高转移潜能细胞外泌体共培养促进肝癌侵袭能力的研究
Promoting invasive ability of hepatocellular carcinoma by co-culture of exosomes from tumor cells with high metastatic potential
目的 观察高转移潜能肝癌(HCC)细胞外泌体与低转移潜能HCC细胞共培养对低转移潜能HCC细胞侵袭能力的影响并探讨其机制.方法 收集4种不同转移潜能肝癌MHCC97H、MHCC97L、SMMC7721、Hep3B细胞上清液和46例HCC患者血浆,提取、纯化和鉴定外泌体,与低侵袭潜能Hep3B细胞共培养,应用侵袭实验检测Hep3B细胞侵袭能力变化、聚合酶链反应检测环状RNA(circRNA)_100338变化及明胶酶谱实验检测基质金属蛋白酶(MMP)活性变化.结果 成功提取、纯化和鉴定相应外泌体.肺转移患者的HCC外泌体中circRNA_100338水平(0.064±0.006)高于无肺转移患者的HCC外泌体中circRNA_100338水平(0.039±0.005),高转移潜能MHCC97H细胞外泌体circRNA_100338水平(0.083±0.005)高于低转移潜能Hep3B细胞外泌体circRNA_100338水平(0.021 ±0.003),MHCC97H细胞外泌体共培养Hep3B细胞,可见Hep3B细胞侵袭性能力增强,穿膜数增加(15.3±2.5比8.7±2.1),共培养组细胞circRNA_100338水平升高(0.019±0.002比0.007±0.001);MMP-9活性逐渐增强(0.062±0.001、0.147±0.002、0.315±0.003),差异均有统计学意义(P<0.05).结论 高转移潜能细胞外泌体促进HCC侵袭能力与外泌体circRNA_100338水平提高及MMP-9活性增强有关.
更多Objective To observe the effect of co-culture of exosomes from tumor cells with high metastatic potential on hepatocellular carcinoma (HCC) cells with low invasive ability and explore the possible molecular mechanism.Methods The supernatant of 4 HCC cell lines of MHCC97H,MHCC97L,SMMC7721 and Hep3B,and plasma of 46 HCC patients were collected to extract,purify and identify exosomes.Hep3B cells with low invasive potential were co-cultured with exosomes.The invasive ability of Hep3B cells was detected by invasive assay,polymerase chain reaction (PCR) was used to test the circular RNA (circRNA) level,and the gelatinase spectrum assay was applied to examine the activity of matrix metalloproteinase (MMP).Results Exosomes were extracted,purified and identified successfully.The circRNA_100338 level in the HCC exosome of patients with lung metastasis (0.064 ± 0.006) was higher than that of HCC exosome of patients without lung metastasis (0.039 ± 0.005);The circRNA_100338 level in the exosome of MHCC97H cells with high metastatic potential (0.083 ±0.005) was higher than that of the exosome of Hep3B cells with low metastatic potential (0.021 ± 0.003);The invasive ability of Hep3B cells was elevated significantly after co-culturue with exosomes from MHCC97H cells,and the number of cells penetrating membranes was increased (15.3 ±2.5 vs.8.7 ±2.1).The circRNA_100338 level was increased in co-culture group (0.019 ±0.002 vs.0.007 ±0.001).The MMP-9 activity was gradually increased (0.062 ± 0.001,0.147 ± 0.002,0.315 ± 0.003).All differences were statistically significant (P < 0.05).Conclusion Exosomes from cells with high metastatic potential promote HCC invasion,which is related to the activated MMP-9 and elevated circRNA_l00338 level in exosomes.
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