甲基转移酶SETDB2对肝癌侵袭迁移的影响及其机制研究

The effect and mechanism of methyltransferase SETDB2 on the invasion and migration of hepatoma carcinoma

目的检测甲基转移酶SETDB2对肝癌侵袭转移的影响并探讨其机制。方法应用Western blot、免疫组化以及Real-time PCR等方法检测37例肝癌及对应癌旁组织标本中SETDB2蛋白及mRNA表达情况。将靶向SETDB2的短发卡RNA(sh-SETDB2)和空载体(sh-NC)分别转染至人肝癌MHCC97H细胞构建稳转细胞系,应用Tanswell侵袭实验、细胞划痕实验检测2组细胞的侵袭和迁移能力。通过基因芯片检测敲低SETDB2后表达有变化的基因。敲低SETDB2后,运用Real-time PCR检测PTEN的mRNA的表达变化以及Western blot检测PTEN和组蛋白H3第9位赖氨酸的三甲基化(H3K9me3)的表达变化,CHIP检测H3K9me3在PTEN的启动子区域的变化。在敲低SETDB2的同时敲低PTEN的表达,Tanswell侵袭实验检测细胞侵袭能力的变化。结果 (1)Western blot和Real-timePCR的结果均表明肝癌组织中SETDB2蛋白及mRNA表达水平均高于癌旁组织;SETDB2表达的高低与肝癌组织学分级和TNM分期有关。(2)转染sh-SETDB2的MHCC97H细胞的侵袭和迁移能力均明显低于sh-NC组。(3)基因芯片、Western blot以及Real-time PCR等实验结果显示敲低SETDB2后PTEN表达上调。(4)在敲低SETDB2后H3K9me3表达下调,PTEN启动子区域的H3K9me3减少。(5)与敲低SETDB1组相比,敲低SETDB2的同时敲低PTEN后细胞的侵袭能力恢复。结论 SETDB2通过下调PTEN的表达促进肝癌细胞的侵袭迁移。

Objective To investigate the effect of methyhransferase SETDB2 on the invasion and metastasis of liver cancer and explore its mechanism. Methods Western blot, immunohistochemistry and Real-time PCR assays were performed to detect the expressions of SETDB2 protein and mRNA in liver cancer and the adjaeent tissue specimens of 37 cases. The short hair pair RNA targeting SETDB2 （sh-SETDB2） and the con＇esponding empty vector （sh-NC） were transfected into MHCC97H cells. The invasion and migration ability of the two groups of cells were detected by Tanswell invasion assay and wound-healing assay. The gene chip was used to detect the expression of the altered genes after knocking down SETDB2. The mRNA expression of PTEN was detected by Real-time PCR. Western blot assay was used to detect the protein expressions of PTEN and H3Kgme3. CHIP was used to detect the change of H3Kgme3 in the promoter region of PTEN. The expression of PTEN was knocked down in shSETDB2-MHCC97H cells, and the invasion ability of cells was detected by the Tanswell invasion assay. Results （1） The results of Western blot and Real-time PCR assays showed that the expression levels of SETDB2 protein and mRNA were higher in the liver cancer tissues than those in the adjacent normal tissues. The expression level of SETDB2 was related to the histological grade and TNM stage of liver cancer. （2） The invasion and migration abilities were significantly lower in MHCC97H cells transfected with sh-SETDB2 than those in sh-NC group. （3） Microarray experiment, Western blot and Real-time PCR assays showed that PTEN was up-regulated after knocking down SETDB2. （4） The expression of H3Kgme3 was decreased after knocking down SETDB2, and the appearance of H3Kgme3 in PTEN promoter region was decreased as well. （5） Compared with the knockdown SETDB1, the invasive ability of cells was restored after both SETDB2 and PTEN were knocked down. Conclusion SETDB2 promotes the invasion and migration of hepatoma cells by downregulating the expression of PTEN.