Screening Of The HOTAIR-EZH2 Inhibitor And Study Of Tumor Targeted Therapy

Increasing studies report that long noncoding RNA(lnc RNA),which is dysregulated in various cancers,plays a critical role in chromatin dynamics and gene regulation by interacting with key epigenetic regulators.To date,HOTAIR has been the most in-depth study as the "star" in lnc RNA.Its 5 ’end domain can bind to the EZH2 protein and recruit PRC2 complex;the 3’ end domain can recruit LSD1 complex.By recruiting the inhibitory protein complex to the target gene site,the level of H3K27me3 at the target gene promoter region is increased,thereby suppressing gene transcription.The purpose of this paper is to study small molecule inhibitors that can specifically block the binding of HOTAIR and EZH2.By interfering with the recruitment of PRC2 by HOTAIR,the role of HOTAIR in tumors can be inhibited,thereby achieving the effect of tumor targeted therapy.In the first part of this study,we first used 3D structural simulation technology to perform models of the HOTAIR’s 5 ’end(212-300bp)and EZH protein.Using molecular docking technology,high-throughput screening was performed in the Small Molecule Database of the American Cancer Institute to obtain 7 candidate compounds with the lowest free energy.Then,using artificial compound synthesis and cytotoxicity tests for preliminary screening of candidate compounds,we identified the potential small molecule AC1Q3QWB(AQB).Additionally,we analyzed the TCGA pan-cancer database and found that HOTAIR and EZH2 were the highest expressed in breast cancer and glioblastoma.Then,through cytotoxicity tests,we found that in glioblastoma and breast cancer,cells with high expression of HOTAIR and EZH2 were more sensitive to AQB,suggesting that the toxicity mechanism of AQB may depend on the expression of HOTAIR and EZH2.In the second part of this study,we used q PCR and Western Blot experiments and found that AQB did not affect the expression levels of HOTAIR,EZH2 or PRC2 core components,but it could reduce the global level of H3K27me3 after 72-hour treatment.The results of RIP experiments found that AQB can specifically inhibit the binding of HOTAIR and EZH2 without affecting the binding of other lnc RNAs to EZH2.Ch IRP experiments further demonstrated that low-dose AQB can significantly reduce HOTAIR-EZH2 binding.Consistently,we found that both the m RNA level and protein level of the target gene increased with the increase of the AQB treatment concentration,and showed an "S" shape.These results suggest that AQB can increase the target gene expression level by inhibiting HOTAIR-EZH2 interaction.Among the up-regulated genes,we identified that APC2 is a target gene of HOTAIR and that it can be significantly up-regulated after AQB treatment.The immunofluorescence and Western Blot experiments showed that up-regulated APC2 resulted in decreased levels of β-catenin and p-β-catenin.Functionally,low doses of AQB(40n M)can inhibit the metastasis of tumor cells,while high doses(40μM)can inhibit the clonal proliferation of tumor cells,and promote apoptosis and cycle arrest.In vivo,we found that AQB(50 mg/kg)can inhibit breast cancer in situ proliferation and lung metastasis.These results indicate that AQB can inhibit the proliferation and metastasis of glioblastoma and breast cancer through the Wnt/β-catenin pathway.In the last part,we explored a combination treatment strategy for AQB.The IC50 curves of AQB,DZNep and AQB combined with DZNep show that AQB can enhance DZNep’s toxicity to tumor cells by 1.5 to 2 times.AQB enhanced the toxicity of DZNep in vitro.In orthotopic breast cancer and glioblastoma patient-derived xenografts(PDX)models,the combination of low doses of AQB and DZNep realized much better killing than DZNep treatment alone.These results suggest that low-dose AQB can enhance the anti-tumor effect of DZNep.In summary,this study identified a small molecule,AC1Q3 QWB,that specifically inhibits the binding of HOTAIR-EZH2.It interferes with the recruitment of PRC2 by HOTAIR and increases the expression of the target genes.APC2,as one of the target genes,inhibits the Wnt/β-catenin pathway by degrading β-catenin,which may contribute to inhibition of tumor growth and metastasis.In addition,low-dose AQB can enhance the anti-tumor effect of EZH2 inhibitor DZNep,which provides a combination strategy for the clinical conversion of AQB.This study first reported a small molecular inhibitor that targets the interaction of long noncoding RNA and protein,providing a new direction for tumor targeted therapy.