ABSTRACT
Although modifier genes are extensively studied in various diseases, little is known about modifier genes that regulate autoimmune diseases. Autoimmune disease caused by the Fas(lpr) mutation depends on the genetic background of mouse strains, suggesting a crucial role of modifier genes. MRL/MpJ-Fas(lpr) (MRL/lpr) and AKR/lpr mice develop severe and mild lupus-like autoimmune disease, respectively, whereas this mutation does not cause disease on C57BL/6 (B6) or C3H background. Both MRL and AKR carry the same haplotype of the Cd72 gene encoding an inhibitory BCR coreceptor (CD72(c)), and CD72(c) contains several amino acid substitutions and a deletion in the extracellular region compared with CD72(a) and CD72(b). To address the role of Cd72(c) locus in the regulation of Fas(lpr)-induced autoimmune disease, we generated B6.CD72(c)/lpr and MRL.CD72(b)/lpr congenic mice. Introduction of the chromosomal interval containing Cd72(c) did not cause disease in B6 mice by itself, but caused development of lupus-like disease in the presence of Fas(lpr) on B6 background, clearly demonstrating that this interval contains the modifier gene that regulates Fas(lpr)-induced autoimmune disease. Conversely, MRL.CD72(b)/lpr congenic mice showed milder disease compared with MRL/lpr mice. We further demonstrated that Cd72(c) is a hypofunctional allele in BCR signal inhibition and that CD72 deficiency induces severe autoimmune disease in the presence of Fas(lpr). These results strongly suggest that the Cd72(c) is a crucial modifier gene that regulates Fas(lpr)-induced autoimmune disease due to its reduced activity of B cell signal regulation.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Gene Expression Regulation , fas Receptor/genetics , Animals , Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Autoantibodies/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Female , Immunophenotyping , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lymph Nodes/cytology , Mice , Mice, Congenic , Mice, Inbred MRL lpr , Mice, Knockout , Mutation , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Spleen/cytology , fas Receptor/immunologyABSTRACT
OBJECTIVE: To investigate the inhibitory effect of siRNA on heparanase expression and invasion ability gastric cancer cells. METHODS: A heparanase mRNA-targeting double-stranded siRNA was designed with the bioinformatics technology. Human gastric cancer cells of the line SGC7901 were cultured and transfected with the siRNA of the concentrations of 5, 10, 20, and 40 nmol/L respectively. Forty-eight hours later RT-PCR and Western blotting were applied to detect the mRNA and protein expression of heparanase. Millicell chamber assay was performed to detect the invasion ability of the SGC7901 cells. Blank control group and negative control group were set. RESULTS: The mRNA expression level of the cells transfected with the siRNA of the concentrations 20 nmol/L and 40 nmol/L were 0.207 +/- 0.095 and 0.200 +/- 0.085 respectively, both significantly lower than that of the control group (0.60 +/- 0.09, both P < 0.05). Western blotting showed that the protein expression of heparanase of the different siRNA subgroups were all decreased dose-dependently; and no heparanase band was seen in the 40 nmol/L subgroup. The invasion rate of the siRNA group was significantly lower than that of the control group with a mean inhibition rate of (61 +/- 36)%. CONCLUSION: RNAi inhibits the expression of heparanase and the invasion ability of human gastric cancer cells. Heparanase may be a new target in treatment of gastric cancer's metastasis.
Subject(s)
Glucuronidase/metabolism , RNA, Small Interfering/genetics , Blotting, Western , Cell Line, Tumor , Cell Movement , Glucuronidase/genetics , Humans , Neoplasm Invasiveness , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/enzymology , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , TransfectionABSTRACT
Chromosome 8p21-p23 harbors tumor suppressor gene(s) implicated in multiple types of cancers. To investigate the involvement of the gene(s) in the carcinogenesis of adenocarcinoma of gastric cardia, loss of heterozygosity (LOH) for microsatellite markers at chromosome 8p21-p23 was examined. Laser capture microdissection (LCM) was used to obtain homogeneous tumor cells from 19 surgical specimens. Subsequently, genomic DNA extracted from the LCM-captured cells was amplified by multiple displacement amplification. Each tumor was assessed for allelic loss using 13 microsatellite markers. An overall LOH frequency of 63.2% (12/19) was observed and the LOH frequency for individual markers varied from 25% to 55.6%. One common deleted region of about 1.2 Mb (8p22GGAA-8p22ATCT) was defined. Our data indicated that the tumor suppressor gene at chromosome 8p22 might play an important role in the development of adenocarcinoma of gastric cardia.