摘要目的:探索髓系相关分化标志物（MYADM）在胆固醇诱导肺腺癌转移中的作用及机制。方法:（1）细胞实验：利用肺腺癌A549和H1975细胞，以0.8 mg/ml胆固醇处理细胞，MYADM敲低组用携带MYADM小干扰RNA的慢病毒转染细胞，MYADM过表达组用MYADM过表达质粒转染细胞。采用Western blot法检测细胞中MYADM、上皮细胞钙黏蛋白（E-cadherin）、β-连环蛋白（β-catenin）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、波形蛋白（vimentin）的表达，平板克隆形成实验检测细胞的增殖能力，Transwell实验检测细胞的迁移和侵袭能力。采用Western blot法和细胞免疫荧光实验检测胆固醇处理的肺腺癌细胞中Akt磷酸化对MYADM、Rac家族小鸟苷三磷酸酶1（Rac1）的表达和骨髓细胞瘤癌基因（c-Myc）磷酸化的影响，采用Western blot法检测c-Myc敲低对肺腺癌细胞中MYADM和单羧酸转运蛋白1（MCT1）表达的影响，采用染色质免疫沉淀（ChIP）实验检测胆固醇对肺腺癌细胞中c-Myc与MYADM和MCT1启动子之间结合的影响。（2）动物实验：将A549细胞或携带抗MYADM慢病毒的A549细胞分别经尾静脉接种于BALB/c裸鼠，再进一步分为正常饲料组和高胆固醇饲料组。使用活体成像仪检测裸鼠体内肿瘤的生长及转移情况。42 d后，取肺组织，免疫组织化学染色检测相关蛋白的变化。结果:经胆固醇处理后，A549细胞中MYADM的表达水平由1.00±0.18增加到3.28±0.28（ P<0.001），H1975细胞中MYADM的表达水平由1.00±0.06增加到2.03±0.10（ P<0.001）。与对照组比较，MYADM敲低组肺腺癌细胞中E-cadherin表达升高（ P<0.01），β-catenin、MMP-2、MMP-9和vimentin表达降低（均 P<0.01）；A549细胞形成的克隆数由（203±23）个下降到（60±18）个（ P=0.001），H1975细胞形成的克隆数由（298±64）个下降到（137±51）个（ P=0.271）；A549细胞的侵袭细胞数由（212±18）个下降到（99±34）个（ P=0.007），H1975细胞的侵袭细胞数由（268±34）个下降到（134±14）个（ P=0.003）；549细胞的迁移细胞数由（353±37）个下降到（124±29）个（ P=0.001），H1975细胞数的迁移细胞数由（279±41）个下降到（79±19）个（ P=0.002）。MYADM过表达组肺腺癌细胞中E-cadherin的表达水平降低（ P<0.01），β-catenin、MMP-2、MMP-9和vimentin的表达水平均升高（均 P<0.01）；A549细胞形成的克隆数由（94±26）个增加到（298±34）个（ P=0.001），H1975细胞形成的克隆数由（83±13）个增加到（331±24）个（ P<0.001）；A549细胞的侵袭细胞数由（118±17）个增加到（193±24）个（ P=0.012），H1975细胞的侵袭细胞数由（156±19）个增加到（321±12）个（ P<0.001）；A549细胞的迁移细胞数由（171±22）个增加到（284±15）个（ P=0.002），H1975细胞的迁移细胞数由（178±7）个增加到（263±12）个（ P<0.001）。分子机制相关实验结果显示，MYADM过表达促进肺腺癌细胞中MCT1的表达（均 P<0.01），胆固醇在促进肺腺癌细胞中MYADM表达的同时还促进Rac1和MCT1的表达以及Akt和c-Myc的磷酸化（均 P<0.05）。免疫共沉淀实验结果显示，与正常的肺腺癌细胞相比，胆固醇处理的肺腺癌细胞中MYADM与Rac1相互作用水平升高［A549细胞：（191.0±26.7）%比（100.0±15.9）%， P=0.007；H1975细胞：（170.0±27.5）%比（100.0±18.2）%， P=0.021］。ChIP实验证实，胆固醇处理能增强c-Myc与MYADM和MCT1启动子之间的结合。动物实验显示，高胆固醇饲料促进肺腺癌细胞在裸鼠体内的转移，可诱导裸鼠肺组织中MYADM、MCT1和Rac1的表达以及Akt和c-Myc的磷酸化，而MYADM敲低可以抑制肺腺癌细胞在裸鼠体内的转移，抑制裸鼠肺组织中MYADM、MCT1和Rac1的表达以及Akt和c-Myc的磷酸化。 结论:胆固醇可能通过调节MYADM/Rac1/Akt/c-Myc/MCT1轴诱导肺腺癌细胞的增殖和转移。

abstractsObjective:To explore the role and related mechanism of myeloid related differentiation markers (MYADM) in lung adenocarcinoma metastasis induced by high cholesterol diet.Methods:(1) Cell experiments: Using lung adenocarcinoma A549 and H1975 cells, the cells were treated with 0.8 mg/ml cholesterol and then transfected with a lentivirus to knock down MYADM. The overexpression of MYADM was achieved by transfecting the cells with an overexpression plasmid. Western blotting was used to detect the expression levels of MYADM, E-cadherin, β-catenin, MMP-2, MMP-9, and vimentin in the cells. The proliferation ability of the cells was assessed using the plate clonal formation assay, while the migration and invasion ability were evaluated using the Transwell assay. Western blot was used to determine the effects of MYADM knockdown or overexpression on these proteins. Western blot and immunofluorescence assays were conducted to investigate the impact of Akt phosphorylation on the expression of MYADM and Rac1 in cholesterol-treated lung adenocarcinoma cells, as well as the phosphorylation of c-Myc. Western blot was also used to assess the effect of c-Myc knockdown on the expression of MYADM and MCT1 in lung adenocarcinoma cells. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the impact of cholesterol on the binding between c-Myc and the promoters of MYADM and MCT1 in lung adenocarcinoma cells. (2) Animal experiment: A549 cells or A549 cells with MYADM knockdown were intravenously inoculated into BALB/c nude mice, which were then divided into a normal diet group and a high cholesterol diet group. Using a live imaging system, the growth and metastasis of tumors in the mice were monitored. After 42 days, lung tissues were collected for immunohistochemical staining to detect changes in relevant proteins.Results:After cholesterol treatment, the expression level of MYADM in A549 cells increased from 1.00±0.18 to 3.28±0.28 ( P<0.001), and in H1975 cells, it increased from 1.00±0.06 to 2.03±0.10 ( P<0.001). Compared with the control group, the expression of E-cadherin in lung adenocarcinoma cells after MYADM knockdown increased ( P<0.01), while the expressions of β-catenin, MMP-2, MMP-9, and vimentin decreased (all P<0.01). After MYADM knockdown, the number of clonal plates decreased in A549 cells (203±23 vs 60±18, t=8.48, P=0.001) and H1975 cells (298±64 vs 137±51, t=3.41, P=0.271). The number of invasive cells also decreased in A549 cells (212±18 vs 99±34, t=5.09, P=0.007) and H1975 cells (268±34 vs 134±14, t=6.31, P=0.003). Additionally, the number of migratory cells decreased in A549 cells (353±37 vs 124±29, t=8.44, P=0.001) and H1975 cells (279±41 vs 79±19, t=7.67, P=0.002). In the lung adenocarcinoma cells overexpressing MYADM, the expression of E-cadherin decreased ( P<0.01), while the levels of β-catenin, MMP-2, MMP-9, and vimentin increased (all P<0.01). The number of plate clonal colonies formed by lung adenocarcinoma cells overexpressing MYADM increased significantly in A549 cells, (94±26 vs 298±34, t=8.26, P=0.001) and H1975 cells (83±13 vs 331±24, t=15.74, P<0.001). The number of invasive A549 cells also increased (118±17 vs 193±24, t=4.41, P=0.012) and (156±19 vs 321±12, t=12.72, P<0.001). Additionally, the number of migrating cells increased in A549 cells (171±22 vs 284±15, t=7.35, P=0.002) and in H1975 cells (178±7 vs 263±12, t=10.6, P<0.001). Experiments related to the molecular mechanism showed that overexpression of MYADM promotes the expression of MCT1 in lung adenocarcinoma cells (all P<0.01). Cholesterol not only enhances the expression of MYADM in lung adenocarcinoma cells, but also boosts the expression of Rac1 and MCT1, as well as the phosphorylation of Akt and c-Myc (all P<0.05). Immunoprecipitation experiments revealed that in A549 cells treated with cholesterol, MYADM-Rac1 interaction levels increased from (100.0±15.9)% to (191.0±26.7)% ( P=0.007), while in H1975 cells, the levels increased from (100.0±18.2)% to (170.0±27.5)% ( P=0.021). ChIP confirmed that cholesterol treatment enhances the binding of c-Myc to the promoters of MYADM and MCT1. In vivo experiments demonstrated that a high-cholesterol diet promotes the metastasis of lung adenocarcinoma cells in mice, inducing the expression of MYADM, MCT1, and Rac1, as well as the phosphorylation of Akt and c-Myc in mouse lung tissue. Conversely, knocking down MYADM inhibits the metastasis of lung adenocarcinoma cells in mice, suppressing the expression of MYADM, MCT1, and Rac1, as well as the phosphorylation of Akt and c-Myc in mouse lung tissues. Conclusion:Cholesterol may induce lung adenocarcinoma cells proliferation and metastasis by regulating the MYADM/Rac1/Akt/c-Myc/MCT1 axis.