The Function And Mechanism Of Non-coding RNA In 5-fluorouracil And Cisplatin Resistance Of Colorectal Cancer

BackgroundColorectal cancer（CRC）is a common malignant tumor of the digestive system,with high morbidity,high mortality and poor prognosis,which become a major disease that endangers human health.For locally unresectable and advanced CRC patients,adjuvant chemotherapy has become an important treatment method,which could reduce the risk of recurrence and metastasis and prolong the survival time of patients.5-Fluorouracil（5-Fu）or cisplatin（DDP）are the classic and basic chemotherapy drugs widely used in the treatment of CRC.However,tumor cells are prone to develop resistance to chemotherapy drugs,resulting in poor efficacy.Therefore,finding hub genes related to drug-resistance is expected to provide a new direction for reversing CRC chemoresistance.In the human genome,only 2%of nucleic acid sequences could be translated into proteins,and more than 70%of the genome were transcribed into non-coding RNA（ncRNA）.The abnormal expression of ncRNA were related to biological processes such as tumor proliferation,migration,invasion,and apoptosis,which have attracted much attention.Moreover,ncRNAs could act as oncogenes or tumor suppressor genes to affect the occurrence and development of tumors,which have become the hope to overcome the problem of tumor chemoresistance.NcRNAs mainly included microRNAs（miRNAs）,long non-coding RNAs（lncRNAs）and circular RNAs（circRNAs）,which can perform biological functions at multiple levels in the form of RNA.At present,only a few studies have reported that specific ncRNAs regulate 5-fluorouracil or cisplatin chemoresistance,but lack of study reported ncRNAs affect both 5-fluorouracil and cisplatin chemoresistance of CRC.Therefore,it is of great significance to find ncRNAs that regulate chemoresistance.ObjectiveThe study searched for ncRNAs closely related to 5-fluorouracil and cisplatin resistance in CRC via whole-transcriptome sequencing,and explored the roles of hub RNAs,and clarified whether hub lncRNAs or circRNAs can serve as competing endogenous RNA（ceRNAs）to combine with miRNAs then regulate the expression of drug resistance genes to affect chemoresistance,thus providing new molecular targets and theoretical basis for reversing CRC chemoresistance.MethodsPart 1.The expression profiles of mRNA,lncRNA,circRNA and miRNA in CRC chemoresistant cells were obtained by whole transcriptome sequencing.Taking the CRC cell line HCT8,the 5-fluorouracil resistant CRC cell line（HCT8/5-Fu）and the cisplatinresistant CRC cell line（HCT8/DDP）as the research objects,the total RNA of the three groups of cells were extracted,and the quality inspection was qualified followed by RNAsequencing.The mRNAs,lncRNAs,circRNAs and miRNAs that were significantly differentially expressed（DE）in the two drug-resistant cells compared with the HCT8 cells were screened based on |log2FoldChange|>1 and P<0.05.Subsequently,to find the RNAs that regulate both 5-fluorouracil and cisplatin chemoresistance,Venn analysis were used to screen out the common differentially expressed mRNAs,lncRNAs,circRNAs and miRNAs in the two drug-resistant cells.The mRNAs,lncRNAs,and circRNAs targeted by miRNAs were identified based on bioinformatics prediction,and competing endogenous RNAs（ceRNAs）networks,including IncRNA-miRNA-mRNA and circRNAmiRNA-mRNA network,were constructed.Finally,Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis were performed on mRNAs with ceRNA relationships.Part 2.The functions and mechanisms of LINC02418/miR-372-3p/EPHA2 in CRC chemoresistance.（1）In the IncRNA-miRNA-mRNA network,the pathways related to chemoresistance were found from the above significantly enriched pathways,the genes related to chemoresistance were further screened out from three most enrichment pathways to construct drug resistance-related ceRNA network,then hub genes were found,and LINC02418/miR-372-3p/EPHA2 or LINC02418/miR-33a-3p/EPHA2 were identified as target ceRNA networks.（2）The biological functions of LINC02418,miR-372-3p,miR-33a-3p and EPHA2 were verified.Quantitative real-time PCR（qRT-PCR）was used to detect the expression of LINC02418,miR-372-3p,miR-33a-3p and EPHA2 in chemo-sensitive and chemoresistant cells.The MTT and flow cytometry were used to detect the cell chemosensitivity and proliferation,apoptosis and cell cycle after interfering their expression in drugresistant cell lines.（3）The molecular mechanism of LINC02418 regulating CRC chemoresistance.LncRNAs often play regulatory roles as ceRNA,thus qRT-PCR was used to detect the expression of miR-372-3p and miR-33a-3p when silencing LINC02418 in drug-resistant cells;qRT-PCR and Western Blot were used to detect the expression of EPHA2 after interfering with LINC02418,miR-372-3p or miR-33a-3p;Dual luciferase experiments were used to verify the targeting binding relationship between LINC02418 and miR-3723p as well as miR-372-3p and EPHA2;The expression levels of EPHA2 after cotransfection of LINC02418 and miR-372-3p were detected by qRT-PCR to verify that LINC02418 could act as a ceRNA for miR-372-3p to regulate the expression of EPHA2.Part 3.The function and mechanism of hsacirc₀02482 in CRC chemoresistance.（1）Enrichment analysis of differentially expressed circRNAs.qRT-PCR to verify the expression levels of common differential circRNAs in the two drug-resistant strains.（1）Enrichment analysis of differentially expressed circRNAs were conducted and the expression levels of common differential circRNAs in the two drug-resistant cell lines were verified by qRT-PCR.（2）In the circRNA-miRNA-mRNA regulatory network,the genes that may related to chemoresistance were screened from the target genes,and the drug resistance-related ceRNA network was constructed,and the hub gene was determined as hsacirc₀02482.（3）The chemosensitivity,cell proliferation and apoptosis of drug-resistant cells were detected after overexpression of the hub gene hsacirc₀02482.qRT-PCR was used to detect the downstream miRNA and mRNA expression of hsacirc₀02482 to explore it functions.ResultsPart 1.Expression profiles of mRNA,lncRNA,circRNA and miRNA.The study verified that HCT8/5-Fu and HCT8/DDP cells were more resistant to chemotherapy than parental cells before RNA-sequencing,which laid a foundation for subsequent studies.The sequencing results showed that 2464 and 2378 mRNAs,597 and 601 lncRNAs,48 and 90 circRNAs,and 98 and 79 miRNAs were differentially expressed in HCT8/5-Fu and HCT8/DDP-resistant cells,respectively.Moreover,1779 mRNAs,295 lncRNAs,11 circRNAs,and 64 miRNAs were common differentially expressed in the two drugresistant cells.Based on the common differentially expressed RNAs,the target gene relationship pairs corresponding to miRNAs were predicted using bioinformatics methods,and then 1844 lncRNA-miRNA-mRNA and 47 circRNA-miRNA-mRNA regulatory networks were constructed.KEGG pathway enrichment analysis showed that the target genes of the lncRNA-miRNA-mRNA regulatory network were mainly enriched in the Ras,PI3K-AKT and MAPK signaling pathway,and the target genes in the circRNA-miRNAmRNA network were mainly enriched in MAPK,AMPK and TNF signaling pathway.Part 2.The biological function and mechanism of LINC02418/miR-372-3p/EPHA2 in CRC chemoresistance.（1）In the lncRNA-miRNA-mRNA network,the target gene related to chemoresistance were screened out in the above three most significantly enriched pathways and then the regulatory networks were constructed.CytoHubba was used to analyze the hub genes in drug resistance-related ceRNA network,then erythropoietinproducing hepatocellular receptor A2（EPHA2）and LINC02418 were regarded as hub genes,LINC02418/miR-372-3p/EPHA2 and LINC02418/miR-33a-3p/EPHA2 became the research object of the study.（2）LINC02418 was significantly highly expressed in CRC drug-resistant cell lines.The chemosensitivity of drug-resistant cells were enhanced,the apoptosis rates were increased and the cell cycle distribution were changed when LINC02418 was knocked down.While miR-372-3p and miR-33a-3p were significantly downregulated in HCT8/5Fu and HCT8/DDP cell lines,overexpression of miR-372-3p and miR-33a-3p could also enhanced the chemosensitivity and induced apoptosis,inhibited cell proliferation and cell cycle progression of CRC chemo-resistant cells.Moreover,EPHA2 was significantly overexpressed in CRC drug-resistant cell lines.The chemosensitivity of drug-resistant cells was enhanced,proliferation ability was decreased,the apoptosis rates were increased,and the cycle distribution were changed when the kinase activity of EPHA2 was inhibited.（3）Knockdown of LINC02418 in drug-resistant cells significantly increased the expression of miR-372-3p and miR-33a-3p,and the up-regulated fold of miR-372-3p was more significant;The expression of EPHA2 was significantly decreased when overexpression of miR-372-3p and miR-33a-3p,and the down-regulated fold caused by miR-372-3p was more significant.Therefore,miR-372-3p was selected as the follow-up mechanism study.The results of dual luciferase assay showed that miR-372-3p was a downstream target of LINC02418,and EPHA2 was a downstream target gene of miR-3723p;the expression level of EPHA2 was significantly downregulated in HCT8/5-Fu and HCT8/DDP cells with LINC02418 knocked down,which were restored by simultaneous transfection of miR-372-3p inhibitors,suggesting that LINC02418 might affect CRC chemoresistance through miR-372-3p/EPHA2 axis.Part 3.The function and mechanism of hsacirc₀02482 in CRC chemoresistance.（1）The differentially expressed circRNAs in HCT8/5-Fu and HCT8/DDP cells were mainly enriched in DNA repair pathway and Hippo signaling pathway,respectively.qRTPCR was used to verify the 11 common differentially expressed circRNAs,and the verification results were consistent with the RNA sequencing results.Hsacirc₀23607 was the most upregulated circRNA in drug-resistant cell lines,but it did not affect the CRC chemosensitivity.（2）Among the 47 circRNA-miRNA-mRNA networks,the hub gene hsacirc₀02482 analyzed by the cytoHubba could regulate target genes involved in drug resistance,such as baculoviral IAP repeat containing 3（BIRC3）.（3）Hsacirc₀02482 was lowly expressed in drug-resistant cells,and its overexpression could significantly enhance the chemosensitivity,inhibite the proliferation and promote cell apoptosis of HCT8/5-Fu and HCT8/DDP cells.In addition,overexpression of hsacirc₀02482 significantly inhibited miR-34b-3p while increased the expression of BIRC3,suggesting that hsacirc₀02482 may play an important role in CRC drug resistance through the miR-34b-3p/BIRC3 axis.ConclusionThe RNA expression profiles of CRC chemo-resistant cell lines were significantly altered compared with chemo-sensitive cell lines,with 1779 mRNAs,295 lncRNAs,11 circRNAs and 64 miRNAs were common differentially expressed in two chemo-resistant cells,and lncRNA/circRNA-miRNA-mRNA were constructed.In the lncRNA-miRNAmRNA network,LINC02418 could act as a ceRNA to competitively combined with miR372-3p,and indirectly regulate the expression of EPHA2 to promote the chemoresistance of 5-fluorouracil and cisplatin in CRC.In the circRNA-miRNA-mRNA network,hsacirc₀02482 was considered as a hub gene,and overexpression of hsacirc₀02482 could increase the chemosensitivity of HCT8/5-Fu and HCT8/DDP cells,which may affect chemoresistance through the miR-34b-3p/BIRC3 axis.This study explored the new mechanism of chemoresistance in CRC,which is expected to provide a new theoretical basis and target for reversing chemoresistance.