Effects Of ABCC1 On Malignant Transformation Of Non-small Cell Lung Cancer And Its Genetic Variation Mechanism Of Uncontrolled Expression

Object:To study the effect of ABCC1 on the malignant transformation and metastasis ability of non-small cell lung cancer,and to preliminary explore the mechanism by which genetic variation in the regulatory region of ABCC1 promotes its expression disorder.Methods:1.q RT-PCR and Western blot were used to detect the expression of ABCC1 m RNA and protein in non-small cell lung cancer tissues and adjacent tissues,respectively.2.In human normal lung epithelial cells（BEAS-2B）,the effect of ABCC1 on the proliferation of BEAS-2B cells was detected by lentiviral pathway overexpression of ABCC1,CCK8 experiments and flow cytometry,and Transwell detected the invasion of BEAC-2B cells by ABCC1 The effect of ABCC1 on the migration ability of BEAS-2B cells was detected by streak experiments.3.In non-small cell lung cancer A549 cells,the lentiviral pathway was used to silence and overexpress ABCC1.Transwell and streak experiments were used to detect the effects of ABCC1 on the invasion and migration ability of A549 cells,respectively.4.Chip experiments verify whether Sp1 can bind to the ABCC1 promoter,and through luciferase experiments,preliminary verification of the genetic variation of the ABCC1 promoter region（SNP rs562009107 A> G）on its binding to transcription factor Sp1.5.Collect blood samples from patients with non-small cell lung cancer and extract genomic DNA from the samples.Using DNA as a template,the sequence including the SNP rs562009107 site was amplified,cloned into the T vector,and sequenced to analyze the rs562009107 A to G gene mutation content and mutation rate.Results:1.The expression of ABCC1 m RNA and protein in non-small cell lung cancer tissues is significantly higher than in adjacent tissues,and the expression of ABCC1 m RNA and protein in lung cancer tissues with metastasis is higher than in adjacent tissues2.Infect the BEAS-2B cells by the lentiviral pathway（Lv-ABCC1）,compared with the control group.The expression of ABCC1 m RNA and protein in Lv-ABCC1 infected cells was significantly enhanced,indicating that the ABCC1 high-expressing BEAS-2B cell line was successfully established;the cell proliferation activity of the Lv-ABCC1 infection group was significantly enhanced（P = 0.002,vs Lv-Control infection group,72h）Flow cytometry analysis showed that the proportion of cells entering the S phase in the Lv-ABCC1 infection group increased significantly（P = 0.004,vs.Lv-Control infection group,72h）;invasion experiments showed that the cell invasion activity of the Lv-ABCC1 infection group was significantly enhanced（P = 0.002,vs.Lv-Control infection group,72h）;Streak test showed that the migration capacity of BEAS-2B cells in the Lv-ABCC1 infection group was significantly enhanced,and the migration trend of each group was consistent with the invasion.3.In non-small cell lung cancer A549 cells,the expression of ABCC1 protein in the silenced and over-expressed groups of ABCC1 established by the lentiviral pathway was0.12 and 4.21 times that of the control group,respectively,indicating that ABCC1 highand low-expressing cell lines were successfully established;ABCC1 gene silencing can significantly inhibit the invasion and migration capabilities of A549 cells,while ABCC1 overexpression can significantly enhance tumor cell invasion and migration capabilities.4.Chip experiments confirmed that there is a Sp1 transcription factor binding site on the ABCC1 promoter.pc DH-Sp1 transfection significantly up-regulated luciferase activity in 293 cells after transfection of p GL3-mt（Pro）-ABCC1（P <0.01,vs p GL3-wt（Pro）-ABCC1）,indicating genetic variation in the ABCC1 promoter region（SNP rs562009107A> G）enhanced its Sp1 binding.5.Of the 26 samples tested,the "A> G" mutation in rs562009107 reached 5 cases,accounting for 19.23%.Conclusions:1.High expression of ABCC1 can promote the malignant transformation of non-small cell lung cancer,and enhance the proliferation,invasion and migration ability of non-small cell lung cancer cells;2.The ABCC1 promoter region can bind to the transcription factor Sp1,and the genetic variation of the ABCC1 promoter region（rs562009107 A> G）can promote the binding of Sp1 to it,thereby promoting the ABCC1 transcriptional activity,which may be the inheritance of uncontrolled ABCC1 expression in non-small cell lung cancer Learning mechanism.