| Cholecystokinin-B/gastrin (CCK-B) receptor belongs to the seven transmembrane G-protein-coupled receptor superfamily. Gastrin, a trophic factor, exerts its physiological and pathological effects on cells after binding CCK-B receptors both in vitro and in vivo. Gastrin/CCK-B receptors in an autocrine pathway may play an important role in the development and progression of gastric cancers.In the study, RT-PCR and sequencing were employed to detect the expression levels of CCK-B receptor, CCKBRi4sv and gastrin mRNAs in 30 human gastric carcinoma and their corresponding normal tissues, 10 gastritis and 2 autopsied normal stomach specimens as well as in a gastric carcinoma cell line SGC-7901 cells. The results showed that human gastric tissues from normal, inflammatory and malignant specimens simultaneously expressed the normal and aberrant splicing cholecystokinin-B /gastrin receptor genes and the expression levels of the two receptor genes were not correlated with the differentiation and metastases of gastric cancers. On the other hand, gastrin mRNA was detected in SGC-7901 cells and in 73.3% of gastric cancer specimens (22/30) but not in other gastric tissues, and its expression was highly associated with the metastases of gastric cancers, (P<0.05). Therefore weproposed that 1) Gastrin/ CCK-B receptor autocrine pathway may possibly play an important role in progression of gastric cancer. 2) CCKBRi4sv may have unknown physiological functions in gastric epithelial cells.To test our viewpoint, we blocked gastrin/ CCK-B receptor autocrine loop by antibodies against gastrin and employed RNA interference technique to suppress CCK-B receptor gene expression, and to study the resulting effects on SGC-7901 cells growth. At first, we demonstrated, by means of RT-PCR, immunocytochemistry method and radioimmunoassay, that SGC-7901 cells not only coexpressed gastrin and CCK-B receptor genes, but expressed and excreted gastrin proteins. Subsequently, SGC-7901 cells were treated with antibodies against gastrin to observe the growth rate of the cells by 3-(4.5-dimethylthiaxol-z-yl)-2,5- dipheny tertraxolium blue (MMT) colorimetric assay, and using FCM to detect the cell cycle kinetics and apoptosis cells. The expression of Ki67 (cell cycle related nuclear protein), caspase-3 (apoptosis-related protein) and MMP-2 (matrix metallporoteinases) proteins were analyzed by Western blot. The results showed that the growth rate, the number of S cells among total cells and proliferation index (PI+) of SGC-7901 cells obviously decreased and the expression levels of Ki67 and MMP-2 proteins also reduced in a antibody concentration-dependent manner. But the number of apoptotic cells among total cells in SGC-7901 cells and the expression levels of caspases-3 proteins apparently increased. This data suggested that when interrupting the gastrin/CCK-B receptor antocrine loop, the proliferation and metastasis of SGC-7901 cells were inhibited,but its apoptosis was enhanced.Finally, we designed a control siRNA (the small interference RNA) and two siRNAs for CCK-B receptor gene cassettes by PCR-basing RNA interference technique, and transfected the cassettes into SGC-7901 cells by the liposomes. The results revealed that one of the siRNAs can knocked down the expression of CCK-B receptor gene, and when the CCK-B receptor genes were silencing the decreases in the growth rate, the number of S cells among total cells and PI+ of SGC-7901 cells were detected, but no changes in apoptosis rate of SGC-7901 cells and the expression levels of caspase-3 in the cells were observed. The Ki67 and MMP-2 expression levels were also reduced. Our experiments indicated that the knockdown of CCK-B receptors gene in SGC-7901 cells transfected by siRNA could inhibit the proliferation and metastasis of the cells, but have no effects on apoptosis of the cells.In conclusion, our present study demonstrated for the first time that in addition to gastric cancers, the normal and inflammatory gastric tissues also expressed alternative splicing CCK-B receptor gene subtype-CCKBRi4sv,...
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