反义CD147分子对人胆管癌细胞株侵袭性影响的实验研究
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摘要
目的探讨反义CD147分子对人胆管癌细胞株QBC939侵袭性的影响。方法构建含反义CD147分子片段的重组真核表达质粒,将人胆管癌细胞株QBC939分为三组:(1)反义CD147组,运用重组真核表达质粒as CD147-pc DNA3.1(-)转染QBC939细胞;(2)空质粒组,运用pc DNA3.1(-)空质粒转染QBC939细胞;(3)对照组,运用DMEM常规处理细胞。采用MTT法检测各组QBC939细胞的生长情况,RT-PCR和Western blotting法分别对三组QBC939细胞中的CD147、MMP-2、MMP-9、TIMP-2的m RNA表达水平及其对应蛋白的表达水平进行检测。结果成功构建了携带反义CD147分子片段的重组真核表达质粒。MTT法检测显示:三组QBC939细胞的生长曲线及对数生长期情况无统计学差异(P>0.05)。RT-PCR法检测显示:反义CD147组QBC939细胞中CD147和MMP-2分子的m RNA表达均低于空质粒组和对照组(P<0.05),且空质粒组与对照组相比无统计学差异(P>0.05);反义CD147组QBC939细胞中MMP-9和TIMP-2分子的m RNA表达与空质粒组和对照组相比无统计学差异(P>0.05)。Western blotting检测发现:与空质粒组和对照组相比,反义CD147组QBC939细胞中CD147和MMP-2分子的蛋白表达明显降低(P<0.05),空质粒组与对照组相比无统计学差异(P>0.05);与空质粒组和对照组相比,反义CD147组QBC939细胞株中MMP-9和TIMP-2分子的蛋白表达无统计学差异(P>0.05)。结论 (1)反义CD147对胆管癌细胞株QBC939的生长无影响;(2)反义CD147对胆管癌细胞株QBC939中MMP-9和TIMP-2的m RNA表达及对应蛋白表达无影响;(3)反义CD147降低胆管癌细胞株QBC939中CD147和MMP-2的m RNA表达及对应蛋白表达,可能会降低胆管癌细胞株的侵袭性。
objective To investigate the effect of antisense CD147 on invasion of human cholangiocarcinoma cell line(QBC939). Methods A eukaryotic expression vector containing the CD147 c DNA in an antisense orientation was reconstructured. Human cholangiocarcinoma cell were divided into either a antisense CD147 treated group, empty vector group, or control group. QBC939 in antisense CD147 treated group was undergone transfection with CD147-pc DNA3.1(-); QBC939 in empty vector group was undergone transfection with pc DNA3.1(-); and control group was treated with DMEM. Three groups were tested with MTT method for cell proliferation. Real-time PCR was used to detect the m RNA expression of CD147, MMP-2, MMP-9 and TIMP-2. Western blotting was used to detect the protein expression of CD147, MMP-2, MMP-9 and TIMP-2. Results A eukaryotic expression vector containing the CD147 c DNA in an antisense orientation was successfully reconstructed. In treated group, no reduction in the growth rate was found after infection with antisense CD147(P>0.05). Compared with empty vector group and control group, the m RNA expression of CD147 and MMP-2 molecule was effectively inhibited in the antisense CD147 treated group(P<0.05), and there was no significant difference between empty vector group and control group(P>0.05). For m RNA expression of MMP-9 and TIMP-2 molecule, there was no significant difference between the antisense CD147 treated group and empty vector group and control group(P>0.05). Compared with empty vector group and control group, the protein expression of CD147 and MMP-2 molecule was effectively inhibited in the antisense CD147 treated group(P<0.05), and there was no significant difference between empty vector group and control group(P>0.05). For protein expression of MMP-9 and TIMP-2 molecule, there was no significant difference between the antisense CD147 treated group and empty vector group and control group(P>0.05). Conclusion(1)Antisense CD147 has no effect on proliferation of QBC939 cell.(2)Antisense CD147 has no effect on the m RNA expression and protein expression of MMP-9 and TIMP-2 molecule in QBC939 cell.(3)Antisense CD147 can inhibit the m RNA expression and protein expression of MMP-9 and TIMP-2 molecule in QBC939 cell, which maybe can influence cell invasion of QBC939.
objective To investigate the effect of antisense CD147 on invasion of human cholangiocarcinoma cell line(QBC939). Methods A eukaryotic expression vector containing the CD147 c DNA in an antisense orientation was reconstructured. Human cholangiocarcinoma cell were divided into either a antisense CD147 treated group, empty vector group, or control group. QBC939 in antisense CD147 treated group was undergone transfection with CD147-pc DNA3.1(-); QBC939 in empty vector group was undergone transfection with pc DNA3.1(-); and control group was treated with DMEM. Three groups were tested with MTT method for cell proliferation. Real-time PCR was used to detect the m RNA expression of CD147, MMP-2, MMP-9 and TIMP-2. Western blotting was used to detect the protein expression of CD147, MMP-2, MMP-9 and TIMP-2. Results A eukaryotic expression vector containing the CD147 c DNA in an antisense orientation was successfully reconstructed. In treated group, no reduction in the growth rate was found after infection with antisense CD147(P>0.05). Compared with empty vector group and control group, the m RNA expression of CD147 and MMP-2 molecule was effectively inhibited in the antisense CD147 treated group(P<0.05), and there was no significant difference between empty vector group and control group(P>0.05). For m RNA expression of MMP-9 and TIMP-2 molecule, there was no significant difference between the antisense CD147 treated group and empty vector group and control group(P>0.05). Compared with empty vector group and control group, the protein expression of CD147 and MMP-2 molecule was effectively inhibited in the antisense CD147 treated group(P<0.05), and there was no significant difference between empty vector group and control group(P>0.05). For protein expression of MMP-9 and TIMP-2 molecule, there was no significant difference between the antisense CD147 treated group and empty vector group and control group(P>0.05). Conclusion(1)Antisense CD147 has no effect on proliferation of QBC939 cell.(2)Antisense CD147 has no effect on the m RNA expression and protein expression of MMP-9 and TIMP-2 molecule in QBC939 cell.(3)Antisense CD147 can inhibit the m RNA expression and protein expression of MMP-9 and TIMP-2 molecule in QBC939 cell, which maybe can influence cell invasion of QBC939.
引文
[1]SIA D,HOSHIDA Y,VILLANUEVA A,et al.Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2classes that have different outcomes[J].Gastroenterology,2013,144(4):829-840.
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[3]SAMESHIMA T,NABESHIMA K,TOOLE B P,et al.Glioma cell extracellular matrix metalloproteinase inducer(EMMPRIN)(CD147)stimulates production of membranetype matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived fibroblasts[J].Cancer Lett,2000,157(2):177-184.
[4]LI Y,SHANG P,QIAN A R,et al.Inhibitory effects of antisense RNA of HAb18G/CD147 on invasion of hepatocellular carcinoma cells in vitro[J].World J Gastroenterol,2003,9(10):2174-2177.
[5]JUNG Y S,LIU X W,CHIRCO R,et al.TIMP-1 induces an EMT-like phenotypic conversion in MDCK cells independent of its MMP-inhibitory domain[J].PLo S One,2012,7(6):e38773.
[2]KANEKURA T,CHEN X.CD147/basigin promotes progression of malignant melanoma and other cancers[J].J Dermatol Sci,2010,57(3):149-154.
[3]SAMESHIMA T,NABESHIMA K,TOOLE B P,et al.Glioma cell extracellular matrix metalloproteinase inducer(EMMPRIN)(CD147)stimulates production of membranetype matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived fibroblasts[J].Cancer Lett,2000,157(2):177-184.
[4]LI Y,SHANG P,QIAN A R,et al.Inhibitory effects of antisense RNA of HAb18G/CD147 on invasion of hepatocellular carcinoma cells in vitro[J].World J Gastroenterol,2003,9(10):2174-2177.
[5]JUNG Y S,LIU X W,CHIRCO R,et al.TIMP-1 induces an EMT-like phenotypic conversion in MDCK cells independent of its MMP-inhibitory domain[J].PLo S One,2012,7(6):e38773.