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

doi:10.3877/cma.j.issn.1674-0807.2020.02.004

Abstract

Abstract:

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

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.

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