[Establishment of BGC-823/pBaBb-puro-MACC1 gastric cancer cell line stably expressing MACC1 and its tumor-related gene expression profiles].
ABSTRACT To establish a gastric cancer cell line with stable expression of metastasis-associated in colon cancer 1 (MACC1) and detect the changes in tumor-related gene expression profiles for investigating the possible regulation mechanisms between MACC1 and the differentially expressed genes.
The full-length MACC1 cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pBaBb-puro vector. The recombinant pBaBb-puro-MACC1 expression vector, after identification with restriction enzyme digestion, was transfected into 293FT cells, and the expression of fluorescent reporter gene was observed. pBaBb-puro-MACC1 vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/pBaBb-puro-MACC1 cell line stably expressing MACC1. Quantitative RT-PCR and Western blotting were used to detect MACC1 expression in both BGC-823/pBaBb-puro-MACC1 and control BGC-823 cells. High-throughout cDNA microarray was used to screen the effects of MACC1 on the gene expression profiles of gastric cancer cells.
The recombinant pBaBb-puro-MACC1 plasmid was successfully constructed and verified by PCR and sequencing. BGC-823/pBaBb-puro-MACC1 cells showed significantly increased MACC1 mRNA expression as compared with the control cells. The results of cDNA microarray identified 33 up-regulated and 24 down-regulated genes in the cells after MACC1 transfection involved were in various cellular functions.
The established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.
- SourceAvailable from: Andrea Bertotti[show abstract] [hide abstract]
ABSTRACT: : Upon colon cancer metastasis resection in liver, disease outcome is heterogeneous, ranging from indolent to very aggressive, with early recurrence. The aim of this study is to investigate the capability of metastasis associated in colon cancer 1 (MACC1) levels measured in liver metastasis specimens to predict further recurrence of the disease. : Gene expression and gene dosage of MACC1, hepatocyte growth factor (HGF), and hepatocyte growth factor receptor (MET) were assessed using quantitative realtime polymerase chain reaction on a cohort of 64 liver metastasis samples from patients with complete follow-up of 36 months and detailed clinical annotation. The most relevant mutations associated to prognosis in colorectal cancer, KRAS, and PIK3CA were assessed on the same specimens with Sanger sequencing. : Receiver operating characteristic (ROC) analysis revealed that MACC1 mRNA abundance is a good indicator of metastatic recurrence (AUC = 0.65, P < 0.05), whereas no such results were obtained with MET and HGF, nor with gene dosage. Generation of MACC1-based risk classes was capable of successfully separating patients into poor and good prognosis subgroups [hazard ratio (HR) = 5.236, 95% confidence interval (CI) = 1.2068-22.715, P < 0.05]. Also KRAS mutation was significantly associated with higher risk of recurrence (HR = 2.07, 95% CI = 1.048-4.09, P < 0.05). Cox regression multivariate analysis supported the independence of MACC1, but not KRAS, from known prognostic clinical information (Node Size HR = 3.155, 95% CI = 1.4418-6.905, P < 0.001, Preoperative carcinoembryonic antigen HR = 2.359, 95% CI = 1.0203-5.452, P < 0.05, MACC1 HR = 7.2739, 95% CI = 1.6584-31.905, P < 0.01). : MACC1, a new easily detectable biomarker in cancer, is an independent prognostic factor of recurrence after liver resection of colorectal cancer metastasis.Annals of surgery 06/2013; 257(6):1089-95. · 7.90 Impact Factor