The Relationship Between Heparan Sulfate Proteoglycans And Salivary Adenoid Cysitc Carcinoma With Lung Metastasis

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 SACC1HSPGs expression in SACC cell linesIn 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 cultureSACC-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 extractionTotal 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 transcriptionThree microgram of total RNA from the sample were used in reversetranscribed with RevertAid First Strand cDNA Synthesis Kit.1.4Real-Time PCRIn 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 linesIn this part, we choose the high expression HSPG (GPC5) as the studygene, and investigated the GPC5expression in SACC cell lines. 2.1ImmunolocalizationGPC5expression and distribution in SACC-M, SACC-2, SACC-83cellswas tested by Immunofluorescence staining with rabbit anti-GPC5monoclonal antibody.2.2Western blot analysisThe 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 metastasisSixteen 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 vector1Construction of GPC5miRNA expression vectorThe 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 validationThree clonies from each plate were picked and sequenced to verify therecombinational cloning insertion sequence and oligo sequence alignmentdesign.3Plasmid midi-preparation4Evaluation of silencing efficiencyPlasmids 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 vivo1Group of experimentGene 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 GPC52.1SACC-M cell cultrue and definition of the minimal lethal concentration2.2SACC-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 efficiencymRNA 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 vivoEighteen 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 SACC1HSPGs expression in highly and poorly metastatic SACC cell linesBy 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 lines2.1ImmunolocalizationBy 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 analysisWestern 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 metastasisIn16clinical 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 GPC5Successful 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 vivo1Establishment of stable cell lines with silenced GPC5Forty-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 vivoThe4th 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.