ABSTRACT
OBJECTIVE: To investigate the expression of guanylyl cyclase C (GCC) in human gastric cancer (GC) tissues and assess the effect of GCC small interfering RNA (siRNA) on the proliferation and apoptosis of SGC-7901. METHODS: The expression of GCC in 30 specimens and three human GC cell lines (SGC-7901, AGS, NCI-N87) were detected by RT-PCR for messenger RNA (mRNA) by Western blot and immunofluorescence for proteins. Recombinant plasmids containing GCC siRNA and scrambled siRNA were constructed and transfected into SGC-7901 cells, respectively. A cell counting kit-8, flow cytometry (FCM) and terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labeling were used to evaluate cell viability, cell cycle distribution and apoptosis, followed by wound healing assay and cell adherent assay for cell motility and adherent, respectively. RESULTS: The expression of GCC was absent in paracancerous tissues, whereas the GCC mRNA and protein expressions were detected in 20/30 and 19/30 of GC specimens, respectively. Moreover, intestinal GC was statistically different from diffuse GC (P < 0.05). The proliferation of SGC-7901 cells was markedly inhibited by GCC siRNA-3 (P < 0.05) and cell morphological changes including volumetric reduction, karyopyknosis and karyorrhexis were observed. FCM showed that the cell count in the sub-G0/G1 peak increased from 5.47% (48 h after transfection) to 5.63% (72 h after transfection). The wound healing assay and cell adherent assay revealed that GCC gene silencing decreased cell motility and adherent. CONCLUSION: The over-expression of GCC has been detected in intestinal type GC. GCC siRNA can effectively inhibit the proliferation and invasion of SGC-7901 cells and induce cell apoptosis. GCC might be a novel biomarker and therapeutic target for GC.
Subject(s)
Biomarkers, Tumor/genetics , Genetic Therapy/methods , Guanylate Cyclase/genetics , Receptors, Peptide/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/therapy , Apoptosis , Biomarkers, Tumor/metabolism , Blotting, Western , CDX2 Transcription Factor , Cell Adhesion , Cell Division , Cell Line, Tumor , Fluorescent Antibody Technique , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Guanylate Cyclase/metabolism , Homeodomain Proteins/genetics , Humans , Precancerous Conditions/genetics , Precancerous Conditions/pathology , RNA, Messenger/metabolism , RNA, Small Interfering , Receptors, Enterotoxin , Receptors, Guanylate Cyclase-Coupled , Receptors, Peptide/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/pathology , TransfectionABSTRACT
OBJECTIVE: To investigate the expression of guanylyl cyclase-C (GC-C) and caudal type homeobox transcription factor 2 (CDX2) in human gastric mucosa at different stages and the significance thereof. METHODS: An Immunofluorescence method was used to detect the expression of GC-C and CDX-2 in 23 specimens of gastric carcinoma and matching noncancerous tissues. Western blotting was used to detect the protein expression of GC-C and CDX2 in the gastric carcinoma tissues and matching noncancerous tissues too. RESULTS: The GC-C and CDX2 expression rates were 39.1% and 39.1% respectively in the intestinal metaplasia specimens, 55.6% and 55.6% respectively in the dysplasia specimens, and 56.7 % and 60.0% in the gastric carcinoma specimens, all significantly higher than those in the normal mucosa specimens (all P = 0.000) without significant differences in the expression of GC-C and CDX-2 among the 3 pathological groups. The GC-C and CDX-2 expression was positively correlated with Lauren classification, The expression levels of GC-C and CDX-2 were significantly higher in the intestinal-type than in the diffuse-type gastric carcinoma (P < 0.05). The GC-C expression was positively correlated with the expression of CDX-2 in intestinal metaplasia and gastric carcinoma. CONCLUSION: Ectopic expression of GC-C and CDX2 in human gastric mucosa may play an important role in the carcinogenesis of intestinal-type gastric carcinoma. Detection of GC-C and CDX2 helps diagnose gastric carcinoma and precursor lesions.
