| Objective:Lung cancer is one of the most common tumors in the world.Among all malignant tumors,lung cancer has the highest mortality.In the developing countries,the incidence of lung cancer has a rapid upward trend.In recent years,many zincfinger proteins have been found to be closely related to the occurrence and progression of lung cancer,while ZNF326 is a newly discovered C2H2 zinc finger protein.However,the role of ZNF326 in lung cancer has not been reported.It plays a role as a tumor suppressor gene in triple-negative breast cancer.It was found that ZNF326 expression was low in triple-negative breast cancer cell lines.Interfering with the expression of ZNF326 could not only up-regulate the expression of EMT and cancer stem cells genes related proteins,but also promote the ability of tumorigenesis in nude mice subcutaneously.It is well-known that different zinc finger proteins play different roles in promoting or inhibiting cancer,and the same zinc finger protein may have opposite roles in different tumors.The molecular structure of ZNF326 protein(containing transcriptional activation domain and C2H2 zinc finger domain).This structural feature enables ZNF326 to act as a transcription factor;however,its target genes and regulatory mechanisms are unknown.If ZNF326 can act as a transcription factor,what role does it play in the development of lung cancer? Whether it is a promote cancer protein or a tumor suppressor protein as well as its target genes and regulatory mechanisms has become the starting point of our experimental research.This paper aims to reveal the role of ZNF326 as a transcription factor in the development of non-small cell lung cancer and its molecular mechanism.Methods: In this study,Ch IP-seq was used to sequence and analyze DNA fragments associated with the ZNF326 antibody and Myc-tag antibody,and potential target genes in the promoter region of the DNA fragment were screened.After changes in the expression of ZNF326,the m RNA levels of potential target genes were detected by RT-q PCR.EMSA and dual-luciferase assays were used to identify the specific binding sequences.The H1299 and A549 cell line,which expressed ZNF326 stably and which expressed ZNF326 knockdown(selected by G418),was used for tumorigenesis in nude mice to observe its ability of subcutaneous tumorigenesis.The effects of ZNF326 on cell cycle,proliferation,migration ability and cycle and migration-related proteins in H1299 and A549 cell line were detected by flow cytology,MTS,Transwell and Western blot assay in vitro.And to verify whether the action is played by its target gene ERCC1.Results:1.ZNF326 promotes cell proliferation in non-small cell lung cancer in vivo and in vitro.The volume and weight of transplanted tumors in the injection group with cells overexpressing ZNF326 were significantly greater than those in the control group(p<0.05).The volume and weight of transplanted tumors in the ZNF326 knockdown H1299 cells were significantly lower than those in the control group(p<0.05).Overexpression of ZNF326 could promote the proliferation of NSCL cells and clone formation ability while the knock down of ZNF326 expression inhibited proliferation and clone formation(p<0.05).The high expression of ZNF326 was closely related to low differentiation and a high p TNM stage of NSCLC(p<0.05).The migration ability of A549 and H1299 cells was enhanced after transfection of ZNF326 plasmid,but the invasion ability of A549 and H1299 cells was decreased after konck down the expression of ZNF326(p<0.05).2.ERCC1 is a target gene of ZNF326:Ch IP-seq to analyze DNA fragments.In total,3848 DNA fragments were screened using a ZNF326 antibody and 2558 DNA fragments were screened using a Myc-tag antibody.These DNA fragments were located in promoter,exon,intron,andintergenic regions.We found 278 common DNA fragments.ZNF326 overexpression could up-regulate the m RNA level of ERCC1,but down-regulate the ERCC1 m RNA levels of after the knock down of ZNF326.The activity of the ERCC1 luciferase reporter gene was enhanced by transfection of the ZNF326 plasmid.However,the activity of the ERCC1 luciferase reporter gene decreased after ZNF326 knock down.The expression of ERCC1 was positively correlated with the expression of ERCC1 in NSCLC(r = 0.737,p < 0.05).3.ZNF326 regulated the expression of ERCC1 via the(-875bp~-883bp)sequence of the ERCC1 promoter.EMSA showed that(-875bp~-883bp)sequence combined with ZNF326 exhibited a specific migration band.The transcriptional activity of ERCC1 could only be activated by preserving the(-875bp~-883bp)sequence of ZNF326.Luciferase reporter gene experiments showed that the transcriptional activation domain and C2H2 domain were necessary for ZNF326 to activate ERCC1 transcription.4.ZNF326 interacts with ERCC1 to regulate cycle-related protein expression to promote the transition from G2 phase to M phase.In NSCLC cells transfected with the ZNF326 plasmid,Cyclin A2,Cyclin B1,Snail,and Slug were up-regulated,and P21 and E-cadherin were reduced.After the knock down of ZNF326 expression,we obtained the opposite results.ERCC1,Cyclin B1 protein levels down-regulated after transfection with plasmids lacking the transcriptional activation domain of ZNF326 or lacking the C2H2 domain of the ZNF326 plasmid.Cotransfection of ZNF326 and ERCC1 had a stronger effect on the up-regulation of Cyclin B1 than single transfection.When ZNF326 transfection interferes with ERCC1,the effect disappears.A flow cytometric analysis indicated that the overexpression of ZNF326 increased the number of cells entering G2/M phase and decreased the number of G2/M phase cells after ZNF326 knockdown.ERCC1 and ZNF326 both promoted colony formation and strengthened each other(p<0.05).5.ERCC1 promoted Cyclin B1 protein synthesis via Chk1.ZNF326 knockdown significantly inhibited the protein levels of Chk1 and Cyclin B1;transfection of ERCC1 had a synergistic effect on ZNF326 activation of Chk1 and Cyclin B1.Transfection of ZNF326 and simultaneous interference of ERCC1 abolished the synergism.Based on the overexpression of ZNF326,we transfected the sh Chk1 or sh NC and found that the role of ZNF326 in promoting Cyclin B1 protein synthesis was significantly weakened.Conclusion:ZNF326 plays a role as a tumor promoter in NSCLC,and its high expression is correlated with low differentiation and a high TNM stage.The C2H2 structure of ZNF326 can combine with the(-875bp~-883bp)sequence in the ERCC1 promoter region to promote ERCC1 transcription and translation,upregulate Chk1-Cyclin B1,and induced the progression of NSCL cell cycle,promoting the proliferation of NSCLC cells. |
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