Co-expression Patterns And MiRNA Regulation Of Overlapping Genes CD3EAP And PPP1R13L And Their Effect On Lung Cancer Induced By Polycyclic Aromatic Hydrocarbons

Objective: Lung cancer,as one of the most common malignancies,is the highest common cancer and leading cause of cancer death worldwide.Lung cancer is a serious public health problem with rapid development and high mortality.Looking for reasonable and effective biomarkers and achieving effective prevention,early diagnosis and reasonable treatment are of great significance for improving the survival rate and quality of life of patients.Population epidemiology and tumor etiology studies suggest that the occurrence of lung cancer is the result of a combination of environmental and genetic factors.Polycyclic aromatic hydrocarbons B[a]P,a kind of environmental carcinogenic factor,is widely found in cigarette smoke,engine exhaust and petroleum combustion smoke,and even in agricultural soil.B[a]P,as an indirect carcinogen,rapidly distributes in various tissues after entering body and transforms into ultimate carcinogen BPDE by cytochrome P450 enzyme.BPDE is nucleophilic and can form BPDE-DNA adduct with DNA,which can cause mutations in some important genes.For example,the proto-oncogene activation or antioncogene inactivation becomes the initial period of tumor development.PPP1R13L is an important proto-oncogene,which is the third member of the highly conserved apoptosis-stimulating protein of p53 family.It can inhibit anti-carcinoma of ASPP1 and ASPP2 and of wild-type TP53 activity,thus inhibits cell apoptosis and promotes the cell abnormal proliferation.PPP1R13 L competitively binds Keap1 with the transcription factor Nrf2 through DLT sequence,and lead to decreased ubiquitination of Nrf2 and increased accumulation of Nrf2.The iaspp-keap1-nrf2 axis promotes tumor growth in vitro and in vivo.In addition,PPP1R13 L also inhibits RelA,an important member of the transcription factor NF-?B family.NF-?B involves in multiple pathophysiological processes,including cell proliferation,apoptosis and immune response.NF-?B signaling pathway becomes a research hotspot because of its anti-apoptosis and cancer promotion function.CD3EAP overlaps with PPP1R13 L 5 '.CD3 EAP is RNA polymerase I subunit,involves in the process of cell proliferation and T cell receptors CD3? pathways.CD3 EAP 3 'end overlaps with DNA repair gene ERCC1.ERCC1 protein is an important rate-limiting enzyme in nucleotide excision repair system,which is playing a key role in the repair of BPDE-DNA adduct.In our previous work,we found that knocking down CD3 EAP will down-regulate ERCC1 expression.CD3 EAP may participate in DNA repair pathway by regulating ERCC1.However,it is undefined whether there is a similar correlation between CD3 EAP and PPP1R13 L.In our previous studies,we found that CD3 EAP and PPP1R13 L share a common promoter.Therefore,we concluded that there was a co-expression mode between CD3 EAP and PPP1R13 L.Many SNPs related studies have taken CD3 EAP and PPP1R13 L as a whole to predict the risk and prognosis of tumors,but there is still no experimental evidence to prove the functional correlation between the two genes.Through literature mining,it has been found that CD3 EAP protein can physically bind with NF-?B inhibitory protein NFKBIB.However,its mechanism have not been verified.Therefore,we hypothesized that PPP1R13 L and CD3 EAP jointly regulate the NF-?B pathway.MicroRNA is a kind of highly conserved,endogenous,non-coding RNA.miRNAs inhibits target genes translation process or promotes mRNAs degradation,through specifically binding the 3'UTR on mRNAs.miRNA plays an important role in various pathophysiological processes,especially in the process of tumor development.How does the regulation of miRNA on one of the overlapping genes affect the co-expression of the two overlapping genes? Methods: 1.84 lung cancer surgical cases were collected from the First Affiliated Hospital of China Medical University.The inclusion criteria of cases were definite diagnosis of primary lung cancer based on standard clinical and histological criteria.The Institutional Review Board of China Medical University approved the study and informed consent was obtained from all participants prior to the study.Demographic data were obtained using a questionnaire.The tissue was removed during the operation,and stored in liquid nitrogen for subsequent experiments.The paired paracancer tissue was taken at least 5cm away from the carcinoma.The records included tumor type,tumor size,lymphatic metastasis and TNM stage.RNA and protein were extracted from the tissues of the patients,and real-time PCR and western blot were used to detect the levels of PPP1R13 L and CD3 EAP mRNA and protein level in the cancer and paracancer tissues.The mRNA and protein expression of each gene in cancer and adjacent tissues were compared,and the co-expression patterns of the two genes were analyzed.Polycyclic aromatic hydrocarbon environmental carcinogens induced 16 HBE cells to establish a malignant transformed cell model,and detected the expression levels of PPP1R13 L and CD3 EAP mRNA in different generations of cells during the low-dose treatment of malignant transformation,and verified the correlation between PPP1R13 L and CD3 EAP mRNA levels.2.After knocking down PPP1R13 L or CD3 EAP in A549 cells,CD3 EAP or PPP1R13 L mRNA levels were also down-regulated.Conversely,after overexpression of PPP1R13 L or CD3 EAP in A549 cells,the level of CD3 EAP or PPP1R13 L mRNA in the cells was significantly increased.Co-immunoprecipitation showed that the two proteins PPP1R13 L and CD3 EAP did not form a complex.Knockdown of PPP1R13 L or CD3 EAP in A549 cells causes up-regulation of RelA function.3.Screening of miRNAs regulating CD3 EAP and PPP1R13 L by bioinformatics methods,and verifying the direct regulation of miRNAs on target genes CD3 EAP and PPP1R13 L by dual luciferase reporter gene assay.Real-time PCR was used to detect the expression of miRNA in cancer and adjacent tissues and malignant transformed cells,and the relationship between CD3 EAP and PPP1R13 L mRNA and miRNA expression was verified.After transfection of miRNA mimics in A549 cell line,the changes of target gene CD3 EAP and PPP1R13 L mRNA and protein,and the change of cell RelA function were detected.Results: 1.The expression of CD3 EAP mRNA in tumor tissues was lower than that in adjacent tissues,P<0.05.PPP1R13 L was not significantly different between cancer and adjacent tissues.The stratified analysis showed that PPP1R13 L mRNA levels were associated with smoking and staging.In lung cancer and adjacent tissues,there is a co-expression relationship between PPP1R13 L and CD3 EAP mRNA.There was no significant difference in the expression of PPP1R13 L and CD3 EAP protein between cancer and paracancerous tissues,but there were also co-expression relationships between PPP1R13 L and CD3 EAP protein levels.When low-dose BPDE was used to treat different generations of malignant cells,the correlation between CD3 EAP and PPP1R13 L mRNA levels was the strongest in the 0th generation cells.While it was till related until CD3 EAP and PPP1R13 L in the 20 th and 40 th generation cells mRNA levels are no longer relevant.2.After knocking down PPP1R13 L or CD3 EAP in A549 cells,CD3 EAP or PPP1R13 L mRNA levels were also down-regulated.Conversely,after overexpression of PPP1R13 L or CD3 EAP in A549 cells,the level of CD3 EAP or PPP1R13 L mRNA in the cells was significantly increased.Co-immunoprecipitation showed that the two proteins PPP1R13 L and CD3 EAP did not form a complex.Knockdown of PPP1R13 L or CD3 EAP in A549 cells causes up-regulation of RelA function.3.miR-182-5p can directly down-regulate the expression of PPP1R13 L,while miR-125a-3p can directly down-regulate the expression of CD3 EAP.The miR-125a-3p mimetic can reduce the expression of CD3 EAP and PPP1R13 L,thereby increasing the function of the cell RelA.Conclusion: 1.Molecular patterns of co-expression were found in the mRNA and protein levels of the inverted overlapping genes CD3 EAP and PPP1R13 L,which are co-located at 19q3.3.Co-expression of CD3 EAP and PPP1R13 L mRNA levels in tumor tissues is easily disrupted.2.In vitro transfection experiments,CD3 EAP and PPP1R13 L mRNA and protein levels change with the same chage,may be through the formation of mRNA-mRNA complexes to protect each other from degradation.However,the two proteins CD3 EAP and PPP1R13 L do not interact by forming a complex.3.The NF-?B family is the cooperative pathway of CD3 EAP and PPP1R13 L.CD3EAP and PPP1R13 L have inhibitory effects on the nuclear translocation ability of RelA protein.4.miR-125a-3p mimetic can directly reduce the expression of PPP1R13 L by directly reducing CD3 EAP mRNA level,thereby enhancing the function of cell RelA.