JIB-04 Inhibits The Proliferation Of Colon Cancer Cells By Regulating Cell Cycle Proteins

Objective Colon cancer is a common malignant digestive system tumor with high morbidity and mortality.At present,great progress has been made in the treatment of colon cancer,but its prognosis is still not optimistic.It has been reported that Histone demethylase inhibitor(JIB-04)inhibits the occurrence and development of tumors,but the specific mechanism is still unclear.In this paper,the effect of histone demethylase inhibitor JIB-04 on the proliferation of colon cancer cells was explored and its mechanism was explained.Methods In this study,colon cancer cells HCT116 and HT29 were used as main targets.Human colon cancer cells HCT116,HT29 and human normal colon mucosal cells FHC were routinely cultured in vitro.Different concen-trations of JIB-04 were selected to treat intestinal cancer cells HCT116,HT29 and human normal colon mucosal cells FHC.MTT assay and plate cloning experiment were used to observe and evaluate the evaluate the growth and proliferation of colon cancer cells and human normal colon mucosal cells.qRT-PCR and Western blotting were used to detect the effects of JIB-04 on colon cancer cell cycle progression and apoptosis,and its effect on histone demethylase.Results MTT assay and plate cloning formation experiments showed that JIB-04 reduced the proliferation ability of HCT116 and HT29 cells in a concentration-and time-dependent manner,with the half-inhibitory concen-trations of 661.7 nmol/L and 226.1 nmol/L,respectively.And the number of cell clones also decreased significantly.qRT-PCR and Western blot results showed that compared with HCT116 and HT29 cells not treated with JIB-04,the mRNA levels of the cyclinDl,cyclinE1,and CDK4,CDK6,and cyclinDl proteins levels were reduced to different degrees.At the same time,CDK inhibitor p21 protein expression increased by about 1.99-fold and 2.37-fold,respectively(P<0.001).Detection of apoptosis-related factors revealed that the mRNA and protein levels of p53,caspase-3,caspase-9,Bax,JNK and PUMA were reduced in HCT116 and HT29 cells.Mechanism analysis found that JIB-04 reduced histone demethylase LSD1 mRNA levels by about 56.8%and 75.5%(P<0.01,P<0.001),and its protein levels was reduced by about 27.12%and 15.32%(P<0.05).48h after transfection of siRNA-LSD1 into HCT116 and HT29 cells,qRT-PCR showed that compared with the control siRNA-NC group,the siRNA-LSD1-3 group was the most significant in HCT116 cells,with a decrease of approximately 78.41%(P<0.01).In HT29 cells,siRNA-LSD1 did not significantly inhibit the expression of LSD1 gene.Detection of the cyclin mRNA level of colon cancer cell HCT116 showed that cyclinDl and cyclinE1 mRNA levels were reduced by approximately 51.38%and 45.61%,respectively(P<0.01).The mRNA level of cyclin D3 was in-creased(P<0.01),while the mRNA levels of cyclinA1 and cyclinB1 had no significant effect.Conclusion JIB-04 can change the activity of histone demethylase LSD1 in colon cancer cells,regulate the cyclin and related factor mRNA and protein expression levels,induce changes in its cycle arrest and apoptotic proteins,therefore,it can inhibit the proliferation and apoptosis of colon cancer cells,and can be used as a potential drug candidate for treating colon cancer.