MyD88 mediates colorectal cancer cell proliferation, migration and invasion via NF‑κB/AP‑1 signaling pathway

Abstract

The role of myeloid differentiation factor 88 (MyD88) in malignant tumors is largely unknown. Therefore, in this study, we aimed to examine the function and underlying mechanism of MyD88 in colorectal carcinoma in vitro using SW480 and HCT116 cell lines and in vivo using a nude mouse model. SW480 and HCT116 cells were infected with a lentiviral‑based effective MyD88 siRNA virus. CCK‑8 and colony formation assay were used to assess cell proliferation. Transwell and scratch assays were used to test the migration of colorectal cancer cells, and the Transwell assay was further used to analyze the invasiveness of colorectal cancer cells. Western blotting was performed to analyze the underlying mechanism of MyD88 regulation. In vitro experiments demonstrated that silencing MyD88 in SW480 and HCT116 cells markedly suppressed growth and invasion. Furthermore, MyD88 knockdown affected the MyD88‑NF‑κB/AP‑1 signaling pathways in SW480 and HCT116 cells. In vivo, MyD88 knockdown inhibited tumor growth in a HCT116 cell subcutaneous nude model. We found that knockdown of the MyD88 gene can affect proliferation, invasion, and migration of colorectal cancer cells. We further verified that MyD88 knockdown can reduce the activity of NF‑κB and AP‑1 pathways. These results show that MyD88 gene plays an important role in promoting colorectal cancer, and thus can be exploited as a potential diagnostic and prognostic biomarker for colorectal cancer.

Keywords: myeloid differentiation factor 88; colorectal cancer; proliferation; migration; invasion.

Figure 1

Knockdown of MyD88 in the SW480 and HCT116 cells. (A) Total RNA from SW480 and HCT116 cells after transfection using NC, siRNA-1, siRNA-2 and siRNA-3 sequences were assessed by RT-qPCR using MyD88 primers. GAPDH was amplified for an internal control. The siRNA-1 and siRNA-3 sequences resulted in higher suppression of the levels of MyD88 mRNA. (B) MyD88 protein in the SW480 and HCT116 cells after transfection using NC, siRNA-1, siRNA-2 and siRNA-3 sequences were detected by western blotting. GAPDH protein was used an internal control. (C) Semi-quantitative analysis showed that MyD88 protein levels in siRNA-1 and siRNA-3 cells were markedly lower compared with NC and siRNA-2 cells. The mRNA expression of MyD88 in SW480 NC group and HCT116 NC group cells were taken as 1. Error bars represent mean ± SEM, representative of three experiments, ^*P<0.05, ^**P<0.01.

Figure 2

Downregulation of MyD88 mRNA and protein expression by lentivirus-mediated MyD88 siRNA in SW480 and HCT116 cells. (A) pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 lentivirus infected the SW480 and HCT116 cells. After 72 h infection, the green fluorescent protein (GFP) as demonstrated by fluorescence microscopy. (B) The mRNA and (C) protein expression was analyzed using RT-qPCR and western blot analysis after infection with lentivirus in the SW480 and HCT116 cells. GAPDH was used as an internal control. (D) Semi-quantitative analysis showed that the level of MyD88 protein were signifi-cantly inhibited by pLKO.1-sh1 and pLKO.1-sh3 in the SW480 and HCT116 cells. The mRNA expression of MyD88 in SW480 pLKO.1 group and HCT116 pLKO.1 group cells were taken as 1. Error bars represent mean ± SEM, representative of three experiments. ^*P<0.05, ^**P<0.01.

Figure 3

Effects of siMyD88 on the viability of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 in the SW480 and HCT116 cells. (A and B) Downregulation of MyD88 reduced the colony number in colony formation assays in the SW480 and HCT116 cells. (C) CCK-8 assays revealing that MyD88 knock-down inhibits the proliferation of SW480 and HCT116 cells. Error bars represent mean ± SEM, representative of three experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 4

Effects of siMyD88 on the migration of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 in the SW480 and HCT116 cells. (A) In the SW480 cells, representative images of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 cell wound healing and microscopic observations were photographed 0 and 48 h after scratching the cell surface. (B) The width of the scratch was analyzed using a histogram in the SW480 cells. (C) In the HCT116 cells, representative images of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 cells wound healing and microscopic observations were photographed 0 and 48 h after scratching the cell surface. (D) The width of the scratch was analyzed using a histogram in the HCT116 cells. Error bars represent mean ± SEM, representative of three experiments. ^*P<0.05, ^***P<0.001.

Figure 5

Effects of siMyD88 on SW480 and HCT116 cells migration and invasion. (A) Migration ability of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 in the SW480 and HCT116 cells. (B) Number of migrated cells in the pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups in the SW480 and HCT116 cells. (C) Invasion ability of pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 in the SW480 and HCT116 cells. (D) Number of invaded cells in the pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups in the SW480 and HCT116 cells. Error bars represent mean ± SEM, representative of three experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 6

Western blot analysis revealed that the silencing of MyD88 markedly inhibited the expression NF-κB (p65), p-NF-κB (p-p65), AP-1 (c-jun) and p-AP-1 (p-c-jun) protein in the SW480 and HCT116 cells. (A) The protein electropherogram showed the protein expression situation of NF-κB (p65), p-NF-κB (p-p65), AP-1 (c-jun), p-AP-1 (p-c-jun) and MyD88 in pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups in the SW480 cells. (B) The protein expression of the pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups was analyzed using a histogram in the SW480 cells. (C) An electropherogram was used to show the protein expression of NF-κB (p65), p-NF-κB (p-p65), AP-1 (c-jun), p-AP-1 (p-c-jun) and MyD88 in pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups in the HCT116 cells. (D) The protein expression of the pLKO.1, pLKO.1-sh1 and pLKO.1-sh3 groups was analyzed using a histogram in the HCT116 cells. Error bars represent mean ± SEM, representative of three experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 7

Suppression of tumor growth by silencing the MyD88 gene in the HCT116 cells subcutaneous xenografts. Three groups (pLKO.1 and pLKO.1-sh3 groups) were subcutaneous xenografted onto nude mice. (A) The tumor growth of pLKO.1-sh3 group was significantly inhibited compared with pLKO.1 group. (B) The size of the primary tumors was measured every 4 days. Mice were sacrificed after 5 weeks. (C) The weights of tumors were weighed. Significant differences were identified between the pLKO.1 and pLKO.1-sh3 groups. (D) Immunohistochemical analysis of the expression of MyD88 in HCT116 cell subcutaneous xenograft tumors. Staining showed expression of MyD88 (×100 and ×200) in pLKO.1 and pLKO.1-sh3 groups. (E) Quantitative evaluation of the expression of MyD88. Bars show the MyD88 staining area ration/per field. Marked differences between the pLKO.1 and pLKO.1-sh3 groups were identified. ^*P<0.05, ^**P<0.01.