis. The overexpression of GPC5was significantlyassociated with lung metastasis (P <0.05). And the GPC5expression chiefly distributed in the tumor cell membrane and cytoplasm, and extracellularmatrix (ECM) deposit in pseudocyte and lumens.
Part II Construction of miRNA expression vector targeting GPC5
Successful construction of miRNA plasmid targeting human GPC5gene:12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2andnegative control plasmid NC. Fourty-eight hours after the transfection of12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2and NCplasmid into SACC-M cells, green fluorescent protein was expressed, and thesilencing efficiency of each plasmid up to80.5%、78.1%、80.3%、89.7%respectively.
Part III The effect of GPC5down-regulation on the lung metastasis ofSACC in vivo
1Establishment of stable cell lines with silenced GPC5
Forty-eight hours after transfection of plasmid targeting GPC5and NCplasmid, stable cell clones GPC5-silenced and GPC5-NC cell were acquiredby using Blasticidin S HCl. Compared with SACC-M cell, the GPC5expression of GPC5-silenced cell was down-regulated by89.7%at mRNAlevel, and96.5%at protein level.
2The inhibition of lung metastasis of SACC in vivo
The4th week after injection, The mice were anesthetized and killed andweighed, the lung wet weight of group GPC5-silenced (0.507±0.223g) wassignificantly lower than that of group GPC5-NC (1.177±0.342g) and groupSACC-M (1.045±0.539g), P <0.05. No significant difference betweengroup SACC-M and group GPC5-NC, P>0.05.
The lung metastasis of group GPC5-silenced decreased to33.33%(2/6),significantly lower than that of group SACC-M (100%,6/6) and groupGPC5-NC (100%,6/6), P <0.05.
The histopathological analysis of the lung samples of group SACC-Mand GPC5-NC showed that the normal structures of lungs in group werealmost destroyed. The lungs were full of metastasis nodules with differentsizes. The tumor cells were round or polyhedral with eosinophilic cytoplasm and round nucleus, arranged in dense clumps of solid, many mitoses could beobserved. Pulmonary vascular cavities were in condition of hyperaemia. Onlya few metastasis nodules were found in the2lung samples of groupGPC5-silenced and the normal structure of them was still remained. The sizeof the metastasis nodle in group GPC5-silenced was much smaller than that ofgroup SACC-M and group GPC5-NC.
Conclusion:
The high expression of heparan sulfate proteoglycan (GPC5) promotesthe metastasis ability of SACC.
涎腺腺样囊性癌（salivary adenoid cystic carcinoma, SACC）是最常见的涎腺恶性肿瘤，约占涎腺恶性肿瘤的22%。此肿瘤无包膜，侵袭性很强。嗜神经生长和远处转移是涎腺腺样囊性癌的两大恶性生物学特性，其中肺脏是涎腺腺样囊性癌远处转移最常发生的部位，也是临床上涎腺腺样囊性癌患者死亡的主要原因之一。
涎腺腺样囊性癌由肿瘤性肌上皮细胞（neoplastic myoepithelial cells,NMCs）和肿瘤性腺上皮细胞构成。肿瘤性肌上皮细胞具有双向分化的特征，它不仅具有正常肌上皮细胞的收缩功能，还具有平滑肌分泌蛋白多糖（proteoglycans, PGs）的功能。蛋白多糖分布于涎腺腺样囊性癌肿瘤细胞团块中，形成大小不等的囊样腔隙，呈筛孔状分布于肿瘤实质区，形成了涎腺腺样囊性癌特征性的组织学结构。蛋白多糖为涎腺腺样囊性癌的生长提供必要的营养来源。
硫酸乙酰肝素蛋白多糖（heparan sulfate proteoglycans, HSPGs）是一类由核心蛋白和（乙酰）硫酸肝素（heparan sulfate, HS）多糖链组成的蛋白多糖的总称。根据核心蛋白结构的不同，HSPGs主要包括位于细胞膜表面的磷脂酰肌醇蛋白多糖家族（glypicans, GPCs）和多配体蛋白多糖家族（syndecans, SDCs），以及位于细胞外基质中的基膜蛋白多糖（perlecan/HSPG2）、XVIII型胶原和集聚蛋白多糖（agrin）。GPCs家族包括六个成员：GPC1、GPC2、GPC3、GPC4、GPC5、GPC6；SDCs家族包括四个成员：SDC1、SDC2、SDC3、SDC4。以往的研究表明，HSPGs广泛分布于涎腺腺样囊性癌肿瘤细胞及细胞外基质。并可作为受体、共受体参与细胞增殖、细胞粘附、基质聚集、凝血等正常生理过程，也参与伤口愈合、炎症、肿瘤细胞的迁移及侵袭等过程。
本研究拟检测HSPGs，包括细胞膜HSPGs（SDCs家族和GPCs家族）与基膜HSPG（perlecan），在涎腺腺样囊性癌高转移细胞株SACC-M、低转移细胞株SACC-2、涎腺腺样囊性癌细胞株SACC-83及伴或不伴有肺转移涎腺腺样囊性癌组织中的表达，并分析探讨HSPGs异常表达，在涎腺腺样囊性癌肺转移过程中的作用。
方法：
第一部分硫酸乙酰肝素蛋白多糖在涎腺腺样囊性癌中的表达
1HSPGs在涎腺腺样囊性癌细胞株中的表达
本部分研究选择以下细胞株：
涎腺腺样囊性癌高转移细胞株SACC-M；
涎腺腺样囊性癌低转移细胞株SACC-2；
涎腺腺样囊性癌细胞株SACC-83。
1.1涎腺腺样囊性癌SACC-M、SACC-2、SACC-83的细胞培养将SACC-M、SACC-2、SACC-83细胞培养于含15%胎牛血清的RPMI-1640培养液（100U/ml青霉素，100U/ml链霉素，pH值为7.2）中，于37C、5%CO_2饱和湿度培养箱中培养。
1.2细胞总RNA提取
Trizol法分别提取SACC-M、SACC-2、SACC-83细胞总RNA；
分光光度计260nm及280nm波长检测RNA纯度及浓度；
1%琼脂糖凝胶电泳鉴定其完整性。
1.3反转录合成第一链cDNA
应用反转录试剂盒RevertAid First Strand cDNA Synthesis Kit合成第一链cDNA。
1.4Real-Time PCR检测
本研究采用Real-Time PCR（CT相对定量法）检测主要HSPGs，两大细胞膜HSPGs（SDCs家族和GPCs家族）与基膜HSPG（perlecan），在涎腺腺样囊性癌不同细胞株（SACC-M、SACC-2、SACC-83）的相对表达水平。
2检测GPC5蛋白在涎腺腺样囊性癌细胞株中的表达
本部分研究根据前述实验结果，选择高表达的硫酸乙酰肝素蛋白多糖——GPC5作为研究目的基因，检测其在不同转移潜能的涎腺腺样囊性癌细胞株中的表达。
2.1免疫荧光细胞化学检测
采用兔抗人GPC5单克隆抗体，通过免疫荧光细胞化学法检测GPC5蛋白在涎腺腺样囊性癌细胞株SACC-M、SACC-2和SACC-83细胞中的表达与分布。
2.2Western blot检测
采用鼠抗人GPC5单克隆抗体，通过Western blot检测GPC5蛋白在SACC-M、SACC-2、SACC-83细胞中的相对表达量。
3检测GPC5蛋白在伴或不伴有肺转移涎腺腺样囊性癌中的表达
收集临床涎腺腺样囊性癌16例，其中肺转移5例，无肺转移11例。免疫组织化学法检测GPC5蛋白在涎腺腺样囊性癌肿瘤组织中的表达，观察分析伴或不伴有肺转移涎腺腺样囊性癌肿瘤中GPC5蛋白的表达差异。
第二部分靶向沉默人GPC5基因miRNA干扰质粒的构建
1miRNA干扰载体的设计及构建
针对靶基因（human GPC5, Gene ID:2262）设计干扰序列并合成一套miRNA干扰载体。一套miRNA干扰载体包含4个miRNA oligos，以及阴性对照序列Oligo，退火后序列亚克隆到pcDNA6.2-GW/EmGFP-miR载体。
2测序验证
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序，以验证重组克隆中插入片段序列是否与设计的序列一致。
3质粒中量抽提
4质粒靶向沉默效果的鉴定
脂质体LipofectamineTM2000介导质粒转染SACC-M细胞，Real-TimePCR（CT相对定量法）检测4个构建质粒的沉默效率。
第三部分沉默人GPC5基因表达对涎腺腺样囊性癌肺转移作用的研究
1试验分组
基因沉默组：GPC5-silenced，GPC5干扰质粒转染SACC-M细胞组；
阴性对照组：GPC5-NC，阴性对照质粒NC转染SACC-M细胞组；
空白对照组：SACC-M，未转染质粒的SACC-M细胞组。
2建立稳定沉默人GPC5基因细胞株
2.1SACC-M细胞培养，确定杀稻瘟菌素对SACC-M细胞的最小致死浓度及筛选浓度。
2.2通过脂质体LipofectamineTM2000介导靶向沉默GPC5基因的miRNA质粒转染SACC-M细胞，并利用药物（杀稻瘟菌素）筛选单克隆，建立稳定沉默人GPC5基因表达的细胞株（GPC5-silenced细胞），同时转染阴性对照质粒（NC质粒）进入SACC-M细胞株，建立阴性对照细胞株（GPC5-NC细胞）。
2.3质粒靶向沉默效率的鉴定
mRNA水平鉴定：Real-Time PCR检测GPC5基因在GPC5-silenced、GPC5-NC、SACC-M细胞的表达；
蛋白水平鉴定：Western blot检测GPC5蛋白在GPC5-silenced、GPC5-NC、SACC-M细胞的表达。
3涎腺腺样囊性癌肺转移体内抑制试验
选择18只BLAB/C nu裸鼠，4周龄，雄性，随机分为3组，分别进行尾静脉注射GPC5-silenced细胞、GPC5-NC细胞和SACC-M细胞，2×107/ml×0.2ml/只。SPF条件下饲养，第4周麻醉处死，称重新鲜肺组织湿重。10%福尔马林固定，HE染色，光镜下观察，进行统计学分析。
结果：
第一部分硫酸乙酰肝素蛋白多糖在涎腺腺样囊性癌中的表达
1HSPGs在涎腺腺样囊性癌细胞株中的表达
Real-Time PCR相对定量（CT相对定量法）分析结果显示，与SACC-2细胞相比，11种主要HSPGs在SACC-M细胞中的表达，其中SDC2、GPC3、GPC5表达分别上调1.50、2.58、3.24倍；与SACC-83细胞相比，11种主要HSPGs在SACC-M细胞中的表达，其中SDC2和GPC5表达分别上调15.32倍和815.69倍。
GPC5在涎腺腺样囊性癌肺转移细胞株SACC-M细胞中的表达明显上调3倍以上。
2GPC5蛋白在SACC-M、SACC-2、SACC-83中的表达
免疫荧光细胞化学法检测结果显示，GPC5蛋白在涎腺腺样囊性癌细胞株SACC-M、SACC-2、SACC-83中均有表达，其中在涎腺腺样囊性癌肺转移株SACC-M中表达最高，SACC-2表达次之，在SACC-83细胞中呈弱表达。另外，GPC5不仅在细胞膜表面有表达，而且在细胞浆部位亦有表达。
Western blot检测结果显示，GPC5蛋白在涎腺腺样囊性癌肺转移细胞株SACC-M中的表达是SACC-2的3.41倍，是SACC-83表达的675.00倍。
3GPC5蛋白在伴或不伴有肺转移涎腺腺样囊性癌原发肿瘤中的表达
免疫组织化学检测结果显示，GPC5蛋白在涎腺腺样囊性癌肿瘤组织中表达，主要沉积在肿瘤细胞的细胞膜、细胞浆。另外，在部分假性囊腔和腺腔内细胞外基质中也有沉积。且GPC5蛋白在伴有肺转移的涎腺腺样囊性癌组织表达明显高于不伴有肺转移的肿瘤，差别具有显著性（P <0.05）。
第二部分靶向沉默人GPC5基因miRNA干扰质粒的构建
1构建靶向沉默GPC5基因的miRNA干扰质粒
成功构建靶向沉默GPC5基因的miRNA干扰质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2及阴性对照质粒NC，并进行测序验证；
2质粒靶向沉默效率的鉴定
脂质体介导质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2及NC转染SACC-M细胞。质粒转染48小时后，均可见有绿色荧光蛋白表达，分别收集细胞并提取细胞总RNA，Real-Time PCR检测结果显示，质粒12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2对靶基因GPC5的沉默效率分别达到80.5%、78.1%、80.3%、89.7%。
第三部分沉默人GPC5基因表达对涎腺腺样囊性癌肺转移作用的研究
1建立稳定沉默GPC5基因的细胞株
通过脂质体介导miRNA质粒成功转染SACC-M细胞株，在基因转染48小时，加入杀稻瘟菌素，进行药物筛选，建立靶向沉默GPC5基因的稳定细胞株GPC5-silenced，以及阴性对照细胞株GPC5-NC。
mRNA水平检测结果显示沉默效率达到89.7%；
蛋白水平检测结果显示沉默效率达到96.5%。
2涎腺腺样囊性癌肺转移体内抑制试验
裸鼠肺转移试验结果显示，GPC5基因沉默组（GPC5-silenced）裸鼠肺转移2例（转移率33.33%），阴性对照组（GPC5-NC组）裸鼠发生肺转移6例（100%），空白对照组SACC-M组裸鼠肺转移6例（100%），GPC5-silenced组与GPC5-NC组、SACC-M组相比，差异具有显著性（P <0.05）；GPC5-NC组与SACC-M组比较，差异没有统计学意义（P>0.05）。
新鲜肺组织湿重，GPC5基因沉默组（GPC5-silenced组）肺湿重（0.507±0.223g）明显低于阴性对照GPC5-NC组（1.177±0.342g）和空白对照SACC-M组（1.045±0.539g），差异具有显著性（P <0.05）；GPC5-NC组与SACC-M组比较，差别无显著性（P>0.05）。
形态学观察发现，SACC-M组和GPC5-NC组中正常肺组织结构消失，双肺内充满肿瘤细胞转移灶，肿瘤灶呈圆形或椭圆形。肿瘤细胞排列成致密的实性团块。肿瘤细胞圆形或多边性，排列紧密，胞浆嗜伊红染色，着色较浅，细胞胞核呈圆形，核分裂像多见，细胞核异型性明显。另外，肺组织内可见大量毛细血管扩张、充血。GPC5-silenced组2个肺转移标本中，正常肺组织结构清晰，偶见散在分布的肿瘤转移灶，且体积较SACC-M组及GPC5-NC组转移灶小，包膜不完整包膜。其余4个肺组织未见转移，肺组织结构正常。
结论：
硫酸乙酰肝素蛋白多糖GPC5基因高表达，能够增强涎腺腺样囊性癌肺转移的能力。
Objectives:
Salivary adenoid cystic carcinoma (SACC) is one of the most commonsalivary malignant tumors without envelope and strongly aggressive. Itaccounts for about22%of the salivary gland malignanies. SACC has a highpropensity of perineural invasion and distant metastasis, the most common siteof distant metastases is the lung which is also the chief cause of clinical death.
SACC is composed of duct epithelial cells and neoplastic myoepithelialcells (NMCs) which are the chief proliferative cells of the tumor. Because ofthe bilateral differentiation, NMCs hold the function of constractile andsecreting proteoglycans which is different from the normal myoepithelial cells.Abundant proteoglycans produced by NMCs make SACC typical cribriformstructures full with proteoglycans and provide nutrition and microenvironmentfor the biological behavior of SACC.
Heparan sulfate proteoglycans (HSPGs), consist of a protein core towhich heparan sulphate glycosaminoglycan chains are attached are abundantcell-surface and extra cellular matrix (ECM). Secreted extra cellular matrixHSPGs include perlecan (HSPG2), agrin and type XVIII collagen. Syndecansand glypicans represent the two major membrane HSPGs, in mammals fourseparate syndecans (SDC1, SDC2, SDC3, and SDC4) and six separateglypicans (GPC1, GPC2, GPC3, GPC4, GPC5, and GPC6) have beenidentified. The syndecans have a transmembrane and cytoplasmic domain,whereas the glypicans are anchored to the extracytoplasmic face of the plasmamembrane via glycosylphosphatidylinositol (GPI).
HSPGs have been shown to act as receptors, co-receptors and playcentral role in many biological process such as cell proliferation, celladhesion/anti-adhesion, inflammation, wound healing, coagulation, matrix assembly, embryo development, tumor metastasis.
In present study, the two major membrane HSPGs and perlecanexpression were analyzed in SACC with and without lung metastasis toexplore the role of HSPGs in the lung metastasis of SACC.
Methods:
Part I HSPGs expression in SACC
1HSPGs expression in SACC cell lines
In this study, the following cell lines with different metastatic abilitywere used:
Highly metastatic SACC cell line: SACC-M cell line;
Poorly metastatic SACC cell lines: SACC-2;
SACC cell lines: SACC-83cell lines.
1.1Cell culture
SACC-M, SACC-2, SACC-83cells were routinely cultured inRPMI-1640medium containing15%fetal bovine serum, penicillin (100U/ml),and streptomycin (100U/ml) at37C in humidified air containing5%CO2.
1.2Total RNA extraction
Total RNA was extracted from the tumor cells by using TRIzol reagent(Invitrogen, USA) following the manufacturer’s instructions. RNAconcentration was obtained through spectrophotometric readings at260and280nm, and quality was evaluated by1%agarose gel electrophoresis.
1.3Reverse transcription
Three microgram of total RNA from the sample were used in reversetranscribed with RevertAid First Strand cDNA Synthesis Kit.1.4Real-Time PCR
In this study, Real-Time PCR (CTRelative quantification) was used totest the expression of HSPGs, the two major membrane HSPGs and perlecan,in SACC cell lines with highly and poorly metastatic ability.
2GPC5protein expression in SACC cell lines
In this part, we choose the high expression HSPG (GPC5) as the studygene, and investigated the GPC5expression in SACC cell lines.
2.1Immunolocalization
GPC5expression and distribution in SACC-M, SACC-2, SACC-83cellswas tested by Immunofluorescence staining with rabbit anti-GPC5monoclonal antibody.
2.2Western blot analysis
The relative quantification of GPC5expression level in SACC-M,SACC-2, SACC-83cells was tested by Western blot with Mouse Anti-GPC5Monoclonal Antibody.
3GPC5expression in SACC with and without lung metastasis
Sixteen cases of salivary adenoid cystic carcinoma (SACC) werecollected from Department of Oral Pathology and collected from Departmentof Oral and Maxillofacial Surgery, College&Hospital of Stomatology, andthe Fourth Hospital, Hebei Medical University. Five out of16cases with lungmetastasis, the other11cases without lung metastasis. Immunohistochemistrystaining was used to investigate the GPC5expression in SACC with andwithout lung metastasis.
Part II construction of GPC5miRNA expression vector
1Construction of GPC5miRNA expression vector
The human gene GPC5(glypican5, Gene ID:2262) was selected fromGenBank. Go to the BLOCK-iT RNAi Designer and input the accessionnumber (NM-004466), the Designer will automatically generate highprobability DNA duplexes miRNA oligos. Hybridize the oligos to form a60-bp duplex with4-nt5′overhangs. Cohesively ligate the duplex into theBLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP.
2Sequence validation
Three clonies from each plate were picked and sequenced to verify therecombinational cloning insertion sequence and oligo sequence alignmentdesign.
3Plasmid midi-preparation
4Evaluation of silencing efficiency
Plasmids were transfected into SACC-M cells through LipofectamineTM2000, and the silencing efficiency was tested by Real-TimePCR.
Part III The effect of GPC5down-regulation on the lung metastasis ofSACC in vivo
1Group of experiment
Gene silenced group: Group GPC5-silenced, SACC-M cells transfectedwith miRNA vector targeting GPC5;
Negative control group: Group GPC5-NC, SACC-M cells transfectedwith negative control miRNA vector;
Black control group: Group SACC-M, SACC-M cells withouttransfection.
2Establishment of stable cell lines with silenced GPC5
2.1SACC-M cell cultrue and definition of the minimal lethal concentration
2.2SACC-M cells were transfected with miRNA plasmid targeting GPC5according to the manufacturer’s of LipofectamineTM2000, transfected with NCmiRNA plasmid as negative control. Three weeks after transfection, stable cellclones (GPC5-silenced and GPC5-NC cell lines) were aquired by usingBlasticidin S HCl.
2.3Evaluation of silencing efficiency
mRNA level: the GPC5mRNA expression was evaluted by Real-TimePCR.
Protein level: the GPC5protein expression was evaluted by Western blot.4The inhibition of lung metastasis of SACC in vivo
Eighteen four-week-old,14~15g, male BALB/C nude mice were dividedinto3groups of6mice each randomly. GPC5-silenced cells, GPC5-NC cellsand SACC-M cells were collected respectively,0.2ml cell suspensioncontaining1×107cells per milliliter was injected into the vena caudalis. Themice were anesthetized and killed at the4th week after injection. The freshlung samples were harvested and weighed. Formalin fixed/paraffin-embeddedspecimens were prepared by ordinary procedures,2.5μm thick sections werestained with hematoxylin and eosin (HE), and then examined by the microscope to evaluate the morphological changes.
Results:
Part I HSPGs expression in SACC
1HSPGs expression in highly and poorly metastatic SACC cell lines
By relative-quantitive Real-Time PCR, HSPGs were expressed atdifferent levels in the three kinds of SACC cell lines, with no expression ofGPC4. Compared with SACC-2, the mRNA expression of SDC2, GPC3andGPC5in SACC-M cell line increased1.50-flod,2.58-fold and3.24-foldrespectively. And two HSPGs (SDC2and GPC5) expression increased(15.32-fold and815.69-fold) in SACC-M compared with SACC-83, onlyGPC5increased significantly (more than3-fold), while others decreased inSACC-M compared with both SACC-2and SACC-83cells.
2GPC5protein expression in highly and poorly metastatic SACC cell lines
2.1Immunolocalization
By Immunofluorescence staining, GPC5expressed not only on cellmembrane, it also be found in cytoplasm of SACC cells. GPC5expressed atdifferent levels in the SACC-M, SACC-2and SACC-83cell lines, weaklypositive in SACC-83cells, and strong positive in SACC-M cells.
2.2Western blot analysis
Western blot analysis showed that the expression level of GPC5proteinin SACC-M was3.41-flod as SACC-2and675.00-flod as SACC-83cells.This is consistent with the results of Real-Time PCR and immunofluorescencestaining.
3GPC5expression in SACC with and without lung metastasis
In16clinical cases of SACC which were confirmed by pathologicaldiagnosis as SACC with description as cribriform, tumular and solid type, theexpression of GPC5showed strong positive in4cases out of5, and1caseshowed positive in SACC with lung metastasis, whereas negative in3out of11cases,7cases with weakly positive and1case with positive in SACCwithout lung metastasis. The overexpression of GPC5was significantlyassociated with lung metastasis (P <0.05). And the GPC5expression chiefly distributed in the tumor cell membrane and cytoplasm, and extracellularmatrix (ECM) deposit in pseudocyte and lumens.
Part II Construction of miRNA expression vector targeting GPC5
Successful construction of miRNA plasmid targeting human GPC5gene:12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2andnegative control plasmid NC. Fourty-eight hours after the transfection of12MR0055-1-1、12MR0055-2-1、12MR0055-3-1、12MR0055-4-2and NCplasmid into SACC-M cells, green fluorescent protein was expressed, and thesilencing efficiency of each plasmid up to80.5%、78.1%、80.3%、89.7%respectively.
Part III The effect of GPC5down-regulation on the lung metastasis ofSACC in vivo
1Establishment of stable cell lines with silenced GPC5
Forty-eight hours after transfection of plasmid targeting GPC5and NCplasmid, stable cell clones GPC5-silenced and GPC5-NC cell were acquiredby using Blasticidin S HCl. Compared with SACC-M cell, the GPC5expression of GPC5-silenced cell was down-regulated by89.7%at mRNAlevel, and96.5%at protein level.
2The inhibition of lung metastasis of SACC in vivo
The4th week after injection, The mice were anesthetized and killed andweighed, the lung wet weight of group GPC5-silenced (0.507±0.223g) wassignificantly lower than that of group GPC5-NC (1.177±0.342g) and groupSACC-M (1.045±0.539g), P <0.05. No significant difference betweengroup SACC-M and group GPC5-NC, P>0.05.
The lung metastasis of group GPC5-silenced decreased to33.33%(2/6),significantly lower than that of group SACC-M (100%,6/6) and groupGPC5-NC (100%,6/6), P <0.05.
The histopathological analysis of the lung samples of group SACC-Mand GPC5-NC showed that the normal structures of lungs in group werealmost destroyed. The lungs were full of metastasis nodules with differentsizes. The tumor cells were round or polyhedral with eosinophilic cytoplasm and round nucleus, arranged in dense clumps of solid, many mitoses could beobserved. Pulmonary vascular cavities were in condition of hyperaemia. Onlya few metastasis nodules were found in the2lung samples of groupGPC5-silenced and the normal structure of them was still remained. The sizeof the metastasis nodle in group GPC5-silenced was much smaller than that ofgroup SACC-M and group GPC5-NC.
Conclusion:
The high expression of heparan sulfate proteoglycan (GPC5) promotesthe metastasis ability of SACC.
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