Establishment And Application Of A Screening Model For Anti-tumor Compounds Targeting NR1H3

Background:Cancer is the leading killer of human health,so it is urgent to develop drugs based on the key targets of tumor occurrence and development.It has been reported that nuclear receptor subfamily 1 group H member 3(NR1H3)activated by ligands can inhibit tumorigenesis and promote apoptosis of tumor cells,which makes NR1H3 as a potential target for cancer treatment.At the same time,the development of NR1H3 active ligands has also been a research hotspot in recent years.Coptidis Rhizoma,as a traditional Chinese medicine,has a long history of medication.It not only has antibacterial,antiviral,hypoglycemic,lipid-lowering and other pharmacological effects,but also has a wide range of anti-tumor effects.However,there are few studies on drug screening based on key targets.Our previous study found that NR1H3 has a potential transcriptional regulation effect on the solute carrier family 16 member 3(SLC16A3),and SLC16A3,a member of the monocarbamate transporter family,plays an important role in maintaining the stability of tumor homeostasis.Therefore,a screening model was established based on the transcriptional activity of NR1H3,and it was expected that NR1H3 ligand compounds with antitumor activity would be screened from Coptidis Rhizoma.Purpose:This study aimed to establish a dual-luciferase screening model targeting NR1H3based on its transcriptional regulatory activity.By using this model to screen the active ingredients in Coptidis Rhizoma,it is expected to obtain the ligand compounds targeting NR1H3 with anti-tumor activity.Method:1.Establishment of a dual-luciferase screening modelTotal RNA was extracted from colon cancer cell HCT116,and then reverse-transcribed into c DNA,which was used as a template to further amplify the coding region of the NR1H3 gene and construct a recombinant pc DNA3.1(-)-NR1H3 overexpression plasmid.The genome of colon cancer cell HCT116 was extracted,the promoter region of human SLC16A3 gene was amplified,and a recombinant p GL3 basic-SLC16A3 luciferase reporter plasmid was constructed.The overexpression plasmid,reporter gene plasmid and internal reference plasmid were transfected into human renal epithelial cells 293T,and the model construction was successful according to the luciferase activity.2.Screening model condition optimizationThe number of cells,transfection time,ratio of overexpressed plasmid to reporter plasmid and other conditions were optimized,and the sensitivity and stability of the screening model were investigated by the agonist T0901317 of NR1H3,and the statistical parameter Z’factor was used as the evaluation index,and Z’>0.5,and the model could be used for subsequent compound screening.3.Screening of candidate ligand compoundsFirstly,the software Discovery Studio was used to connect NR1H3 protein with the active ingredients of Coptidis Rhizoma in the pharmacological database analysis platform of the Chinese medicine system,and the active ingredients in Coptidis Rhizoma were preliminarily screened.According to the computer virtual screening results,the diluciferase screening model was used to rescreen the initial screening results.By comparing luciferase signal intensities,potential NR1H3 ligand compounds were screened.4.Validation of antitumor activity of candidate ligand compoundsThe effect of candidate compounds on tumor cell proliferation was explored by MTT method,the effect of candidate compounds on SLC16A3 gene expression by q RT-PCR method,and the effect of candidate compounds on SLC16A3 protein expression by Western Blot method.Result:1.Establishment of NR1H3 targeted dual luciferase screening modelAfter successfully constructing human pc DNA3.1(-)-NR1H3 overexpression plasmid and p GL3 basic-SLC16A3 reporter plasmid,the constructed overexpression plasmid,reporter plasmid and internal reference plasmid were co-transfected into human renal epithelial cells 293T,and the co-transfection of pc DNA3.1(-)-NR1H3overexpression plasmid and p GL3 basic-SLC16A3 reporter plasmid group was significantly increased(P<0.01)compared with the negative control group,and the model was successfully established.2.Optimization of dual-luciferase screening model conditionsThe number of cells of 6×10~3cells/well,the transfection time of 24 h,the ratio of overexpressed plasmid to reporter plasmid of 2.5:1,and the ratio of reporter plasmid to internal control plasmid of 100:1 were finally determined by double luciferase reporter gene experiment.NR1H3 agonist T0901317 was used to investigate the stability and sensitivity of the model,and the statistical parameter Z’factor was used as the evaluation criterion(Z’=0.58),which was greater than 0.5,indicating that the sensitivity and stability of the screening system were ideal,and it could be used for the screening of subsequent compounds.3.Screening of candidate ligand compoundsThrough the Discovery Studio software docking calculation,the 34 active ingredients of Rhizoma coptidis in the TCMSP database can be combined with NR1H3,among which the binding scores of Coptisine,Epi Berberine,Palmatine,Berberine and Columbamine had higher binding scores.Combined with the results of the previous computer virtual screening and the existing resources of the laboratory,the secondary screening of Coptisine,Epiberberine,Palmatine,Berberine and Columbamine were carried out based on the luciferase activity.screening model constructed.The results showed that Coptisine(P<0.01),Berberine(P<0.01),Palmatine(P<0.01),and Columbamine(P<0.01)significantly increased luciferase activity,and there was no significant change in luciferase activity after Epiberberine.4.Verification of antitumor activity of candidate compoundsWhen exploring the effect of candidate ligand compound concentrations on tumor proliferation,MTT results showed that compared with the control group,Coptisine,Berberine,and Palmatine could inhibit the proliferation of colon cancer HCT116,SW480and HT29 cells,and the greater the compound concentration,the more obvious the inhibitory effect.When exploring the relationship between tumor cell proliferation and compound action time,MTT results showed that compared with the control group,the proliferation ability of colon cancer HCT116,SW480 and HT29 cells was significantly inhibited after 24 h of treatment with Coptisine,Berberine,and Palmatine,and with the extension of the compound action time,the proliferation inhibitory effect of tumor cells also increased.q RT-PCR results showed that Berberine(P<0.05)and Palmatine(P<0.05)inhibited SLC16A3 gene expression levels after acting on tumor cells,and SLC16A3gene expression levels decreased with the increase of Berberine and Palmatine concentrations.According to the results of Western Blot experiments,Berberine and Palmatine inhibited the expression level of SLC16A3 protein.Conclusion:In this study,the dual luciferase screening model targeting NR1H3 was successfully constructed,which can be used to screen ligand compounds targeting NR1H3transcriptional activity.Through this model,Palmatine and Berberine were screened from the active components of Coptidis Rhizoma as ligands targeting NR1H3.Therefore,this model can be used as an efficient screening model for NR1H3 ligand compounds,and also provide a basis for further development of anti-tumor active drugs.