Nei Endounuclease VⅢ-like1(NEIL1) Inhibits Apoptosis Of Human Colorectal Cancer Cells

Background:Colorectal cancer is one of the most common cancers in the world,and its occurrence and progression can be formed by the accumulation of tumor suppressor genes and oncogene mutations.Defects in the DNA damage repair system lead to increased gene mutation rates and promote tumorigenesis and progression.As an important DNA repair enzyme,Nei endounuclease VIII-like1（NEIL1）is involved in the development of various cancers,but its exact role in CRC progression is unclear.Through big data analysis,we found that CRC patients with high expression of NEIL1 survived poorly.Through siRNA silencing and recombinant plasmid overexpression experiments in human colon cancer cells,we found that NEIL1 can inhibit colon cancer cell apoptosis and promote cell viability.Through big data and targeted software analysis,we found that NEIL1 may be potentially targeted by miR-7-5p and verified.The effect of miR-7-5p regulation on NEIL1 inhibition of cell apoptosis and cell viability was also explored.Our data indicate that NEIL1 promotes cell viability by inhibiting apoptosis of human colon cancer cells,which is regulated by the negative regulation of miR-7-5p.This study provides novel insights into the network of NEIL1 regulating colon cancer development and provides a basis for its potential therapeutic targets for colorectal cancer.Objective:This study is to investigate the role of Nei endounuclease VIII-like1（NEIL1）in the pathogenesis of colorectal cancer（CRC）.Methods:7 Design and synthesize siNEIL1,transfect it with control siNC into human colon cancer cells HCT116 and SW480,q RT-PCR and Western blotting to detect transfection efficiency MTT assay was used to detect changes in colon cancer cell viability;PI staining was used to detect changes in apoptosis;Western Blotting was used to detect changes in apoptosis-related proteins.In addition,an NEIL1 overexpression plasmid was constructed,and the control plasmid was transfected into human colon cancer cells,and the above changes were observed.8 Bioinformatics software to analyze the correlation and targeting of miR-7 and NEIL1 expression.The miR-7 expression plasmid was constructed,and miR-7-5p inhibitors were designed and synthesized.The above methods were used to observe changes in human colon cancer cell viability and apoptosis.9 Change the level of miR-7 and observe the effect of NEIL1 on the viability and apoptosis of human colon cancer cells.Results:9.2 Extract the data of NEIL1 expression from colorectal cancer tissues from TCGA database,and use long rank analysis to find that CRC patients with high expression of NEIL1 have poor survival.Real-time quantitative PCR and Western blotting showed that the m RNA and protein expression of NEIL1 in cells were significantly down-regulated.MTT analysis of cell viability showed that compared with the control group,the down-regulation of NEIL1 expression resulted in a significant decrease in cell viability.Flow cytometry detected apoptosis,and found that early and late withered cells were increased in cells with downregulated NEIL1 expression.In addition,Western blotting results showed that transfection of siNEIL1 increased Bax and Bcl-2 in human colon cancer cells,and the balance of Bax / Bcl-2 was pro-apoptotic,and the content of caspase-9 activated by cleavage also increased.9.3 Recombinant plasmids expressing NEIL1 and control plasmids were transfected into HCT116 and SW480 cells,respectively.Real-time quantitative PCR and Western blotting results showed that m RNA and protein expression of NEIL1 were significantly up-regulated in the cells.MTT analysis showed that compared to the control group,high expression of NEIL1 promoted cell viability.Flow cytometry analysis of apoptosis revealed that high expression of NEIL1 reduced apoptotic cells in the cells,and high expression of NEIL1 biased the balance of Bax / Bcl-2 in human colon cancer cells to anti-apoptosis and was cleaved and activated Caspase-9 The content decreases.These are all contrary to the results obtained by down-regulation of NEIL1 expression.9.4 Micro RNAs can negatively regulate target gene expression after transcription by binding to the 3’-UTR of the target gene.We passed two commonly used public databases Target Scan（http://www.targetscan.org/vert₇1）and miRDB（http://mirdb.org/miRDB）and used an online cross-check tool（http: //bioinfogp.cnb.csic.es/tools/venny/index.html）find that miR-7-5p can target the 3’-UTR of NEIL1.And in colon cancer tissues and human colon cancer cells,miR-7-5p and NEIL1 expression levels showed a negative correlation.Therefore,we constructed a recombinant plasmid that highly expressed miR-7,and designed and synthesized an inhibitor of miR-7-5p.The miR-7 high expression plasmid and control plasmid were transfected into HCT116 and SW480 cells,respectively.Real-time quantitative PCR results showed that miR-7-5p was no significant change in the cells;miR-7-5p inhibitor miR-7-5p-inhibitor and The inhibitor-NC control was transfected into HCT116 and SW480 cells,respectively.Realtime quantitative PCR showed that miR-7-5p was down-regulated in the cells.Importantly,the results of real-time quantitative PCR and Western blotting also showed that compared with the control group,overexpression of miR-7 significantly reduced the expression of NEIL1 m RNA and protein in HCT116 and SW480 cells,while transfection with miR-7-5p-inhibitor Up-regulated m RNA and protein expression of NEIL1 in HCT116 and SW480 cells.9.5 The role of miR-7-5p in human colon cancer cells.MTT analysis showed that compared with the control group,the high expression of miR-7 significantly reduced the viability of HCT116 and SW480 cells.Flow cytometry analysis of apoptosis revealed that high expression of miR-7 increased the number of early and late withering cells,and biased the balance of Bax / Bcl-2 in the cells to promote apoptosis,which was cleaved by activated caspase-The increase of 9 indicates that increasing the expression of miR-7 in human colon cancer cells can promote apoptosis and reduce cell viability,which is consistent with the experimental results of transfection with siNEIL1.In contrast,compared with the control group,transfection of miR-7-5p-inhibitor promoted the cell viability of HCT116 and SW480,reduced apoptotic cells,and the balance of Bax / Bcl-2in the cells was biased towards anti-apoptosis,which was cut and activated The content of Caspase-9 decreased,which was consistent with the results obtained by high expression of NEIL1.9.6 The above results indicate that miR-7-5p can promote apoptosis and inhibit the vitality of human colon cancer cells in human colon cancer cells,which is the opposite of NEIL1,suggesting that miR-7-5p can be negative in human colon cancer cells.Regulates NEIL1 and has an effect on apoptosis and cell viability.Next,we overexpressed NEIL1 and miR-7 in HCT116 and SW480 cells,respectively or simultaneously,and found that overexpression of miR-7 significantly reduced NEIL1-improved human colon cancer cell viability（Figure 7A）and apoptosis inhibition（Figure7B and 7C）.These results further illustrate that in human colon cancer cells,changes in the expression of miR-7 can negatively regulate NEIL1,leading to changes in apoptosis and cell viability.Conclusions:2)Decreasing the expression of NEIL1 in human colon cancer cells can promote apoptosis and reduce cell viability.3)NEIL1 can inhibit apoptosis and improve cell viability in human colon cancer cells,which leads to poor survival of CRC patients with high expression of NEIL1.4)NEIL1 promotes the proliferation of CRC cells,which is negatively regulated by miR-7-5p.5)The above results indicate that miR-7-5p can promote apoptosis and inhibit the viability of human colon cancer cells in human colon cancer cells,which has the opposite effect to NEIL1,suggesting that miR-7-5p can be used in human colon cancer cells.Negatively regulates NEIL1,and affects apoptosis and cell viability.