The Study Of LAPTM4B-35 In Salivary Adenoid Cystic Carcinoma And Mucoepidermoid Carcinoma

Adenoid cystic carcinoma(ACC)and mucoepidermoid carcinoma(MEC)are the most common salivary gland malignancies,occuring most frequently in the parotid glands and small salivary glands in the head and neck region.As most adenoid cystic carcinoma and poorly differentiated mucoepidermoid carcinomas are resistant to chemotherapy and radiotherapy,the prognosis of these patients remains dismal,thus identification of more prognostic biomarkers which can predict the response of ACC/MEC to targeted therapy and/or be used as a target for individual therapy need to be encouraged.Lysosome-associated protein transmembrane 4 beta(LAPTM4B),a newly identified oncogene(NM＿018407,gene ID: 55353),was first cloned by Professor Zhou Ruoli at the Department of Cell Biology of Basic Medical College of Peking University.It is located on chromosome 8q22.1,spanning at least 50 kb,including seven exons and six introns.The full-length c DNA of LAPTM4 B contains two translational initiation codons(ATG)with an interval of 273 base pair,and encode at least two protein isoforms,LAPTM4B-35 and LAPTM4B-24,a type III transmembrane protein with four transmembrane regions.Previous studies have shown that LAPTM4B-35 protein is up-regulated in in a number of clinically aggressive cancers.It is closely associated with histological grade of hepatocellular carcinoma,and negatively correlated with overall survival and disease-free survival of patients in various tumors.So far there are no studies on the association analysis between LAPTM4B-35 expression and clinicopathological features of salivary ACC and MEC.In the present study,we aimed to investigate LAPMT4B-35 expression in normal salivary glands,as well as ACC and MEC tissues to identify the potential relationship with clinicopathological features.RNA interference was used to explore the effect of LAPMT4B-35 on the growth of H292 mucoepidermoid carcinoma cell line.MATERIALS AND METHODS:1.The expression of LAPTM4B-35 in adenoid cystic carcinoma and mucoepidermoid carcinoma of salivary gland: Cases were recruited from the the Department of pathological at the Second Affiliated Hospital and the First Hospital of Soochow University and other institutes.The clino-pathogical data were also collected.By immunohistochemistry analysis,LAPTM4B-35 expression was evaluated in 106 ACC and 85 MEC tissues,their adjacent noncancerous tissues and 20 normal salivary glands.The relationship between the expression of LAPTM4B-35 and the clinicopathological characteristics of these two tumors was analyzed.The correlation of LAPTM4B-35 expression with clinicopathological parameters of ACC and MEC was assessed using Chi-square or Fisher’s exact test.2.Mucoepidermoid carcinoma H292 cell line was selected for in vitro experiments.The interfering RNA(Si RNA)was synthesized and transfected into H292 cell line.The transfection efficiency was determined by fluorescence inversion microscopy.The expression of LAPTM4B-35 gene and protein after RNAi was detected by real-time fluorescence quantitative PCR and Western Blot.Sequences with high silence efficiency were further selected.3.The effects of silence of LAPTM4B-35 in H292 cell line: After transient silence of LAPTM4B-35,CCK-8 assay was utilized to investigate the effect on cell proliferation,and transwell chamber assay was used to investigate the effect on tumor invasion,while flow cytometry was performed to explore the effect on apoptosis and cell cycle.RESULTS:1.The level of LAPTM4B-35 expression was variant among different cell types of normal salivary gland.It was expressed fairly low in serous and mucous acini,weak in intercalated duct and excretory duct cells,moderate in secretory/striated ducts.2.LAPTM4B-35 overexpression was associated with high histological grade and advanced clinical stage of ACC and MEC.3.Small interfering RNA transfection: Si RNA and negative control were transiently transfected into H292 cell line respectively.After successful transfection,the expression of LAPTM4B-35 gene at RNA and protein level decreased significantly.4.The effect of LAPTM4B-35 on H292 cells: After knock down of LAPTM4B-35,the proliferation and invasion ability decreased significantly,the apoptosis rate was increased and the cell cycle distribution was changed.CONCLUSIONS:1.LAPTM4B-35 is differentially expressed in normal salivary gland cells,and is related to histological grade and clinical stage of ACC and MEC.It is valuable to study the expression of LAPTM4B-35 in these two salivary gland tumors.2.LAPTM4B-35 affects the proliferation,migration,apoptosis and cell cycle distribution in inmucoepidermoid carcinoma H292 cells,thus may play an important role in the development of MEC.Our results indicates that LAPTM4B-35 plays an important role in salivary ACC and MEC may serve as a diagnostic marker and a target for individual therapy.