Effect of TLR2 on the proliferation of inflammation-related colorectal cancer and sporadic colorectal cancer

Abstract

Background: Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD) with a poor prognosis because it is often diagnosed in advanced stages with local progression or metastasis. Compared with the more common polyp-induced sporadic colorectal cancer (sCRC), CAC has different molecular mechanisms. Toll-like receptor 2 (TLR2) expression is not limited to cells related to inflammation and immune function. High levels of TLR2 expression in tumor tissues of colorectal cancer (CRC) patients have been reported. This report is to investigate the effects of knockout and knockdown of the TLR2 gene on the proliferation of CAC and sCRC.

Methods: Twelve C57BL/6 J wild-type mice (WT) and 12 TLR2 knockout mice (TLR2-/-) were used to rapidly establish a colitis-associated cancer (CAC) model via the 1,2-dimethylhydrazine-dextran sodium sulfate (DMH-DSS) method and were divided into the normal WT control group (NC), TLR2 knockout control group (KC), normal wild-type tumor modeling group (NT), and TLR2 knockout tumor modeling group (KT), with 6 mice in each group. The general performance of the mice during modeling, the gross changes of the colon and the rectum, and the pathological score of HE staining were used to observe tumor growth. The expression of TLR2 was detected by immunohistochemistry, and tumor proliferation was detected by Ki67 labeling. Lentivirus carrying TLR2-RNAi was used to stably infect colorectal cancer cells (HCT116 and HT29) to knock down TLR2 gene expression. The experimental groups included the uninfected control group, negative control group, and gene knockdown group. After infection, the expression of TLR2 protein was detected by Western blot, and cell proliferation and the cell cycle were detected by the CCK-8 method and fluorescence-activated cell sorting. Western blot was used to detect the expression levels of p- NF-κβ, cyclin D1 and cyclin D3 protein in each group of cells.

Results: TLR2 knockout in the CAC model resulted in greater changes in body weight and more severe diarrhea and colorectal hemorrhage. However, knocking out the TLR2 gene reduced the shortening of colorectal length, the number of tumors, and the total tumor volume and inhibited the growth of CAC. Knocking out the TLR2 gene also reduced the pathological score and tumor severity. TLR2 was localized in the cell membrane of the colorectal epithelium of the NC group and of the colorectal tumors of the NT group and was highly expressed in the NT group, while antigen Ki67 was localized in the nucleus of the colorectal tumor cells of the NT group and the KT group, and its expression was reduced in the KT group. In an in vitro sporadic colorectal cancer cell experiment, TLR2 protein in the TLR2 knockdown group was significantly downregulated, and TLR2 knockdown significantly inhibited the proliferation of HCT116 and HT29 colorectal cancer cells, resulting in G1 phase arrest. The expression levels of p-NF-κβ, cyclin D1 and cyclin D3 proteins in TLR2 gene knockdown group cells were significantly reduced.

Conclusion: Knockout and knockdown of TLR2 can inhibit the proliferation of inflammation-related colorectal cancer and sporadic colorectal cancer.

Keywords: Cell proliferation; Inflammation-related colorectal cancer; Sporadic colorectal cancer; Toll-like receptor 2.

Fig. 1

Protocol for the induction of the animal model. Control: WT and TLR2−/− mice without DMH-DSS; tumor: WT and TLR2−/− mice with DMH-DSS

Fig. 2

General symptoms such as body weight, total days of diarrhea, and total days of bleeding. NC: WT without DMH-DSS; KC: TLR2−/− without DMH-DSS; NT: WT with DMH-DSS; KT: TLR2-/- with DMH-DSS. ***P < 0.001, KT vs NT group (n = 6)

Fig. 3

General pathological changes such as shortening of colorectal length, number of tumors, and total volume of the tumor. NC: WT without DMH-DSS; KC: TLR2-/- without DMH-DSS; NT: WT with DMH-DSS; KT: TLR2−/− with DMH-DSS. *P < 0.05, KT vs NT group (n = 6); ***P < 0.001, KT vs NT group (n = 6)

Fig. 4

Mouse colorectal HE staining (microscope ×100) and tumor pathological score. NC: WT without DMH-DSS; KC: TLR2−/− without DMH-DSS; NT: WT with DMH-DSS; KT: TLR2−/− with DMH-DSS. Blue represents the nucleus and pink represents the cytoplasm. These arrows represent infiltrating leukocytes. ***P < 0.001, KT vs NT group (n = 6); **P < 0.01, KT vs NT group (n = 6)

Fig. 5

Mouse colorectal immunohistochemistry (microscope ×100) and rate of cells with positive expression. NC: WT without DMH-DSS; KC: TLR2−/− without DMH-DSS; NT: WT with DMH-DSS; KT: TLR2−/− with DMH-DSS. Arrows represent positive staining. ^&&&P < 0.001, NT vs NC group (n = 6); ^###P < 0.001, NT vs KC group (n = 6);  ^※※※P < 0.001, NT vs KT group ( n = 6). ***P < 0.001, KT vs NT group (n = 6); ^△△△P < 0.001, KT vs NC group (n = 6); ^aaaP < 0.001, KT vs KC group (n = 6)

Fig. 6

The morphological images of HCT116 and HT29 cells at day 5 after infection with lentivirus under a fluorescence microscope (×40). CON: HCT116 or HT29 cells without lentiviral infection; NC: HCT116 or HT29 cells infected with negative-RNAi lentivirus; KD: HCT116 or HT29 cells infected with TLR2-RNAi lentivirus (RNA interference). Green represents FITC fluorescence

Fig. 7

The expression levels of TLR2 protein in HCT116 and HT29 cells infected with lentivirus were detected by Western blotting. CON: HCT116 or HT29 cells without lentiviral infection; NC:  HCT116 or HT29 cells infected with negative-RNAi lentivirus; KD: HCT116 or HT29 cells infected with TLR2-RNAi lentivirus (RNA interference). GAPDH was used as the internal reference. **P < 0.01 KD vs CON group (n = 3); ^△△P < 0.01 KD vs NC group (n = 3)

Fig. 8

The expression levels of proteins in HCT116 and HT29 cells in various groups detected by Western blotting method. CON: HCT116 or HT29 cells without lentiviral infection; NC: HCT116 or HT29 cells infected with negative-RNAi lentivirus; KD: HCT116 or HT29 cells infected with TLR2-RNAi lentivirus (RNA interference). GAPDH was used as the internal reference. **P < 0.01 KD vs CON group (n = 3); ^△△P < 0.01 KD vs NC group (n = 3)

Fig. 9

The effect of TLR2 gene knockdown on the proliferation of HCT116 and HT29 cells was determined by CCK-8 assay. CON: HCT116 or HT29 cells without lentiviral infection; NC: HCT116 or HT29 cells infected with negative-RNAi lentivirus; KD: HCT116 or HT29 cells infected with TLR2-RNAi lentivirus (RNA interference). *P < 0.05 KD vs CON group (n = 3); ^△P < 0.05 KD vs NC group (n = 3)

Fig. 10

The effect of TLR2 gene knockdown on the cell cycle of HCT116 and HT29 cells was determined by FACS analysis. CON: HCT116 or HT29 cells without lentiviral infection; NC: HCT116 or HT29 cells infected with negative-RNAi lentivirus; KD: HCT116 or HT29 cells infected with TLR2-RNAi lentivirus (RNA interference). *P < 0.05 KD vs CON group (n = 3); ^△P < 0.05 KD vs NC group (n = 3)