| 陈 睿,杨 欣,赵伟庆.组蛋白三甲基化转移酶SETD2对肺腺癌上皮—间充质转化的影响[J].中国肿瘤,2020,29(2):140-147. |
| 组蛋白三甲基化转移酶SETD2对肺腺癌上皮—间充质转化的影响 |
| Effect of Histone Methyltransferase SETD2 on the Epithelial?鄄Mesenchymal Transition of Lung Adenocarcinoma |
| 中文关键词 修订日期：2019-09-09 |
| DOI：10.11735/j.issn.1004-0242.2020.02.A010 |
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| 中文关键词: 组蛋白三甲基化转移酶SETD2 肺腺癌 增殖 迁移 侵袭 上皮—间充质转化 |
| 英文关键词:histone methyltransferase SETD2 lung adenocarcinoma cell proliferation cell migration cell invasion epithelial-mesenchymal transition |
| 基金项目:国家自然科学基金（81501971）；中国博士后科学基金（2018M630603）；江苏省自然科学基金（BK20150252）；江苏省“六大人才高峰”基金（WSW-142）；江苏省青年医学人才基金（QNRC2016279） |
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| 中文摘要: |
| 摘 要：［目的］ 研究组蛋白三甲基化转移酶SETD2与肺腺癌的临床相关性及其在肺腺癌组织中的表达，探讨SETD2对肺腺癌细胞株A549和H1975增殖、迁移、侵袭能力及上皮—间充质转化（EMT）过程的影响。［方法］ 结合癌症基因组图谱数据库（TCGA），分析SETD2的表达水平与肺腺癌患者总生存期（OS）的相关性。分别应用Real-time PCR和Western blot方法检测SETD2mRNA和蛋白在人肺腺癌组织芯片（包含12对肺腺癌组织标本）中的表达情况。构建慢病毒载体过表达Lv-SETD2-A549、Lv-SETD2-H1975稳转细胞株，沉默Si-SETD2-A549、Si-SETD2-H1975稳转细胞株及阴性对照组Lv-NC、Si-NC稳转细胞株。通过细胞计数试剂盒（CCK-8）法、细胞划痕和Transwell小室实验，分别检测过表达和沉默SETD2对细胞增殖、迁移和侵袭能力的影响。采用Western blot方法比较4组细胞株中EMT相关蛋白上皮型钙黏附蛋白（E-cadherin）、神经性钙黏附蛋白（N-cadherin）、波形蛋白（Vimentin）及正常对照组（GADPH）的表达水平差异；应用免疫荧光法检测4组间E-cadherin、Vimentin的表达水平。［结果］ （1）SETD2表达与肺腺癌患者OS成正相关（P＝0.014）。（2）SETD2mRNA和蛋白在肺腺癌组织中表达明显下降，且与临床分期明显相关（P＝0.001）。（3）与Lv-NC组相比，A549和H1975在SETD2过表达组中SETD2mRNA和蛋白显著升高，细胞相对增殖活性（A549-0h：0.18±0.02，A549-24h：0.22±0.04，A549-48h：0.35±0.06，A549-72h：0.55±0.05；H1975-0h：0.21±0.02，H1975-24h：0.31±0.04，H1975-48h：0.43±0.03，H1975-72h：0.71±0.09），细胞穿膜数(A549：54.00±11.23；H1975：57.13±10.28)均显著下降，迁移间距(A549：0.65±0.09；H1975：0.51±0.10)降低；这些指标，在SETD2沉默组中恰恰相反。（4）与阴性对照组相比，过表达SETD2可上调A549、H1975细胞株中E-cadherin的蛋白表达水平，而下调N-cadherin、Vimentin的蛋白表达水平；这些指标在SETD2沉默组中则相反；免疫荧光实验结果显示过表达SETD2的A549和H1975组E-cadherin的红色荧光强度较Lv-NC组增强，而Vimentin的绿色荧光较Lv-NC组明显减弱。 ［结论］ SETD2在肺腺癌组织中表达下降，SETD2低表达组患者OS较高表达明显下降。上调SETD2表达水平能够有效抑制肺腺癌细胞的增殖、迁移和侵袭能力，且逆转其发生EMT，抑制SETD2的表达则可诱导上述过程；SETD2可能发挥抑癌作用。 |
| 英文摘要: |
| Abstract：［Objective］ To investigate the effects of histone methyltransferase SETD2 expression on prognosis of patients with lung adenocarcinoma，and to investigate the effects of SETD2 on proliferation，migration，invasion and epithelial-mesenchymal transition(EMT) in lung adenocarcinoma cell line A549 and H1975. ［Methods］ The association of SETD2 expression with overall survival(OS) of patients was analyzed upon The Cancer Genome Alta(TCGA) database. The expression of SETD2mRNA and protein in lung adenocarcinoma tissue microarray(including 12 pairs of lung adenocarcinoma tissue samples) were detected by Real-time PCR and Western Blot respectively. Lentivirus-mediated SETD2 over-expression or silence in lung adenocarcinoma cell line A549(Lv-SETD2-A549 or Si-SETD2-A549) and H1975(Lv-SETD2-A549 or Si-SETD2-H1975) were constructed. The in vitro functional analysis，including proliferation，migration and invasion assays were performed by CCK-8，wound healing and transwell assays. The expression of EMT related proteins including E-cadherin，N-cadherin and Vimentin were compared with GADPH in 4 groups by Western Blot. The immunofluorescent assay was performed to detect the expres-sion of E-cadherin and Vimentin in 4 groups. ［Results］ (1)The expression level of SETD2 was positively related to the long-term prognosis of lung adenocarcinoma patients(P＝0.014). (2)The expression of SETD2 mRNA and protein in lung adenocarcinoma were significantly lower and correlated with the clinical stage(P=0.001). (3)Compared to the negative control Lv-NC，the expression level of SETD2mRNA and protein in Lv-SETD2 A549 and H1975 cells significantly increased，the relative cell proliferation activity(A549-0h：0.18±0.02，A549-24h：0.22±0.04，A549-48h：0.35±0.06，A549-72h：0.55±0.05；H1975-0h：0.21±0.02，H1975-24h：0.31±0.04，H1975-48h：0.43±0.03，H1975-72h：0.71±0.09) and the number of membrane penetration(A549：54.00±11.23；H1975：57.13±10.28) decreased，the length of cell migration spacing(A549：0.65±0.09；H1975：0.51±0.10) was shortened after SETD2 over-expression，the results were reversed when SETD2 silence. (4)The expression level of E-cadherin in A549 and H1975 cell lines was up-regulated by SETD2 over-expression，and the expression level of N-cadherin and Vimentin were down-regulated，the results were on the contrary after SETD2 silence. The immunofluorescent assay indicated that the E-cadherin red fluorescence intensity in Lv-SETD2-A549 and Lv-SETD2-H1975 groups were stronger than that in NC groups，whereas the Vimentin green fluorescence intensity in both 2 groups was significantly weaker than that in NC groups. ［Conclusion］ The expression of SETD2 in lung adenocarcinoma decreased and the low expression of SETD2 had an adverse influence on the prognosis of patients. The up-regulation of SETD2 can inhibit the cell proliferation，migration，invasion，and EMT of lung adenocarcinoma. SETD2 may act as a tumor suppressor. |
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