The M6A Reader HNRNPC Enhances The Mechanism Of LncRNA AC145207.5 Stability-mediated Ferroptosis In Colorectal Cancer

Objective:Study of the m6A reader HNRNPC regulates the expression of Lnc RNA AC145207.5 in colorectal cancer cells by enhancing its stability and may affect the development of colorectal cancer via Nrf2/GPX4 signaling axis-mediated ferroptosis.Methods:1.Extract and screen the expression profiles of colorectal cancer and control from TCGA-GTEx-COADREAD database to identify Lnc RNAs with significant differential expression.ROC-receptive curve to predict their potential as tumor markers.RT-q PCR to detect the expression of AC145207.5 in colorectal cancer tissues and normal colorectal tissues.2.To detect the effects of AC145207.5 knockdown or overexpression on the malignant phenotype of tumor cells such as proliferation,migration and invasion,respectively;to detect the localization of AC145207.5 in colorectal cancer cells by fluorescence in situ hybridization assay（FISH assay）.Lentiviral constructs of NC,sh-AC145207.5,a stably transfected HCT116 cell line were injected into the axillary region of nude mice for subcutaneous ectopic tumorigenesis assay and tumor size was examined.3.The expression of AC145207.5 in colorectal cancer tissues and normal tissues was analyzed by the ENCORI database and the TCGA database;after silencing HNRNPC in colorectal cancer cells,the expression of AC145207.5 was detected by RT-q PCR to verify the regulatory role;RIP-q PCR assay verified the direct binding of HNRNPC to AC145207.5;RNA half-life assay verified whether HNRNPC maintained the stability of AC145207.5;Biosign database predicted the presence of m6A modification sites in AC145207.5 sequence recognized by m6A reader;Me RIP-q PCR verified that the m6A reader protein HNRNPC played the role of recognizing the methylation-modified AC145207.5;Revert experiments verify that overexpression of AC145207.5 restores the malignant phenotype of knockdown HNRNPC tumors.4.The expression level of AC145207.5 was knocked down in HCT116 cells,and total RNA was extracted for transcriptome sequencing（n=3）to search for differentially expressed genes and perform functional enrichment analysis of the differential genes in the signaling pathway;Control,NC,si-AC145207.5,si-AC145207.5+Fer-1experimental groups were set up,and the survival rate of each group was detected by MTS.Fe²⁺concentration,reactive oxygen species（ROS）level,malondialdehyde（MDA）content,total glutathione content and reduced glutathione/oxidized glutathione（GSH/GSSG）levels to verify the correlation between AC145207.5 and ferroptosis,and WB to detect the expression of Nrf2 and GPX4,key factors of AC145207.5 in the regulation of ferroptosis.Results:1.Analysis of the expression profiles of a total of 742 normal control and colorectal cancer tissues in the TCGA-GTEx-COADREAD database（Tumor=383,Normal=359）revealed that AC145207.5 was significantly highly expressed in colorectal cancer tissues;secondly,the area under the ROC curve showed AUC=0.826（CI=0.797-0.855）,which means that AC145207.5 could be a marker for colorectal cancer diagnosis.This result was subsequently verified using RT-q PCR as well.2.AC145207.5 knockdown and overexpression assays were performed on HCT116 and SW480 cells,respectively,and its effects on proliferation,migration and invasion of colorectal cancer cells were verified by MTS,plate cloning,cell scoring and Transwell assays.The results showed that the si RNA significantly inhibited the proliferation and invasion and migration of HCT116 and SW480 cells compared with the NC group,while ov-AC145207.5 showed a promotion effect compared with the Vector group.The localization of AC145207.5 in colorectal cancer cells was examined by fluorescence in situ hybridization assay（FISH assay）.Laser confocal observation showed that AC145207.5 was distributed in the cytoplasm and nucleus of colorectal cancer cells HCT116 and SW480,and mainly in the nucleus.Measuring the tumor size of different groups,the tumor-forming size of sh-AC145207.5 group of nude mice was significantly reduced compared with the NC group,indicating that it could play an in vivo role in animals.3.Four RBP proteins（FMR1,ELAVL1,HNRNPC,IGF2BP2）were found to bind with the highest confidence to AC145207.5 prediction by ENCORI database,and the expression of these four RBP proteins was found to be higher in colorectal cancer tissues than in normal tissues by analysis of TCGA database.The results of subsequent experiments showed that only interference with HNRNPC could down-regulate the expression of AC145207.5.The predictive ability of HNRNPC had some accuracy（AUC=0.936,CI=0.917-0.955）,HNRNPC could potentially be a marker for colorectal cancer diagnosis.The RT-q PCR results showed that the expression level of AC145207.5 was significantly decreased after silencing HNRNPC,indicating that it was positively regulated.The results of RIP-q PCR showed that the expression of AC145207.5 was significantly increased in the anti-HNRNPC group compared with the anti-Ig G group,indicating that HNRNPC could directly act on and bind to AC145207.5.RNA half-life assay The results showed that knockdown of HNRNPC accelerated the degradation of AC145207.5,thus suggesting that the binding of HNRNPC maintained the stability of AC145207.5.The AC145207.5 sequence was found to have an m6A motif that could be recognized by the m6A reader by prediction from the Biosign database.The results of Me RIP-q PCR showed that AC145207.5 could be enriched by m6A antibody,but the enriched AC145207.5 was significantly reduced after knockdown of HNRNPC.The above results showed that AC145207.5 has m6A modification site,and HNRNPC can stabilize AC145207.5 by recognizing the m6A modification site of AC145207.5 and binding to it.Compared with Vector+sh-HNRNPC and sh-NC（HNRNPC）+ov-AC145207.5 groups,sh-HNRNPC+ov-AC145207.5 group was statistically significant,indicating that overexpression of AC145207.5 responded to the inhibitory effect of silencing HNRNPC on colorectal cancer cells.4.The transcriptome sequencing results showed that there were differentially expressed genes in the AC145207.5 low expression group of cells compared to the NC group.GO functional enrichment analysis was performed on the differentially expressed genes in the above sequencing results.The enrichment results showed that AC145207.5 was involved in cholesterol biosynthesis;DNA-template;regulation of transcription;steroid metabolic process;and lipid metabolism.To further investigate the distribution of the above differential genes in gene annotation functions,enrichment analysis of KEGG functional signaling pathways was performed,and the results showed that the most important pathway involved in the differential genes in AC145207.5 low-expressing cells was the ferroptosis pathway.Compared with knockdown of AC145207.5 alone,knockdown of AC145207.5 with the addition of ferroptosis inhibitor（Fer-1）significantly increased the cell survival rate;Fe²⁺concentration,MDA and ROS content were significantly higher in sh-AC145207.5group compared with NC group,while they were significantly lower in ov-AC145207.5 group.The total glutathione content and GSH/GSSG levels were significantly lower in the sh-AC145207.5 group compared with the NC group,while they were significantly higher in the ov-AC145207.5 group.The expression levels of Nrf2 and GPX4 were significantly lower in the sh-AC145207.5 group compared with the NC group,while they were significantly higher in the ov-AC145207.5 group.Conclusion:Lnc RNA AC145207.5 is highly expressed in colorectal cancer tissues and promotes proliferation,invasion and migration of colorectal cancer cells.m6A reader protein HNRNPC positively regulates AC145207.5 expression in colorectal cancer cells by recognizing and binding to methylation-modified AC145207.5,which enhances its stability and inhibits degradation.AC145207.5 may affect the development of colorectal cancer through the Nrf2/GPX4 signaling pathway-mediated ferroptosis.