| Background:Colorectal cancer(CRC)is one of the most common malignant tumors in our country.Although advances in surgical techniques and the use of new anticancer drugs have led to significant improvements in patient outcomes,CRC remains an incurable disease in most cases.Recent studies have confirmed that long non-coding RNAs(lnc RNAs),as an important participant in the tumor progression mechanism,are involved in the initiation,progression and metastasis of CRC,and can also be used as a diagnostic and prognostic marker of CRC.Metabolic reprogramming is closely related to behavioral changes of tumor cells,and lnc RNAs also play an important role in tumor metabolic reprogramming,including lnc RNAs mediated metabolic enzymes,metabolism-related transcription factors and post-translational modifications of other proteins in related signaling pathways.lnc RNA ELFN1-AS1(ELFN1-AS1)is highly expressed in a variety of tumors and can promote tumor progression.However,whether ELFN1-AS1 is involved in metabolic reprogramming in CRC cells remains unclear.Therefore,this study explores the specific mechanism of ELFN1-AS1 in CRC,and provides better ideas for the treatment of CRC.Objective: Focusing on ELFN1-AS1,this study further explored the role and mechanism of YY1/ELFN1-AS1/TP53/G6 PD axis in the progression of CRC,providing new ideas for the diagnosis and treatment of CRC.Methods:(1)GEPIA database was used to analyze the expression profile of ELFN1-AS1 in various common tumors,and then the expression of ELFN1-AS1 in CRC tissues and normal tissues in TCGA and GEO databases was analyzed.The relationship between ELFN1-AS1 and prognosis was evaluated.(2)The expression level of ELFN1-AS1 in CRC tissues and normal adjacent tissues was detected by q RTPCR,and the clinical correlation of ELFN1-AS1 was analyzed.(3)The expression level of ELFN1-AS1 in CRC cells and normal colorectal mucosal cells was detected by q RTPCR.The knockdown model of ELFN1-AS1 was constructed by plasmid and lentivirus.(4)The effects of ELFN1-AS1 on the malignant biological function of CRC cells were evaluated by CCK-8 assay,plate cloning assay and transwell chamber assay,respectively.(5)The relationship between ELFN1-AS1 and pentose phosphate pathway was analyzed by GSEA,and the contents of lactic acid,glucose,NADPH and the activity levels of G6 PD in different cell models were detected by corresponding detection kits.(6)The effect of ELFN1-AS1 on G6 PD was detected by q RT-PCR and western blot,and the correlation between G6 PD and ELFN1-AS1 in clinical samples was analyzed.(7)Rescue experiments verified that ELFN1-AS1 affected the proliferation,migration,invasion,reactive oxygen species(ROS)and apoptosis of CRC cells by G6 PD.(8)Proteins that might bind to ELFN1-AS1 directly were predicted by RNAInter,STRING and cat RAPID databases,and the interaction between ELFN1-AS1 and the target protein was verified by RNA immunoprecipitation(RIP)and RNA pull down experiments.(9)To investigate whether ELFN1-AS1 mediates ubiquitination regulation of TP53 through ubiquitination experiments.(10)The effect of ELFN1-AS1 on tumor growth was detected in vivo,and epithelial-mesenchymal transition(EMT)and apoptosis-related proteins were detected by immunohistochemical(IHC)assay and western blot assay.(11)Chromatin immunocoprecipitation(Ch IP)and dual luciferase reporter assays were used to detect interactions between the promoter region of ELFN1-AS1 and transcription factors.Results:(1)The expression profile of ELFN1-AS1 in various common tumors was analyzed by GEPIA database,and the results showed that the differential expression of ELFN1-AS1 was the most significant in CRC tissues.The results of TCGA and GEO database analysis showed that ELFN1-AS1 was highly expressed in CRC tissues,and subsequent analysis showed that the high expression of ELFN1-AS1 was associated with poor prognosis of patients.(2)q RT-PCR assay confirmed that the expression level of ELFN1-AS1 in CRC tissues was higher than that in normal adjacent tissues.Clinical correlation analysis of ELFN1-AS1 showed that the expression of ELFN1-AS1 was correlated with lymph node metastasis and TNM stage.(3)q RT-PCR assay showed that the expression level of ELFN1-AS1 in CRC cells was significantly higher than that in normal colorectal mucosal cells.(4)The results of CCK-8 assay,plate cloning assay and transwell chamber assay showed that down-regulation of ELFN1-AS1 significantly inhibited the proliferation,migration and invasion of CRC cells compared with the control group.(5)The results of GSEA analysis showed that ELFN1-AS1 was involved in the regulation of pentose phosphate pathway.Further studies showed that down-regulation of ELFN1-AS1 significantly inhibited glucose consumption,lactic acid and NADPH production levels in CRC cells.(6)The expression of G6 PD in cancer tissues was significantly higher than that in normal adjacent tissues,and the expression level of ELFN1-AS1 was moderately correlated with the protein expression level of G6 PD.Further experiments showed that downregulation of ELFN1-AS1 could significantly inhibit the expression and activity level of G6 PD protein.(7)Rescue experiments verified that down-regulated ELFN1-AS1 inhibited the proliferation,migration and invasion of CRC cells through G6 PD,and inhibited the expression of N-cadherin and MMP-9,and promoted the expression of Ecadherin.In addition,down-regulation of ELFN1-AS1 also promoted ROS production and apoptosis of CRC cells through G6 PD,and promoted cleaved caspase-3 and CytC expression,and inhibited Bcl-2 expression.(8)It was predicted by RNAInter,STRING and cat RAPID databases that TP53 directly bound to ELFN1-AS1 to regulate the expression of G6 PD,and the experiment verified that down-regulation of ELFN1-AS1 indeed inhibits the expression of G6 PD by TP53.In addition,the interaction between ELFN1-AS1 and TP53 was verified by RIP and RNA pull down experiments.(9)Down-regulation of ELFN1-AS1 reduced ubiquitination of TP53,while overexpression of MDM2 attenuated this inhibition.(10)The results of subcutaneous tumor formation in nude mice showed that down-regulation of ELFN1-AS1 inhibited tumor growth by down-regulating the expression of G6 PD,and related protein detection showed that down-regulation of ELFN1-AS1 inhibited the expression of Ki-67,N-cadherin,G6 PD and Bcl-2 and promoted E-cadherin and cleaved caspase-3expression,while up-regulation of G6 PD reversed these results.(11)YY1 knockdown inhibited the transcription of ELFN1-AS1 and the expression of G6 PD,and the interaction between the promoter region of ELFN1-AS1 and YY1 was verified by Ch IP and dual luciferase reporter.Conclusions:(1)ELFN1-AS1 activates the pentose phosphate pathway of CRC cells by regulating the TP53/G6 PD axis,thereby promoting the proliferation,migration and invasion of CRC cells and inhibiting ROS production and apoptosis.(2)The transcription factor YY1 can bind to the promoter region of ELFN1-AS1,and promote the expression of ELFN1-AS1 in CRC cells... |
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