Study On The Role And Mechanism Of CircHOMER1 In The Therapy Of DVDMs-PDT On Colorectal Cancer

Objective:Photodynamic therapy(PDT)is a novel treatment with the characteristics of high efficiency,low toxicity,combination other therapy and tumor-specific accumulation,which makes PDT to be a potential treatment for coloreactal cancer.In recent years,researchers have found that noncoding RNA(miRNAs and lnc RNAs)plays an important role in photodynamic therapy of colorectal cancer by regulating downstream target genes or affecting signaling pathway,and increases or inhibits sensitivity of colorectal cancer to photodynamic therapy.CircRNAs as a hotspot in the cancer research fields,however,the mechanism and function role of circRNAs in the PDT treatment of colorectal cancer have little reports.In this study,we aim to explore the role and mechanism of circRNAs in photodynamic therapy of colorectal cancer.Methods:(1)Firstly,we detected the killing effects of DVDMs-PDT on colorectal cancer cells and colorectal cancer stem cells by cytotoxicity assays.Subsequently,RNA-seq was performed to detect the different expression circRNAs in RKO cells or RKO cells that under the DVDMsPDT treatment.Fluorescence in situ hybridization(FISH)and qPCR assay were used to analyze the expression of circHOMER1 in colorectal cancer tissues and adjacent tissues.The biological characteristics of circHOMER1 were verified by Sanger sequencing,actinomycin D and RNase R digestion,nucleocytoplasmic separation and cell FISH assays.(2)Secondly,we constructed circHOMER1 overexpression vector and explore its role in treatment of colorectal cancer by DVDMs-PDT through CCK8 and apoptosis flow cytometry assays.Bioinformatics analysis,western blot and qPCR assays were used to screen the combination miRNAs and targets of circHOMER1.Luciferase assay and AGO2-RIP assay was used to prove the binding of circHOMER1 to miR-942-5p and DLC1.(3)In addition,rescue experiments were used to conform that circHOMER1 improve the DVDMs-PDT treatment in colorectal cancer by regulating DLC1.(4)Finally,we constructed transplanted tumor models in nude mice with DVDMs-PDT treated to further verify the efficacy of DVDMs-PDT treatment in vivo and detected the expression of circHOMER1 and DLC1 in tumor tissues.Results:(1)DVDMs-PDT inhibits tumour progression in colorectal cancer cells and cancer stem cells(CSCs)DVDMs-PDT has higher effective treatment than Photosan-PDT by cell viability assays.DVDMs-PDT could significantly inhibit DNA synthesis of CRC cells compared and cell migration via Ed U assay and Transwell assay.Through accumulation of colorectal cancer stem cells and treated with DVDMs-PDT,we found that DVDMs-PDT could also effectively induce colorectal cancer stem cells death and decrease the protein levels of molecular markers on the surface of tumour stem cells(CD44 and CD133).By using western blotting,we found that the protein levels of p-AKT,mTOR and p-mTOR,EMT related molecules MMP2,9and EGFR were down-regulated,while the expression level of E-cad protein was up-regulated.The key molecues of Notch 1 signaling pathway,were also down regulated.(2)CircHOMER1 expression is induced by DVDMs-PDT in colorectal cellsRNA-seq was found that circHOMER1 was upregulated in colorectal cells after treated with DVDMs-PDT,and then verified by qPCR assay.Sanger sequencing was applied to confirm that circHOMRE1 was reverse spliced from exon 2-5 of the parent gene HOMER1.Rnase R digestion and actinomycin D intervention assays were proved that circHOMER1 was more stable than that of its parent gene HOMER1.FISH and nucleocytoplasmic separation assays showed that circHOMER1 was mainly located in the cytoplasm,which may indicate its ce RNA mechanism in this study.We also detect the expression of circHOMER1 in colorectal cancer tissues and adjacent tissues by qPCR and FISH assay.(3)CircHOMER1 promote the therapy efficacy of DVDMs-PDT on colorectal cancer cellsIn order to explore the function of circHOMER1 in PDT treatment for colorectal cancer cells,we constructed circHOMER1 overexpression vector to upregulate circHOMER1 expression in RKO and CX-1 cells.By using CCK8 and apoptosis flow cytometry assay,we found that circHOMER1 could significantly improve the inhibition of DVDMs-PDT on colorectal cells.Through western blot assay,circHOMER1 overexpression could decrease the protein levels of BCL2,and increase the protein levels of BAX.In addition,circHOMER1 could inhibit the expression of PI3 K,p-AKT,EGFR.(4)CircHOMER1 serves as a sponge for miR-942-5p to activate Deleted in Liver Cancer Gene 1(DLC1)Since circHOMER1 could improve the therapeutic effect of DVDMsPDT on colorectal cancer,we analyzed the circHOMER1 binding miRNAs by using the online analysis software(RNAhybrid,targetscan and Miranda).Using qPCR and luciferase assay,miR-942-5p could bind to the sequence of circHOMER1 and was downregulated in colorectal cells treated with DVDMs-PDT.Besides,AGO2-RIP assay also was found that circ HOEMR1 could be enriched in the complex of miR-942-5p overexpression cells.Subsequently,online analysis suggested that DLC1 may be the downstream targets of miR-942-5p.To detect the binding of miR-942-5p and DLC1,luciferase assay was applied to confirm that miR-942-5p could target the 3’UTR of DLC1.The resuce experiments were designed to verify that circHOMER1 could regulate DLC1 expression by sponging miR-942-5p.DLC1 protein levels were increased in circHOMER1 overexprssion cells,while DLC1 was rescued by miR-942-5p mimics.In addition,DLC1 was upregulated by DVDMs-PDT,which was consistent with the expression of circHOMER1.By using GEO database,miR-942-5p was upregulated in colorectal cancer tissues in GSE126093,while DLC1 was downregulated in colorectal cancer tissues in GSE126092.There was a negative correlation between DLC1 and miR-942-5p in 450 colorectal cancer tissues analyzed in Starbase database.(5)CircHOMER1 could enhance the inhibition effect of DVDMsPDT oncolorectal cancer cell proliferation by regulating DLC1 expressionSince circHOMER1 could restore the expression level of DLC1 by sponging miR-942-5p,we designed biological function experiments to conform circHOMER1 affect the therapeutic effect of DVDMs-PDT on colorectal cancer by regulating DLC1.CCK8 assay and Colony formation assay were applied to found that interference of DLC1 could diminishthe inhibitory effect of circHOMER1 on cell proliferation.In addition,interference of DLC1 could rescue the expression of BCL2,PI3 K,p-AKT,EGFR,which inhibited by circHOMER1.(6)DVDMs-PDT could effectively treat tumors in vivoWe have confirmed that DVDMs-PDT could induce colorectal cell death and significantly elevated the expression of circHOMER1 and DLC1 in vitro.Then,we investigated the theraprutic effect of DVDMs-PDT on the model of translanted tumor in nude mice.HE assays suggested that DVDMs-PDT could effectively decrease tumor cells and induce cell degeneration,apoptosis and fibrosis.Tunnel and IHC assays indicated that DVDMs-PDT could increase cell apoptosis and decrease cell proliferation.Subsequently,we found that circHOMER1 and DLC1 was upregulated in PDT groups,and circHOMER1 was mainly located in cytoplasm of tumor cells.Through qPCR and western blot assay,the results showed that DVDMs-PDT could increase the expression level of DLC1 in tumor tissues.Summary:(1)DVDMs-PDT could effectively kill colorectal cancer cells and colorectal cancer stem cells.(2)CircHOMER1 and DLC1 expression were upregulated in colorectal cancer cells,while miR-942-5p were downregulated under DVDMs-PDT treatment.(3)CircHOMER1 and DLC1 expression were upregulated in colorectal cancer cells,while miR-942-5p were downregulated under DVDMs-PDT treatment.(4)CircHOMER1 enhance killing efficacy of DVDMs-PDT via modulating miR-942-5p /DLC1.