Effect of insulin-like growth factor-2 mRNA-binding protein 3 on proliferation,migration of diffuse gastric cancer cells,and M2 macrophage polarization
Objective To investigate the impact of insulin-like growth factor-2 mRNA(mRNA)-binding protein 3(IGF2BP3)on the proliferation,migration of diffuse gastric cancer cells,and the polari-zation of alternatively activated macrophages(M2).Methods The cells utilized in this study were sourced from the Zhejiang Meisen Cell Repository and the Shanghai Cell Bank of the Chinese Academy of Sciences.We employed short hairpin RNA lentivirus and lentiviral packaging plasmids to infect the human diffuse gastric cancer cell lines NUGC-4 and SNU-668.This approach was used to establish cell lines with knock-down and overexpression of IGF2BP3,along with their respective control cell lines.The expression levels of IGF2BP3 and the transfection efficiency in each diffuse gastric cancer cell line were analyzed using re-verse transcription quantitative polymerase chain reaction(RT-qPCR)and Western blotting.To investigate the effects of IGF2BP3 on the proliferative and migratory capabilities of diffuse gastric cancer cells,we con-ducted assays including the Cell Counting Kit-8(CCK-8),colony formation,and scratch wound healing tests.To induce macrophage differentiation from human monocytes THP-1,phorbol myristate acetate was used.Conditioned media(CM)from control and experimental gastric cancer cells were collected and used to treat macrophages.The expression of M2 macrophage markers,including cluster of differentiation 163(CD163),CD206,transforming growth factor beta 1(TGF-β1),interleukin 10(IL-10),and peroxisome proliferator activated receptor gamma(PPARG),was assessed using RT-qPCR.Student's t-test was em-ployed for the comparison of mean values between the two groups.Results The CCK-8 assay revealed that the absorbance at 450 nm(A450)of the control group was significantly higher than that of the IGF2BP3 knockdown group at various time points(24 h:0.22±0.10 vs.0.10±0.09,t=6.806,P＜0.05;48 h:0.46±0.21 vs.0.30±0.10,t=8.372,P＜0.05;72 h:0.71±0.38 vs.0.51±0.11,t=7.827,P＜0.05;96 h:1.01±0.44 vs.0.79±0.09,t=4.539,P＜0.05).Conversely,the A450 of the control group was lower than that of the IGF2BP3 overexpression group(24 h:0.25±0.02 vs.0.38±0.01,t=12.074,P＜0.05;48 h:0.60±0.02 vs.0.87±0.04,t=9.273,P＜0.05;72 h:0.93±0.02 vs.1.36±0.02,t=27.955,P＜0.05;96 h:1.40±0.05vs.1.88±0.08,t=9.057,P＜0.05).Colony formation assay results indicated that the rate of colony formation in the control group was higher than that in the IGF2BP3 knockdown group[(74.73±7.92)％vs.(52.90±6.25)％,t=3.747,P＜0.05].In contrast,the rate of colony formation in the control group was lower than that in the IGF2BP3 overexpres-sion group[(23.57±3.96)％vs.(41.47±3.26)％,t=6.040,P＜0.05].The scratch assay showed no statistically significant difference in the rate of wound healing between the control group and the IGF2BP3 knockdown group[(3.42±1.68)％vs.(2.33±1.66)％,t=0.802,P＞0.05].After treat-ment with CM in the control and experimental groups,the relative mRNA expression levels of M2 macro-phage markers were higher in the control CM-treated group than the IGF2BP3 knockdown CM-treated group(CD163:1.00±0.02 vs.0.18±0.01,t=86.622,P＜0.05;CD206:1.00±0.07 vs.0.60±0.08,t=6.303,P＜0.05;TGF-β1:1.00±0.01 vs.0.81±0.05,t=7.007,P＜0.05;IL-10:1.00±0.01 vs.0.83±0.03,t=9.798,P＜0.05;PPARG:1.00±0.02 vs.0.39±0.01,t=41.025,P＜0.05).Furthermore,the relative mRNA expression levels of M2 macrophage markers were lower in the control CM-treated group than the IGF2BP3 overexpression CM-treated group(CD163:1.00±0.05 vs.2.31± 0.09,t=22.020,P＜0.05;CD206:1.00±0.01 vs.2.07±0.12,t=15.553,P＜0.05;TGF-β1:1.00±0.02vs.1.25±0.03,t=11.128,P＜0.05;IL-10:1.00±0.05vs.1.30±0.13,t=3.623,P＜0.05;PPARG:1.00±0.01 vs.1.50±0.02,t=30.753,P＜0.05).Conclusion IGF2BP3 pro-motes the proliferation of diffuse gastric cancer cells and stimulates the polarization of macrophages towards the M2 phenotype,but it has no effect on the migratory capacity of diffuse gastric cancer cells.
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