Gene Expression Profile Of Lacrimal Gland Adenoid Cystic Carcinoma And Expression And Function Study Of Genes Associated With Extracellular Matrix

ObjectivesThe present study aims to(1) use large-scale microarray analysis to characterize the expression profiles of lacrimal gland adenoid cystic carcinoma(LGACC) and normal lacrimal gland,and to specifically identify those genes differentially expressed between them;(2) validate laminin(LN) and typeâ…£collogen at mRNA and protein levels,and to identify the relationship between these markers and histological subtypes;(3) examine the ultrastructure of ACC and normal lacrimal gland,and determine the appearance of basement membrance(BM) of different histological substypes;(4) study the cellular biological effect of LAMB1 by RNA interference(RNAi) in ACC-2 cell line.Methods(1) Total RNAs were isolated from LGACC tissues and normal lacrimal glands from respective 5 cases.The aRNA prepared from in vitro transcription were hybridized with the Phalanx Human Onearray and general gene expression profiles were obtained.Genes with significant differential expression were identified by bioinformatics analytic tools.(2) Real-time quantitative PCR(RT-qPCR) was used to detect expression of LAMB1 and COL4A1 in LGACC and normal larcrimal gland at mRNA level.Immunohistocheical analysis was used to determine the expression of LN and typeâ…£collagen,which were the proteins of targets genes,and to explore the correlation between the distribution of these proteins and histological subtypes.(3) Materials for electron microscopy were obtaind from 6 cases with LGACC and 2 cases with normal lacrimal gland.The ultrastructural features and changes of BM in different histologic patterns were focused. (4) PCR examination was used to validate the expression of targets genes in ACC-2 cells. RNAi-mediated RNA silencing was used to downregulate LAMB1 expression in ACC-2 by transfection with cationic lipid complexes.MTT colorimetric assay was used to detect the changes of cell proliferation after RNAi.Results Camparing with normal tissues,the gene expression profile of LGACC showed that expression of 1103 genes was significantly increased,while expression of 1001 genes was significantly decreased.The functions of these genes were involved in cellular metabolism,communication,localization,cell cycle,cell adhesion and proliferation,etc.RT-qPCR showed there were a 10.61-fold increase of LAMB1 transcript,a 12.91-fold increase of COL4A1 transcript and a 0.27-fold descrease of PIGR transcript in LGACC, as compared to normal lacrimal gland,which were consistent with the results of genechips.Both LN and type IV collagen were more intensively expressed in LGACC than in normal tissue.Besides in cytoplasm of tumor cells,immunostaining was in the inner border of pseudocysts,the surrounding areas of tumor nests and BM of cribriform and tubular types,which showed continuous or noncontinuous streak.The solid type showed noncontinuous,irregular immunopositivity in the surrounding areas of massive tumors.(3) In ultrastructure,the BM of tubular and cribirform types showed obvious thickening,multi-layered and reticular structure.Pseudopodia of tumor cells were seen extending into BM and making it discontinuation and dissolution.The massive dissoluted extracellular matrix was revealed in the intercellular space of solid pattern.The intact BM was unusual.(4) The ACC-2 cell line,derived from salivary glands,was proved expression of LAMB 1 by PCR test LAMB1 expression was downregulated to 9%after transfection of siRNA.MTT test showed cell growth was obviously reduced 72 hours after transfection.Conclusions(1) Microarray,an important biological technique,can win the aim to screen out the genes related to tumorigenesis.Through it,we confirmed the gene expression profile of LGACC was significant different from that of normal lacrimal gland.There were obvious overexpression of LAMB 1 and COL4A1 and oversynthesis of LN and type IV collagen in LGACC.Cell growth was decreased by knockdown of LAMBl,which suggested extracellular matrix(ECM) synthesized by LGACC promote tumor proliferation.Further study maybe lead to new therapeutic targets for this carcinoma.(2) The thickening and structural disorder of BM were important features of LGACC,dissolution and discontinuation of which could be the evidence of tumor invasive growth.More findings might serve as one of the explanations on invasion and metastasis mechanism of LGACC.