Reduction Of P120ctn Isoforms 1 And 3 Is Significantly Associated With Lymph Node Metastasis Of Lung Cancer

Introductionp120-catenin(p120ctn) belongs to the Catenin family of proteins.It plays an inportant role in cell adhesion and signaling transduction.The human p120ctn gene comprises 21 exons,potentially encoding up to 32 protein isoforms as the result of alternative product splicing.All p120ctn isoforms share the central Armadillo repeat domain but have a divergent N- and C-terminal end.Human p120ctn isoforms, designated from 1 to 4,differ from each other by the start codon used.However,little is known about the biological functions of the different p120ctn isoforms.At present, there is little study about p120ctn in lung cancer and its isoforms expression in lung cancer is unclear.In this study,we used immunofluence,Western blot and reverse transcription-polymerase chain reaction(RT-PCR) to examine the expression of p120ctn isoforms in lung squamous cell carcinomas(SCCs),adenocarcinomas and lung cancer cells,and investigate their relationship to clinicopathological factors.Materials and Methods1.Patients and specimensThe primary tumors specimens were from patients with lung SCC and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA. 2.Cell cultureBE1 and LH7 cell lines derived from the PG cell line were established from human pulmonary giant cell carcinoma(a gift from Dr Jie Zheng,Medical College of Beijing University,China).BE1 cells have a high metastatic potential(94%),whereas LH7 cells have a low metastatic potential(50%).The cells(2×10⁵)were cultured in 35cm² plastic flasks,grown on coverslips,with RPMI 1640 medium(GiBCO Inc,Los Angeles,CA,USA) containing 10%fetal calf serum(GiBCO Inc,Los Angeles,CA, USA),2g/L NaHCO3,100 U/ml penicillin G and 100 U/ml streptomycin at 37℃in a humidified atmosphere(5%CO₂ and 95%air).The coverslips were then fixed with dimethyl ketone for 10 min at 4℃.3.ImmunofluenceAll the sections and lung cancer cells cultured on coverslips were treated with 0.2%Triton X-100 for 15 min and blocked with normal goat serum at 37℃for 30 min.The sections of tissue samples and cell samples were then incubated with p120ctn mouse monoclonal antibody pp120 that detects all p120ctn isoforms(1:400,BD Transduction Laboratories,Franklin Lakes,NJ,USA)overnight at 4℃.Samples were then washed in PBS three times,each for 5 min,and incubated with anti-mouse secondary antibody conjugated to rhodamine(1:200,Zhongshan Golden Bridge, Beijing,China)for 30 min at 37℃.The nuclei were counterstained with DAPI(1:200, SIGMA,St Louis,MO,USA) for 30 min at 37℃.The samples were examined by fluorescence microscopy at _rhodamine =527nm and _dapi=372nm,respectively.4.Western BlotEach sample(50μg) was separated by 7%SDS-PAGE for 90 min and then transferred to a polyvinylidene fluoride membrane(100V,2h).The membranes were then blocked with 1%bovine serum albumin(BSA) at 37℃for 1h.After washing in TTBS(20 mmol/L Tris-HCl,500 mmol/L NaCl,0.05%Tween-20),the transferred samples were incubated with anti-p120ctn antibody(pp120,1:400 dilution,BD Transduction Laboratories,Franklin Lakes,NJ,USA) overnight at 4℃.After incubating with peroxidase-coupled anti-mouse IgG(SABC,Beijing,China) at 37℃for 1h,washing in TTBS three times and the protein bands were visualized by 3,3'-diaminobenzidine and the band intensity was semiquantified using BioImaging Systems(UVP,Upland,CA,USA).The relative expression quantity was scored as ratios of p120ctn protein and P-actin staining intensities.5.RT-PCRTotal RNA was extracted from samples with Trizol Reagent(Invitrogen,Carlsbad, CA,USA).RT-PCR was performed with the RNA PCR Kit(AMV,Ver 3.0,TaKaRa, Dalian,China) according to the manufacturer's protocol.The PCR cycle conditions of pl20ctn and P-actin were:amplification for 30 cycles at 94℃for 40s;53℃(55℃for P-actin) for 40s;72℃for 40 s;and extension at 72℃for 5min.After electrophoresis with 1.5%agars gel,the bands of PCR products were visualized using Biolmaging Systems(UVP,Upland,CA,USA).The greyscales of pl20ctn were measured.The relative expression quantity was scored as ratios of pl20ctn and P-actin staining intensities.6.Statistical analysisAll statistical calculations were performed by SPSS version 11.5 for Windows software.p values less than 0.05 were considered statistically significant.ResultsCompared with corresponding normal lung tissues,lung cancer tissues have significantly lower levels of pl20ctn proteins and mRNA.The isoforms 1(120 kD) and 3(100 kD) proteins were major isoforms of p120 catenin expressed in normal lung tissues,which were significantly reduced in lung cancer samples.The mRNA of p120ctn isoforms 1.2,1.3,2.3,3.1 and 3.3 was detected in corresponding normal lung tissues,but was significantly absent in lung cancer samples.Furthermore,p120ctn isoform 1 is negatively associated-whereas pl20ctn isoform 3 is positively associated-with lymph node metastasis.ConclusionsIn normal bronchial epithelial cells,pl20ctn isoform 1A and 3A were expressed, which is reduced in lung cancer cells.These pl20ctn isoforms contribute differently to cancer cell adhesion and migration.