Figure 1.
Relationship between the expression of Bmi1 and levels of TAMs.
(A) Immunohistochemistry of Bmi1, CD68, and CD163 expression in 83 gastric cancer tissues. Scale bars, 100um. (B) The percentage of CD68/163 positive specimens in high Bmi1 expressing gastric cancer. There was a significant correlation between Bmi1 expression and CD68/163 expression (*P < 0.05, ***P < 0.001, respectively). (C) Immunohistochemistry of Bmi1, CD68, and CD163 expression in 49 colon cancer tissues. Scale bars, 100um. (D) The percentage of CD68/163 positive specimens in high Bmi1 expressing colon cancer. There was a significant correlation between these two groups (**P < 0.01, **P < 0.01, respectively).
Figure 2.
Bmi1 expression and sphere assay in gastrointestinal cancer cell lines co-cultured with M1- or M2-polarized THP-1 macrophages.
(A) Cytokine production profile of M1- and-M2 polarized THP-1 macrophages. (B) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in AGS cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (C) qRT-PCR analysis of Bmi1 expression in HCT116 cells co-cultured with M1- and M2-polarized THP-1 macrophages, compared with Bmi1 expression in HCT116 cells only as a control group. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, ***P < 0.001, respectively). (D) Microscopic images of 3D sphere cultured AGS cells co-cultured with macrophages, compared with 3D sphere cultured AGS cells only as a control group. Scale bars, 100um. (E) Microscopic images of 3D sphere cultured HCT116 cells co-cultured with macrophages, compared with 3D sphere cultured HCT116 cells only as a control group. Scale bars, 100um. (F) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in AGS cells (*P < 0.05, *P < 0.05, respectively). (G) Significantly higher sphere numbers were detected in co-cultured groups compared with the control group in HCT116 cells (*P < 0.05, **P < 0.01, respectively).
Figure 3.
miR-30e* suppresses Bmi1 expression in gastrointestinal cells.
(A) Western blot analysis of Bmi1 expression in 6 gastrointestinal cancer cell lines. (B) Western blot analysis of Bmi1 expression in high Bmi1-expressing AGS cell lines transfected with negative control (NC) and miR-30e* mimics. (C) Western blot analysis of Bmi1 expression in high Bmi1-expressing HCT116 cell lines transfected with NC and miR-30e* mimics. (D) Western blot analysis of Bmi1 expression in low Bmi1-expressing NUGC4 cell lines transfected with NC and miR-30e* inhibitors. (E) Western blot analysis of Bmi1 expression in low Bmi1-expressing COLO201 cell lines transfected with NC and miR-30e* inhibitors.
Figure 4.
miR-30e* downregulates Bmi1 expression by directly targeting its 3′ UTR.
(A) 3D sphere culture grown in serum-free non-adherent culture with AGS cells co-cultured with macrophages and transfected with mimic miR-30e*, compared with 3D sphere culture with AGS cells co-cultured with macrophages and transfected with mimic NC as a control group. Scale bars, 100um. (B) Significantly low sphere numbers were detected in mimic miR-30e* transfected groups compared with the control group (*P < 0.05, *P < 0.05, respectively). (C)The putative miR-30e* target site or a mutated sequence of the 3′ UTR of Bmi1 was cloned immediately downstream of the luciferase gene. (D) Luciferase activity of AGS cells co-transfected with plasmids containing the wild-type miR-30e* target sequence in the 3′ UTR of Bmi1 or control plasmids along with the mRNA mimic NC and mimic miR-30e*. (E) Luciferase activity of AGS cells co-transfected with plasmids containing the wild-type or mutant miR-30e* target sequence in the 3′ UTR of Bmi1 along with the mRNA mimic NC and mimic miR-30e*.
Figure 5.
miR-30e* expression in human gastrointestinal cancer tissues.
(A) The levels of miR-30e* expression in 16 gastric cancer tissues and their matched adjacent normal gastric epithelia as assessed by qRT-PCR. (B) The levels of miR-30e* expression in 29 of high and 24 of low Bmi1-expressing gastric cancer tissues as assessed by qRT-PCR. (C) The levels of miR-30e* expression in 37 colon cancer tissues and their matched adjacent normal colon epithelia as assessed by qRT-PCR. (D) The levels of miR-30e* expression in 20 of high and 17 of low Bmi1-expressing colon cancer tissues as assessed by qRT-PCR.
Figure 6.
Expression of miR-30e* and Bmi1 co-cultured with M1- and M2-polarized macrophages purified from human monocytes.
(A) qRT-PCR analysis of miR-30e* expression in AGS cells co-cultured with M1- and M2-polarized macrophages. Significantly lower miR-30e* expression was detected in co-cultured groups compared with the control group (***P < 0.001, *P < 0.05, respectively). (B) qRT-PCR analysis of miR-30e* expression in HCT116 cells co-cultured with M1- and M2-polarized macrophages. Significantly lower miR-30e* expression was detected in co-cultured groups compared with the control group (***P < 0.001, **P < 0.01, respectively). (C) qRT-PCR analysis of Bmi1 expression in AGS cells co-cultured with M1- and M2-polarized macrophages. Significantly higher Bmi1 expression was detected in co-cultured groups compared with the control group (***P < 0.001, **P < 0.01, respectively). (D) qRT-PCR analysis of miR-30e* expression in HCT116 cells co-cultured with M1- and M2-polarized macrophages. Significantly higher Bmi1 expression was detected in M1 macrophage co-cultured groups compared with the control group (**P < 0.01). (E) Schematic representation of miR-30e*-Bmi1 signaling mediated by TAMs.