Identification Of A Novel Inhibitor For Histone Methyltransferase G9a And Its Efficacy On NSCLC Progression

ObjectiveEpigenetic phenotypes caused by chromosomal changes without altering the DNA sequence.The most common epigenetic changes include DNA methylation,histone modifications,and non-coding microRNAs(miRNAs).A number of studies have reported that abnormal changes in epigenetic regulation can lead to a variety of diseases,such as cardiovascular disease,liver disease,etc.,especially cancer.Treating or reversing diseases through regulating epigenetic changes has become a hot topic in research.G9a,a kind of histone methyltransferase encoded by the EHMT2 gene and having a SET domain,is a key epigenetic enzyme.G9a is highly expressed in many cancerous species,such as lung cancer,breast cancer,liver cancer,etc.,and influences tumorigenesis.Thus,G9a inhibitors have obvious therapeutic effects on tumors with high expression of G9a.Unfortunately,Ccurrently developed G9a inhibitors are notboth non-specific and limited to preclinical studies.Therefore,developing G9a-specific inhibitors that can be used in clinical,is of great significance.In addition,lung cancer incidence and mortality rank first in the world cancer statistics.Studies have found that G9a is highly expressed in lung cancer,but its mechanism in regulaiting lung cancer is still unclear.Therefore,the development of G9a-specific small molecule inhibitors is urgently needed.It is of great clinical significance to study the effect of G9a on the functional phenotype of NSCLC cells and the mechanism of action of G9a-specific small molecule inhibitors on NSCLC.Methods1.Evaluation of basal expression of G9a in multiple cancer cells.NSCLC cell lines A549,H1299 and H1975,breast cancer cell lines MDA-MB-231 and MCF-7,ovarian cancer cell lines SK-OV-3 and OVCAR3,colon cancer cell line HT29,liver cancer cell lines Huh7 and HepG2 and Kidney cancer cell line 786-O-R were selected,and Western blot was used to detect the expression of G9a protein in different tumor cells.At the level,immunofluorescence experiments were further used to confirm the basal expression of G9a in tumor cells.2.Effect of G9a on the migration of NSCLC cell line H1299.H1299 were transfected with plasmid for 6-8h,and the expression of G9a in H1299 was knocked down.After 24h of culture,the effect of G9a on the migration ability of H1299 was detected by wound healing.3.Effect of G9a on proliferation in H1299.H1299 were transfected for 6-8 h after plasmid transfection,and the expression of G9a in H1299 cells was knocked down.The cells were cultured for 24 h and inoculated into 6well plates(very low density,200 cells/well).Colony formation assay was performed to observe the effect of G9a on cell viabilities of H1299.4.Effect of G9a on dryness in H1299.H1299 were transfected with plasmid for 6-8 h,knockdown the expression of G9a in H1299 cells,and cultured for 24 h.The expression of CD44+and CD 133+ were detected by flow cytometry to investigate the effect of G9a on the dryness.5.Effect of G9a on autophagy in H1299.H1299 were transfected with plasmid for 6-8 h,and the expression of G9a in H1299 was knocked down.After incubation for 24 h,the cells were incubated with the dye of CytoID kit for half an hour,and the cells were harvested by flow cytometry.6.Efficacy of first-line chemotherapy drugs on H1299 cells that knock down G9a expression.H1299 were transfected and the expression of G9a was knocked down.After 24 hours of culture,the cells were collected and seeded in 96-well plates.After treatment with cisplatin and paclitaxel for 72h,the cell survival was detected by SRB assay.7.To investigate the ability of natural small molecule compounds and structurally modified compounds to bind to G9a by CETSA.H1299 cells were collected,and total protein was extracted by repeated freezing and thawing of liquid nitrogen combined with RIPA cleavage protein.After incubation with total protein and natural small molecule compounds and structurally modified compounds or solvents,the soluble components were extracted at different temperatures and examined by western blot to screen the compounds which bind to G9a.8.To further screen the ability of natural small molecule compounds and structurally modified compounds to specifically bind to G9a by SPR.The solution containing the compound was injected onto the surface of G9a by a microfluidic system by immobilizing the G9a protein on the GLH chip.When the compound binds to the surface of G9a,an increase in the SPR signal(in response units,RU)can be observed,thereby screening for a compound directly binding to G9a,and calculating the binding constant KD of the compound binding to G9a.9.Effect of target compounds on the viability of NSCLC cells by SRB assayNSCLC cell lines H1299 and A549 were treated with target compounds(0-50μM)for 72h.Cell viabilities was detected by SRB assay.Cell viability rate and IC50 were calculated.10.Effects of the compounds LZL072 and LZL082 on the proliferation of NSCLC cells by EdU methodCompounds LZL072 and LZL082 were treated with H1299 and A549 cells for 72h,and diluted with EdU for 2h.Apollo staining was performed.The incorporation rate of EdU was detected by fluorescence microscopy.The effects of compounds LZL072 and LZL082 on the proliferation of NSCLC cells were evaluated.Results1.The highest expression of G9a in NSCLC cell line H1299.According to western blot and immunofluorescence results,the expression of G9a in cancer cells was observed.It was found that H1299 had the highest expression of G9a,and H1299 was selected for subsequent experimental.2.Low expression of G9a significantly inhibited the migration of H1299.The expression level of G9a protein and gene were decreased in H1299 by plasmid transfection.Further wound healing results showed that the migration ability of H1299 with low expression of G9a was significantly decreased compared with wide type H1299(P<0.001).The inhibition rate of migration reached 30%.3.Low expression of G9a significantly inhibited the proliferation of H1299.The expression level of G9a protein and gene were decreased in H1299 by plasmid transfection.The results of colony formation showed that the proliferation of H1299 with low expression of G9a was significantly inhibited compared with wide type H1299(P<0.001).The inhibited rate of proliferation was up to 61%.4.Low expression of G9a has no significant effect on the dryness in H1299.The plasmid transfection was used to reduce the G9a protein and gene levels in H1299,and the ratio of CD133+and CD44+was detected by flow cytometry.The results showed that the ratio of CD133+and CD44+in H1299 with low expression of G9a was higher than that of wide type H1 299.The impact is not obvious.5.Low expression of G9a promoted autophagy in H1299.The plasmid transfection was used to reduce the G9a protein and gene levels in H1299.The results of CYTO-ID kit combined with flow cytometry showed that H1299 with low expression of G9a significantly increased autophagy(P<0.001),and the incidence of autophagy was increased by 115%compared with wide type H1299.6.Low expression of G9a enhanced the efficacy of first-line chemotherapy drugs in vitro.The 72h-IC50 values of cisplatin and paclitaxel in normal H1299 were 7.29±0.79 μM and 11.66 ± 2.8 nM,respectively,while in H1299 with low expression of G9a were 2.64 ±0.44 μM and 8.97± 3.9 nM,respectively.Cisplatin and paclitaxel had enhanced efficacy against H1299 cells with low expression of G9a.7.The CETSA screened 5 natural small molecule compounds and 6 structurally modified compounds to bind to G9a.CETSA results showed that the expression of G9a in H1299 cell lysate after incubation with five natural small molecule compounds(35558、28990、20710、80814)and seven structurally modified compounds(LZL087、LZL030、LZL103、LZL026、LZL082 and LZL025)was higher than control group,indicating that these compounds may have a binding effect with G9a.8.SPR assay screened structurally modified compounds LZL033,LZL072,LZL082 combined with G9aFirstly,10 compounds with the highest response value were screened by l0μM concentration,including LZL007>LZL007-2>LZL082>L2L033>LZL079>LZL015>LZL072>LZL024>LZL078>LZL025.Then the concentrations of the compounds were further investigate the binding constant of the compound to G9a.The results showed that 3 structurally modified compounds with medium strength binding to G9a were obtained and the binding constant KD was calculated,which were LZL033(KD=15.7 μM),LZL072(KD=6.79 μM),and LZL(KD=22.4 μM),respectively.9.Structurally modified compounds LZL033,LZL072,LZL072 significantly inhibited the viability in A549 and H1299The 72h-IC50 values of structurally modified compounds LZL033,LZL072 and LZL082 in H1299 were 5.75 μM,3.54 μM,11.09 μM,respectively,while in A549 were 6.31μM,7.92 μM,6.90 pM.10.Compounds LZL072 and LZL082 significantly inhibited the proliferation of H1299 and A549 cells.As the concentration of the drug is increased,the number of fluorescent particles is gradually decreased,and the doping ratio of EdU is lowered.These results indicated that the compounds LZL072 and LZL082 significantly inhibited the DNA synthesis of H1299 and A549 cells,thereby inhibiting cell proliferation(P<0.001).ConclusionsIn this study,the effect of G9a on the function of H1299 cells in non-small cell lung cancer was studied.It was found that the low expression of G9a significantly inhibited the migration,cell proliferation of H1299,promoted autophagy,and enhanced the effect of firstline chemotherapeutic drugs on the viability,confirming G9a played an important role on the occurrence and development of lung cancer.The compounds LZL033,LZL072 and LZL082 screened by CETSA and SPR experiments can specifically bind to G9a while having antitumor activity.In summary,LZL033,LZL072,and LZL082 can be used as G9a epigenetic modifiers to bind to G9a and inhibit its expression,thereby regulating NSCLC.The study provided experimental evidence for the development of G9a inhibitors and new insights for epigenetic to prevent and treat cancer.