Identification Of The Expression Profile Of CircRNA In Chemotherapy-induced Liver Injury In Breast Cancer And Its Role As A "Sponge"

Objectives Through the screening and identification of circ RNAs in the serum of patients with breast cancer chemotherapy-induced liver injury,it was determined that circ RNAs were differentially expressed during the disease process,the circ RNA-mi RNAm RNA regulatory network was constructed,and the biological functions of circ RNAs were analyzed and explored.The mechanism of damage regulation lays the foundation for further research on the development mechanism of chemotherapy drug-induced liver injury in breast cancer.Methods 1 Sample collection: the peripheral blood serum of breast cancer patients undergoing chemotherapy at Tangshan People’s Hospital from May 2019 to November2019 was collected.According to Danan criteria,newly treated breast cancer patients receiving chemotherapy were divided into liver injury group and non-liver injury group.2Circ RNA chip screening and regulatory function analysis: three cases were selected from each of the two groups,and their serum samples were screened by Agilent chip,the prediction of the differentially significantly expressed circ RNA target mi RNA and downstream target genes were predicted by mi Randa database,the predicted target gene and the significantly different expression m RNA screened by the chip were crossed,and Cytoscape software constructed a circ RNA-mi RNA-m RNA co-regulatory network.3Verification of circ RNA in serum: according to the results of chip screening and functional analysis,the q RT-PCR method was used to verify the hsa＿circ＿0030051 and hsa＿circ＿0080408 in the serum of patients with liver injury,the primer design of circ RNA and internal reference(GAPDH)was completed with Prime Premier 5.0.4 Validation of the regulatory mechanism of hsa＿circ＿0080408: Through co-expression network and bioinformatics analysis,the mechanism of the differentially downregulated hsa＿circ＿0080408 was studied.Three cell models were established by adding paclitaxel,epirubicin,paclitaxel and epirubicin to HL-7702 hepatocytes respectively;after overexpressing hsa＿circ＿0080408 and inhibiting mi RNA,CCK-8 method,ALT and AST were used to determine the status of hepatocytes;Gene expression levels were detected by q RT-PCR and Western Blot;the binding sites of hsa＿circ＿0080408 and mi R-6756-5p,mi R-6756-5p and CDKN2 A were verified by dual luciferase reporter gene assay.Results 1 Results of clinical data analysis: The basic information of the research subjects was collected by reviewing the clinical medical records of the patients,and the general data,tumor information,chemotherapy regimens,etc.of the patients in the liver injury group and the non-liver injury group were analyzed for the relationship with breast cancer chemotherapy drug-induced liver injury.In addition,the use of different chemotherapy drugs has been linked to liver damage.We selected the three chemotherapy regimens with the most liver damage(paclitaxel group,anthracycline group,and combination drug group)for follow-up experiments.2 Circ RNA expression profile analysis and regulatory network construction: the results of the circ RNA chip showed that there were 25 significantly differentially expressed circ RNAs in the serum of patients(|log FC|≥2.0,P<0.05),of which8 were up-regulated and 17 were down-regulated.Since the differential expression fold of the hsa＿circ＿0030051 target gene DEFA1 is>10 times,which is much higher than that of other m RNAs;the hsa＿circ＿0080408 target gene CDKN2 A is involved in the P53 signaling pathway,and the P53 signaling pathway can regulate the occurrence and development of liver diseases,so hsa＿circ＿0030051,hsa＿circ＿0080408 were selected to construct co-regulatory network.In the hsa＿circ＿0030051 regulatory network,its target gene functions mainly include cellular processes,single biological processes and so on;participating in signaling pathways include MAPK signaling pathway,NOD-like receptor signaling pathway and so on.In the hsa＿circ＿0080408 regulatory network,its target gene functions are concentrated in neuron differentiation,neuron generation and so on;participating in signaling pathways include P53 signaling pathway,oxytocin signaling pathway and so on.3 Validation results of hsa＿circ＿0030051 and hsa＿circ＿0080408 in serum: in the serum of patients with breast cancer chemotherapy drug-induced liver injury and non-liver injury patients whose chemotherapy regimens were paclitaxel group,anthracycline group and combination group,hsa＿circ＿0030051 was significantly differentially expressed in serum of patients with liver injury Up-regulated,the differential expression of hsa＿circ＿0080408 was significantly down-regulated,and the expression trend was consistent with the microarray results.4 Preliminary study on the regulatory mechanism of hsa＿circ＿0080408: The mechanism of hsa＿circ＿0080408-mi R-6756-5pCDKN2A-MDM2 functional axis activating the P53-BAX-BCL-2 pathway was selected for cell experiments.The study found that by adding paclitaxel,anthracycline,paclitaxel +anthracycline to HL-7702 human normal hepatocytes,a cell model of breast cancer chemotherapy-induced liver injury could be successfully established.In the paclitaxel drug group cell model,hsa＿circ＿0080408 or mi R-6756-5p may regulate the change trend of each gene through the P53 signaling pathway,but the regulatory mechanism is unclear.In the anthracycline group cell model,hsa＿circ＿0080408 or mi R-6756-5p may not affect the development of breast cancer chemotherapy-induced liver injury through the P53-mediated apoptosis pathway.In the paclitaxel and anthracycline group cell model,there may be a regulatory mechanism that hsa＿circ＿0080408 competes with mi R-6756-5p to bind CDKN2 A,and activates the P53-BAX-BCL-2 apoptosis pathway through MDM2.The results of q RT-PCR and Elisa verification of each gene in the serum of breast cancer patients with combined chemotherapy were consistent with the results of cell verification.Conclusions 1 There are differentially expressed circ RNAs in breast cancer chemotherapeutic liver injury,which may affect the occurrence and development of the disease.2 Paclitaxel and anthracycline combination drugs,breast cancer chemotherapy drug-induced liver injury may have a regulatory mechanism of hsa＿circ＿0080408-mi R-6756-5p-CDKN2A-MDM2 function axis activating the P53-BAX-BCL-2 apoptosis pathway.Figure 57;Table 35;Reference 265...