Effect Of CD147 Antisense RNA On Invasion And MDR Of Human Gallbladder Carcinoma Cell Line GBC-SD In Vitro

Part I Expressions and clinicopthological significance of MMP-2, TIMP-2, CD147 and P-gp in human gallbladder carcinomaObjective To investigate the expression of CD147,TIMP-2,MMP-2 and P-gp in human primary gallbladder carcinoma tissue and the relationship among them, and to evaluate its clinicopathological significance in the progression of gallbladder carcinoma. Methods:Immunohistochemical method of SP was used to examine the expression of CD 147, TIMP-2, MMP-2 and P-gp in 60 cases of gallbladder carcinoma and 40 cases of chronic cholecystitis tissue (control group). Results In gallbladder carcinoma tissue, each positive expression rate of CD 147, MMP-2 and P-gp was significantly higher than that in control group (66.7% vs 20.0%,75.0% vs 10.0%,76.7% vs 7.5%, respectively, P<0.01), whereas the positive expression rate of TIMP-2 was remarkably lower than that in control group (40.0% vs 90%, P<0.01).There was a correlation between the expression of CD147,MMP-2,TIMP-2 as well as P-gp and Nevin stage, differentiated degree, with or without metastasis. The expression of CD 147 was positively correlated with that of MMP-2 and P-gp in gallbladder carcinoma (CD 147 vs MMP-2, r=0.277, P=0.015; CD147 vs P-gp, r=0.464, P=0.026).There was a negative correlation between the productions of TIMP-2 and MMP-2 (r=-0.583, P=0.000). Conlusions The enhanced expression of CD147, MMP-2 and P-gp might coordinately promote the invasion and metastasis of gallbladder carcinoma. TIMP-2 might inhibit the invasion and metastasis of gallbladder carcinoma by inhibiting the activation of MMP-2. Over expressed CD147 might promote the invasion, metastasis and MDR of gallbladder carcinoma via stimulating the productions of MMP-2 and P-gp.Part II Construction of eukaryotic antisense RNA expression vector of CD147 and clone identificationObjective To construct eukaryotic antisense RNA expression vector of CD 147, to transfect the antisense RNA vector to human gallbladder carcinoma cell line GBC-SD and to select the positive clones. Methods PCI-CD147 was constructed by inserting CD 147 cDNA reversely to eukaryotic expression vector PCI-neo. The human gallbladder carcinoma cell line GBC-SD was transfected by PCI-CD147. After selected by G418,positive clones were identified by western blot. Results It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic antisense RNA expression vector PCI-CD147 was correct. And the expression of CD 147 in GBC-SD cells transfected by PCI-CD147 was significantly lower than that in the cells untransfected or transfected by empty vector PCI-neo. Conlusions CD 147 antisense RNA expression vector PCI-CD147 was successfully constructed. CD 147 antisense RNA could effectively inhibit the expression of CD147 in human gallbladder carcinoma cell line GBC-SD. The result of this study paves the way for further studying the molecular mechanism of CD 147 in invasion and metastasis and gene therapy of human gallbladder carcinoma. Partâ…¢Effect of CD147 antisense RNA on invasion and MDR of human gallbladder carcinoma cell line GBC-SD in vitroObjective To explore the inhibitory effect of CD147 antisense RNA on the invasion and multidrug resistance (MDR) of gallbladder carcinoma cell line GBC-SD. Methods Three groups consisted of experimental group (GBC-SD cell line transfected by antisense RNA vector PCI-CD147, named GBC-SD/PCI-CD147), experimental control group(GBC-SD cell line without transfection, GBC-SD) and blank control group(GBC-SD cell line transfected by empty vector PCI-neo, GBC-SD/PCI-neo). RT-PCR and Western-blot were used to testify the mRNA and protein level of CD147,MMP-2,TIMP-2 and MDR1/P-gp in the three groups of cells,respectively. MTT method was used to investigate the growth of each group of cells. Cell cycle and rate of apoptosis of those cells were detected by flow cytometry analysis. Gelatin zymography was employed to analyze the secretions of MMP-2 and MMP-9 in the three groups of cells. transwell invasion assay was performed to evaluate the invasive ability of those cells. Results CD 147 antisense RNA had no effect on the growth of GBC-SD cell line. The secretions of MMP-2 and MMP-9 in the CD147 antisense RNA transfectants were down regulated significantly with respect to parental and empty vector-transfected cells in Gelatin zymography assay(P<0.001). In group GBC-SD/PCI-CD147, each mRNA and protein lever of MMP-2, CD 147 and P-gp was remarkably lower than that in group GBC-SD or group GBC-SD/PCI-neo, whereas TIMP-2 level in group GBC-SD/PCI-CD147 was obviously higher than that in each of the other groups. It was found that the number of cells which had invaded the reconstructed basement membrane in group GBC-SD/PCI-CD147 showed a significant decrease than that in others (P<0.001). The time of river crossing in group GBC-SD/PCI-CD147 was significantly longer than that in the others (P<0.001). After chemotherapeutics with mutidrug, Both survival rate and apoptosis rate of GBC-SD/PCI-CD147 were lower than those of the others. Conclusion The down regulated expression of CD147 via CD 147 antisense RNA could inhibit the invasive ability and could reverse the MDR of gallbladder carcinoma cell line GBC-SD.