Effect of P16INK4A regulating Wnt/β-catenin signaling pathway on biological behaviours of colon adenocarcinoma cells
Objective To observe the changes of P16INK4A expression in colon adenocarcinoma tissues and HT29 cells,and to explore the effects of upregulating P16INK4A expression on the Wnt/β-catenin signaling pathway and the biological behaviours of HT29 cells.Methods The samples of surgically resected colon adenocarcinoma tissues and paracancerous tissues were harvested from 23 colon adenocarcinoma patients in the First Affiliated Hospital of Henan University from January to December 2023.The relative expression of P16INK4A mRNA was detected by real-time fluorescence quantitative PCR.The HT29 cells in logarithmic growth phase were divided into control group(normal culture),pcDNA3.0 group(transfected with pcDNA3.0 plasmid),and pcDNA3.0-P16INK4A group(transfected with pcDNA3.0-P16INK4A plasmid).After 48-h culture,the optical density(OD)value of cell proliferation in three groups was detected by CCK-8 assay,the apoptosis rate was detected by flow cytometry,the migration rate was detected by cell scratch assay,and the number of invasive cells was detected by Trans well chamber assay.The relative expressions of P16INK4A,β-catenin,c-Myc,cyclin D1 and T cell factor-4(TCF-4)mRNAs were detected by real-time fluorescence quantitative PCR,and the relative expressions of P16INK4A,β-catenin,c-Myc,cyclin D1 and TCF-4 proteins were detected by Western blot.Results The relative expression of P16INK4A mRNA was lower in colon adenocarcinoma tissues(0.53±0.08)than that in paracancerous tissues(1.00±0.10)(t=17.604,P＜0.001).After 48-h culture,the OD value,apoptosis rate,migration rate,and the number of invasive cells showed significant differences among three groups(F=39.000,241.449,44.816,24.052;all P values＜0.05),the OD value and migration rate were lower in pcDNA3.0-P16INK4A group[0.47±0.05,(21.38±2.85)％]than those in pcDNA3.0 group[0.85±0.10,(40.79±4.53)％]and control group[0.82±0.09,(41.85±4.97)％](P＜0.05),the apoptosis rate was higher in pcDNA3.0-P16INK4A group[(35.28±3.74)％]than that in pcDNA3.0 group[(10.09±1.13)％]and control group[(9.26±1.02)％](P＜0.05),and the number of invasive cells was less in pcDNA3.0-P16INK4A group(102.33±12.27)than that in pcDNA3.0 group(158.33±16.45)and control group(155.33±17.93)(P＜0.05).The expressions of P16INK4A,β-catenin,c-Myc,cyclin D1,TCF-4 mRNAs and proteins showed significant differences among three groups(F=31.484-83.046,all P values＜0.05).The relative expressions of P16INK4A mRNA and protein were higher in pcDNA3.0-P16INK4A group than those in pcDNA3.0 group and control group(P＜0.05),the relative expressions ofβ-catenin,c-Myc,cyclin D1 and TCF-4 mRNAs and proteins were lower in pcDNA3.0-P16INK4A group than those in pcDNA3.0 group and control group(P＜0.05),and there were no significant differences in the above indexes between pcDNA3.0 group and control group(P＞0.05).Conclusion P16INK4A is lowly expressed in human colon adenocarcinoma tissues,and to upregulate P16INK4A expression can inhibit the cell proliferation,migration and invasion via affecting the activity of Wnt/β-catenin pathway of HT29 cells so as to promote the apoptosis.
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