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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2020, Vol. 14 ›› Issue (02) : 78 -85. doi: 10.3877/cma.j.issn.1674-0807.2020.02.004

所属专题： 文献；

论著

肿瘤相关巨噬细胞中过氧化物酶体增殖物激活受体γ辅助活化因子1α对乳腺癌4T1细胞上皮-间质转化及侵袭、转移能力的影响
靳鑫1, 王园园2, 倪田根2, 王宁2, 罗浩军2, 明佳2,†()   
  1. 1. 408000　重庆市涪陵中心医院重症医学科
    2. 400010　重庆医科大学附属第二医院乳腺甲状腺外科
  • 收稿日期:2018-09-02 出版日期:2020-04-01
  • 通信作者: 明佳
  • 基金资助:
    重庆市渝中区科技计划项目(20160104); 重庆市卫生计生委医学科研计划项目(2016ZDXM012); 重庆技术创新于应用示范（社会民生类）一般项目(cstc2018jscx-msybx0020)

Effect of PPAR γ coactivator-1α in tumor-associated macrophages on epithelial-mesenchymal transition, invasion and metastasis of breast cancer 4T1 cells

Xin Jin1, Yuanyuan Wang2, Tiangen Ni2, Ning Wang2, Haojun Luo2, Jia Ming2,†()   

  1. 1. Department of Critical Medicine, Chongqing Fuling Central Hospital, Chongqing 408000, China
    2. Department of Breast and Thyroid Surgery, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
  • Received:2018-09-02 Published:2020-04-01
  • Corresponding author: Jia Ming
  • About author:
    Corresponding author: Ming Jia, Email:
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引用本文：

靳鑫, 王园园, 倪田根, 王宁, 罗浩军, 明佳. 肿瘤相关巨噬细胞中过氧化物酶体增殖物激活受体γ辅助活化因子1α对乳腺癌4T1细胞上皮-间质转化及侵袭、转移能力的影响[J]. 中华乳腺病杂志(电子版), 2020, 14(02): 78-85.

Xin Jin, Yuanyuan Wang, Tiangen Ni, Ning Wang, Haojun Luo, Jia Ming. Effect of PPAR γ coactivator-1α in tumor-associated macrophages on epithelial-mesenchymal transition, invasion and metastasis of breast cancer 4T1 cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2020, 14(02): 78-85.

目的

探讨小鼠乳腺癌细胞(4T1)和小鼠腹腔巨噬细胞(PMs)共培养构成的肿瘤相关巨噬细胞(TAMs)中，过氧化物酶体增殖物激活受体γ辅助活化因子1α(PGC-1α)促进4T1细胞侵袭的作用机制。

方法

(1)提取PMs并用小发卡RNA(shRNA)敲低PGC-1α蛋白表达：采用原代细胞提取的方法提取PMs，并用低表达PGC-1α的shRNA(sh-PGC-1α-1、sh-PGC-1α-2、sh-PGC-1α-3)转染细胞，然后用Western blot检测PMs[对照组(PMs组)、阴性对照组(sh-control)、sh-PGC-1α-1组、sh-PGC-1α-2组、sh-PGC-1α-3组]中PGC-1α蛋白表达水平，选取抑制效果最好的shRNA用于实验。所得低表达PGC-1α的sh-PGC-1α PMs为本实验所需。(2)验证TAMs中PGC-1α蛋白表达水平：利用Transwell建立PMs/sh-PGC-1α PMs和4T1细胞的共培养体系，得到TAMs/sh-PGC-1α TAMs。收集细胞并提取蛋白，用Western blot检测对照组(PMs组)、TAMs组、sh-PGC-1α TAMs组PGC-1α蛋白表达水平。(3)验证共培养体系中4T1细胞的侵袭、迁移、愈合能力：利用Transwell小室模型观察对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组4T1细胞的侵袭、迁移数；利用创伤愈合实验观察对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组4T1细胞的愈合情况。(4)验证共培养体系中PMs的PGC-1α对4T1细胞发生细胞上皮-间质转化(EMT)的影响：利用Transwell建立PMs /sh-PGC-1α PMs和4T1细胞的共培养体系，采用Western blot检测对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组EMT相关蛋白E-cadherin(上皮标志物)及vimentin(间质标志物)的表达情况。多组实验数据比较采用单因素方差分析，两两比较采用LSD法。

结果

(1)对照组(PMs组)、阴性对照组、sh-PGC-1α-1组、sh-PGC-1α-2组和sh-PGC-1α-3组PGC-1α表达水平分别为1.00±0.00、0.96±0.06、0.43±0.04、0.35±0.07和0.38±0.08，5组比较，差异有统计学意义(F＝32.648，P<0.050)，其中，对照组(PMs组)分别与阴性对照组、sh-PGC-1α-3组比较，PGC-1α表达水平的差异均无统计学意义(P＝0.991、0.065)，但是，sh-PGC-1α-1组、sh-PGC-1α-2组PGC-1α表达水平均低于对照组(PMs组)(P＝0.014、0.045)，并且，sh-PGC-1α-1组、sh-PGC-1α-2组和sh-PGC-1α-3组的PGC-1α表达水平均低于阴性对照组(P＝0.015、0.018、0.035)。因此，选择对PGC-1α抑制效果最好的sh-PGC-1α-2进行后续实验。(2)对照组(PMs组)、TAMs组、sh-PGC-1α TAMs组PGC-1α表达水平分别为1.00±0.00、1.92±0.20和0.90±0.23，3组比较，差异有统计学意义(F＝10.294，P＝0.011)，其中TAMs组PGC-1α蛋白表达水平明显高于对照组(PMs组)和sh-PGC-1α TAMs组(P均<0.050)。(3)Transwell侵袭实验显示，对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组比较，共培养24 h后穿膜的细胞数分别为(41.67±6.01)、(75.33±5.46)和(24.33±1.45)个，3组比较，差异有统计学意义(F＝29.668，P<0.050)，其中TAMs组4T1细胞的侵袭能力显著高于对照组(4T1组)和sh-PGC-1α TAMs组(P均<0.050)。Transwell迁移实验显示，对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组共培养24 h后穿膜的细胞数分别为(55.67±12.13)、(97.67±8.45)和(30.67±6.49)个，3组比较，差异有统计学意义(F＝13.193，P<0.05)，其中TAMs组4T1细胞的迁移能力显著高于对照组(4T1组)和sh-PGC-1α TAMs组(P均<0.050)。创伤愈合实验显示，对照组(4T1组)、TAMs组、sh-PGC-1α TAMs组4T1细胞24 h的愈合率分别为(100.00±0.00)%、(135.98±5.18)%和(68.82±7.28)%，3组比较，差异有统计学意义(F＝42.435，P<0.050)，其中TAMs组4T1细胞的愈合能力显著高于对照组(4T1组)和sh-PGC-1α TAMs组(P<0.050)。(4)Western blot检测发现：对照组(4T1组)、TAMs组和sh-PGC-1α TAMs组E-cadherin表达水平分别为1.00±0.00、0.44±0.11和2.51±1.67, vimentin表达水平分别为1.00±0.00、2.20±0.15和0.36±0.10，3组比较，E-cadherin和vimentin表达水平的差异均有统计学意义(F＝87.295、79.058，P均<0.050)；组间两两比较显示，TAMs组E-cadherin表达水平显著低于对照组(4T1组)和sh-PGC-1α TAMs组(P均<0.050)，vimentin表达水平显著高于对照组(4T1组)和sh-PGC-1α TAMs组(P均<0.050)，而sh-PGC-1α TAMs组E-cadherin表达水平显著高于对照组(4T1组)(P<0.050)，vimentin表达水平显著低于对照组(4T1组)(P<0.050)。

结论

TAMs可促进4T1细胞的侵袭、迁移、愈合以及EMT过程，且该作用与TAMs的PGC-1α相关。

关键词: 乳腺肿瘤, 巨噬细胞, 转录因子, 肿瘤侵润, 肿瘤转移
Objective

To investigate the activation of peroxisome proliferator-activated receptor(PPAR) γ in tumor-associated macrophages (TAMs) composed of mouse breast cancer cells (4T1) and co-cultured mouse peritoneal macrophages (PMs) and explore the mechanism of PPAR γ coactivator-1α (PGC-1α) promoting 4T1 cell invasion.

Methods

(1) Extract PMs and use short hairpin RNA (shRNA) to knock down PGC-1α protein expression. PMs were extracted form primary cells, and transfected with shRNA (sh-PGC-1α-1, sh-PGC-1α-2, sh-PGC-1α-3), and then PGC-1α protein expression was determined in the control group (PMs group), negative control group (sh-control), sh-PGC-1α-1 group, sh-PGC-1α-2 group and sh-PGC-1α-3 group using Western blot. The shRNA with the best inhibitory effect was selected for experiments. The sh-PGC-1α PMs with low expression of PGC-1α were preserved for this experiment. (2) Verify the expression of PGC-1α protein in TAMs. Transwell method was used to establish a co-culture system of PMs/sh-PGC-1α PMs and 4T1 cells to obtain TAMs/sh-PGC-1α TAMs. The cells were collected and the protein was extracted. The expression of PGC-1α protein in the control group (PMs group), TAMs group, and sh-PGC-1α TAMs group was detected by Western blot. (3) Verify the invasion, migration, and healing ability of 4T1 cells in the co-culture system. The numbers of invasive and metastatic 4T1 cells in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group were measured using a Transwell chamber model; the wound healing test was used to observe the healing of 4T1 cells in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group. (4) Verify the effect of PGC-1α in PMs on the epithelial-mesenchymal transition (EMT) of 4T1 cells in the co-culture system. Transwell was used to establish a co-culture system of PMs/sh-PGC-1α PMs and 4T1 cells; the expressions of E-cadherin (epithelial marker) and vimentin (interstitial marker) in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group were detected using Western blot. Group comparison was performed using single factor analysis of variance, and the pairwise comparison was performed using the LSD method.

Results

(1) The expression of PGC-1α was 1.00±0.00, 0.96 ± 0.06, 0.43 ± 0.04, 0.35 ± 0.07 and 0.38 ± 0.08 in the control group (PMs group), negative control group, sh-PGC-1α-1 group, sh-PGC-1α-2 group and sh-PGC-1α-3 group, respectively, indicating a significant difference across five groups (F=32.648, P<0.050). There was no significant difference in PGC-1α expression between the control group (PMs group) and negative control group or sh-PGC-1α-3 group (P=0.991, 0.065). The sh-PGC-1α-1 group and sh-PGC-1α-2 group had significantly lower expression of PGC-1α expression compared with the control group (PMs group) (P=0.014, 0.045), and PGC-1α expression in the three groups (sh-PGC-1α-1 group, sh-PGC-1α-2 group and sh-PGC-1α-3 group) was significantly lower than that in the negative control group (P=0.015, 0.018, 0.035). Therefore, sh-PGC-1α-2 presented the best inhibitory effect on PGC-1α and it was selected for subsequent experiments. (2) The expression of PGC-1α in the control group (PMs group), TAMs group and sh-PGC-1α TAMs group was 1.00 ± 0.00, 1.92 ± 0.20 and 0.90 ± 0.23, respectively, indicating a significant difference across three groups (F=10.294, P=0.011). The expression of PGC-1α in TAMs group was significantly higher than that in the control group (PMs group) or sh-PGC-1α TAMs group (both P<0.050). (3) Transwell invasion experiments showed that the number of cells that passed through the membrane after 24 hours of co-culture were 41.67 ± 6.01, 75.33 ± 5.46 and 24.33 ± 1.45 in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group, respectively, indicating a significant difference (F=29.668, P<0.050). The invasion ability of 4T1 cells in the TAMs group was significantly higher than that in the control group (4T1 group) and sh-PGC-1α TAMs group (both P<0.050). Transwell migration experiments showed that the number of cells that passed through the membrane after the 24 hours of co-culture was 55.67 ± 12.13, 97.67 ± 8.45 and 30.67± 6.49 in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group, respectively, indicating a significant difference (F=13.193, P<0.050). The migration ability of 4T1 cells in the TAMs group was significantly higher than that in the control group (4T1 group) or the sh-PGC-1α TAMs group (both P<0.050). Wound healing experiments showed that the healing rate of 4T1 cells for 24 hours was (100.00 ± 0.00)%, (135.98 ± 5.18)% and (68.82±7.28)% in the control group (4T1 group), TAMs group, and sh-PGC-1α TAMs group, respectively, indicating a significant difference (F=42.435, P<0.050). The healing ability of 4T1 cells in the TAMs group was significantly higher than that in the control group (4T1 group) or sh-PGC-1α TAMs group (P<0.050). (4) Western blot analysis found that the expression of E-cadherin was 1.00 ± 0.00, 0.44 ± 0.11 and 2.51 ± 1.67, and the expression of vimentin was 1.00 ± 0.00, 2.20 ± 0.15 and 0.36 ± 0.10 in the control group (4T1 group), TAMs group and sh-PGC-1α TAMs group, respectively, indicating a significant difference across three groups (F=87.295, 79.058, both P<0.050); pairwise comparison showed that the expression of E-cadherin in the TAMs group was significantly lower than that in the control group (4T1 group) or sh-PGC-1α TAMs group (both P<0.050), and the expression of vimentin in the TAMs group was significantly higher than that in the control group (4T1 group) or sh-PGC-1α TAMs group (all P<0.050), but the expression of E-cadherin in the sh-PGC-1α TAMs group was significantly higher than that in the control group (4T1 group) (P<0.050), and the expression of vimentin in the sh-PGC-1α TAMs group was significantly lower than that in the control group (P<0.050).

Conclusion

TAMs can promote the invasion, migration, healing and EMT of 4T1 cells, which is related to PGC-1α in TAMs.

Key words: Breast neoplasms, Macrophages, Transcription factors, Neoplasm invasiveness, Neoplasm metastasis
图1 采用Western blot检测5组PMs的PGC-1α蛋白表达水平
图2 采用Western blot检测3组细胞的PGC-1α蛋白表达水平
图3 采用普通光学显微镜观察4T1细胞的侵袭能力(结晶紫　×400)　a～c图分别所示对照组(4T1组)、TAMs组和sh-PGC-1α TAMs组细胞的侵袭能力
图4 采用普通光学显微镜观察3组4T1细胞的迁移能力(结晶紫　×400)　a～c图分别所示对照组(4T1组)、TAMs组和sh-PGC-1α TAMs组细胞的迁移能力
图5 采用普通光学显微镜观察3组4T1细胞的愈合能力(×100)　a～c图与d～f图分别所示对照组(4T1组)、TAMs组和sh-PGC-1α TAMs组细胞0 h与24 h的愈合能力
图6 采用Western blot检测3组4T1细胞中E-cadherin和vimentin表达水平
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