The Study On The Mechanism Of Berberine Targeting IGF2BP3 Against Colorectal Cancer

Colorectal cancer(CRC)is one of the major public health burdens in the world due to its high incidence and mortality,so the prevention and treatment of CRC is urgent.Berberine(BBR)has anti-inflammatory,antibacterial,antitumor and other pharmacological activities,especially in anti-CRC.However,the mechanism of action of BBR needs to be further clarified.Insulin-like growth factor 2 RNA binding protein3(IGF2BP3)is highly expressed during the development of a variety of cancers.In CRC cells,multiple oncogenic transcripts have been identified as downstream targets of IGF2BP3 and play vital roles in the proliferation and migration of cancer cells.Our previous work suggested that IGF2BP3 might be a key regulatory gene for BBR.The natural product BBR has great potential in the prevention of CRC,but the target of BBR is still difficult to determine.We have recently reported that IGF2BP3 might be a target of BBR.The purpose of this study is to explore the mechanism by which BBR targeting IGF2BP3 improves CRC,and to provide new strategies for the clinical application of drugs and the design of anticancer drugs based on IGF2BP3.The specific study results are presented as follows:1.Based on CPTAC database and human tissue IHC microarray,it has been demonstrated that IGF2BP3 was highly expressed in CRC tissues,and the high expression of IGF2BP3 may be correlated with poor survival prognosis.By establishing a mouse model of CRC induced by Azomethane/Dextran sodium sulfate(AOM/DSS),we evaluated the regulation of IGF2BP3 by BBR and its therapeutic effect on CRC in mice.The DAI score indicated that BBR could improve the intestinal system in a dose-dependent manner.The results of pathological sections showed that BBR inhibited the development of CRC in vivo in a dosage-dependent manner.After determining the therapeutic effect of BBR,we returned to IGF2BP3 as a possible target.The results of Immunohistochemical analysis and Western blot showed that the expression of IGF2BP3 increased in mouse CRC tissues,and BBR down-regulated the expression of IGF2BP3 in a dose-dependent manner.The down-regulation of IGF2BP3 by BBR echoes the inhibition of CRC by BBR.2.The effect of BBR on the stability of IGF2BP3 m RNA and protein in CRC cells HCT116 and SW620 was examined by incubating actinomycin D and the protein synthesis inhibitor CHX.The results of q PCR and Western blot showed that BBR mainly regulated IGF2BP3 at the protein level and reduced the protein stability,but had no significant effect on the m RNA stability.We also extracted nuclear and cytoplasmic proteins from CRC cells,and found that IGF2BP3 was expressed in both cytoplasm and nucleus,and BBR mainly down-regulated the expression of IGF2BP3 in cytoplasm.To determine whether BBR can directly target IGF2BP3,we first simulated the binding mode of BBR and IGF2BP3 using molecular docking.The results of molecular docking showed that BBR could bind to IGF2BP3 protein and interact with amino acid residues in it.The results of cell thermodynamic stability analysis(CETSA)and drug affinity responsive target stability(DARTS)demonstrated that BBR may bind to IGF2BP3 protein in a dosage-dependent manner to improve the thermal stability and hydrolase resistance of extracellular IGF2BP3.3.Based on StarBase,TCGA,and GSE databases,221 highly expressed transcripts that bind to IGF2BP3 and are associated with CRC were screened,and the functional and pathway enrichment of these transcripts was performed using Meto Scape.We found that these genes are highly correlated with the cell cycle.The effect of BBR on IGF2BP3 function was verified by RNA binding protein immunoprecipitation(R.I.P)assay.The results of R.I.P showed that after BBR treatment,the CDK4/CCND1 m RNA in IGF2BP3 pull-down decreased significantly,indicating that BBR targeting IGF2BP3 may interfere with its stability on downstream m RNA.Western blot and cell cycle assay results showed that BBR inhibited the expression of Cyclin-dependent kinase 4(CDK4)/Cyclin D1(CCND1),resulting in G1→S phase transition arrest of CRC cells.However,overexpression of IGF2BP3 alleviates this inhibition.4.The effect of BBR on the expression of IGF2BP3 in HCT116 and SW 620 cells was tested by incubating the proteasome inhibitors(MG132)and chloroquine(CQ).The results showed that MG132 incubation could save the degradation of IGF2BP3,and BBR degraded IGF2BP3 through ubiquitin-proteasome pathway.In order to identify the target for IGF2BP3 ubiquitination,an E3 ubiquitin ligase called Tripartite motif-containing protein 21(TRIM21)was selected based on Co-IP and liquid chromatography-mass spectrometry(LC-MS/MS).The results of Co-IP showed that BBR promoted IGF2BP3 binding to TRIM21,and TRIM21 induced ubiquitination degradation of IGF2BP3 by K-48 linkage.The expression of TRIM21 was knocked down in HCT116 cells,and the cells were treated with BBR.However,both TRIM21 and ubiquitin pulled down by IGF2BP3 were reduced,and the degradation of IGF2BP3 was also inhibited.The results of Immunofluorescence showed that BBR promoted IGF2BP3 binding to TRIM21 in a dosage-dependent manner in AOM/DSS-inducing CRC tissues.We verified the role of TRIM21 in CRC cells based on RNA interference(RNAi).Clonal formation and Ed U results showed that the knockdown of TRIM21 mitigated the inhibition of BBR on CRC cells proliferation.5.By overexpressing IGF2BP3 in CRC cells,it was verified that BBR inhibited the proliferation of CRC cells by inhibiting the expression of IGF2BP3.The results of colony formation and Ed U showed that the overexpression of IGF2BP3 could promote the proliferation of CRC cells and alleviate the inhibition of BBR on the proliferation of CRC cells.The stable expression of IGF2BP3 cell line HCT116-IGF2BP3 was constructed for subcutaneous tumor grafting and then drug therapy.The results of animal experiments showed that although BBR inhibited tumor growth to a certain extent,the high expression of IGF2BP3 could alleviate this inhibitory effect.Immunohistochemical analysis of these tumor tissues showed that BBR inhibited the expression of IGF2BP3,but overexpression of IGF2BP3 could reduce the inhibition of BBR on the protein expression of IGF2BP3.The results of immunofluorescence showed that overexpression of IGF2BP3 resulted in increased targeting of TRIM21,and BBR promoted IGF2BP3 binding to TRIM21 in a dose-dependent manner.This study shows that BBR could target to IGF2BP3 and promote its degradation by E3 ligase TRIM21 through the ubiquitin-proteasome pathway,which interferes with IGF2BP3’s stabilization of CDK4/CCND1 m RNA and leads to CRC cell cycle arrest,thereby inhibiting the development of CRC.Our work demonstrates the potential of BBR targeting IGF2BP3 to improve CRC and provides new strategies for clinical therapy and anticancer drug design based on IGF2BP3 and TRIM21.