Krüppel like factor 6 splice variant 1 (KLF6-SV1) overexpression recruits macrophages to participate in lung cancer metastasis by up-regulating TWIST1

Abstract

The aim of this study was to investigate the mechanism by which KLF6-SV1 promoted lung cancer metastasis through tumor-associated macrophages (TAMs). Plasmid transfection was used to construct cells that upregulated or silenced gene. Tumor-bearing mouse model was established using A549 cells. SP staining was performed to detect the CD163 and CD68. Six-well plates and Transwell chamber were used for co-culture of lung cancer A549 cells and macrophages. CCK-8 and Transwell assay were applied to detected the cell viability and migration respectively. Protein and mRNA were tested by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR).KLF6-SV1 overexpression promoted the expression levels of TWIST1 and CCL2, and also induce macrophage polarization to M2 and epithelial-mesenchymal transition (EMT). In vitro experiments showed that KLF6-SV1 might regulate the migration of lung cancer cells by regulating the expression of TWIST1 and CCL-2. M2 macrophages did not affect the expression of KLF6-SV1, TWIST1 and CCL-2. The co-culture system could up-regulate the EMT of A549 cells.Overexpression of KLF6-SV1 promoted the expression of TWIST1 and CCL2, and up-regulation of TWIST1 expression might promote the infiltration of M2 macrophages, which promoted the involvement of EMT in the metastasis of lung cancer cells.

Keywords: Krüppel like factor 6; epithelial-mesenchymal transition; lung cancer; non-small cell lung cancer; tumor-associated macrophage.

Figure 1.

Effects of KLF6-SV1 on the expression levels of TWIST1 and CCL2. (a-f) KLF6-SV1, TWIST1 and CCL2 proteins and mRNAs were tested by Western blot and qRT-PCR. (g) CCK-8 assay was used to tested the cell viability. *P < 0.05,**P < 0.01, versus control group; ^{^}P < 0.05, ^{^^}P < 0.01, versus NC group; ^#P < 0.05, ^##P < 0.01, versus KLF6-SV1 group.

Figure 2.

Effects of KLF6-SV1 on TAMs. (a, b) ELISA was used to detect the M-CSF and GM-CSF expressions. (c, d) SP staining was performed to detect the CD163 and CD68 proteins in tumor tissues. *P < 0.05,**P < 0.01, versus control group; ^{^}P < 0.05, ^{^^}P < 0.01, versus NC group.

Figure 3.

Effects of KLF6-SV1 on tumor weigh and EMT. (a-c) Tumor tissue size and weight of mice. (d-e) E-cadherin, N-cadherin and MMP-9 proteins and mRNAs were tested by Western blot and qRT-PCR. *P < 0.05,**P < 0.01, versus control group; ^{^}P < 0.05, ^{^^}P < 0.01, versus NC group.

Figure 4.

Effects of co-transfection of KLF6-SV1 and siTWIST1 on A549 cells. (a) CCK-8 assay was used to tested the cell viability. (b-d) KLF6-SV1, TWIST1 and CCL2 proteins and mRNAs levels at 48 h after tansfection were tested by Western blot and qRT-PCR. (e, f) ELISA was applied to detect the M-CSF and GM-CSF expressions at 48 h after tansfection. (g) Migration rate at 48 h after tansfection was detected using Transwell assay. *P < 0.05,**P < 0.01, versus control group; ^{^}P < 0.05, ^{^^}P < 0.01, versus NC group.

Figure 5.

Effects of co-culture of A549 cells and macrophages on A549 cells. (a, b) CD163 and CD68 proteins and mRNAs were tested by Western blot and qRT-PCR. (c) CCK-8 assay was used to tested the cell viability. (d-f) KLF6-SV1, TWIST1 and CCL2 proteins and mRNAs were tested by using Western blot and qRT-PCR. (g, h) ELISA was applied to detect the M-CSF and GM-CSF expressions. *P < 0.05,**P < 0.01, versus control group.

Figure 6.

Effects of co-culture of A549 cells and macrophages on EMT of A549 cells. (a, b) Migration rate was detected using Transwell assay. (c-e) E-cadherin, N-cadherin and MMP-9 proteins and mRNAs were tested by Western blot and qRT-PCR. *P < 0.05,**P < 0.01, versus control group; ^{^}P < 0.05, ^{^^}P < 0.01, versus NC group; ^#P < 0.05, ^##P < 0.01, versus KLF6-SV1 group; ^&P < 0.05, ^&&P < 0.01, versus siKLF6-SV1 group.