Characteristic Analysis And Mechanism Of PPP1R13L Alternative Splicing In Malignant Transformation Of Lung Cancer Induced Bypolyaromatic Hydrocarbon

Objective:In recent years,lung cancer,with its rising morbidity and mortality,has rapidly become one of the malignant diseases to threaten human health worldwide.Therefore,it is important to find effective genetic biology markers.The alternative splicing of pre-mRNA is an important mechanism of gene expression and protein diversity,and is also a natural source of carcinogenesis.Many oncogenes are regulated byalternative splicing.One or some alternative splicing may play a key role in tumorigenesis and development,and become the specific mode of cancers.PPP1R13L,as new oncogene,has two common alternative splicing PPP1R13L-L and PPP1R13L-SV,which may play different biological functions in the occurrence and development of lung cancer caused by environmental carcinogens,presenting a certain type of lung cancer specific alternative splicing.In this study,lung cancer-related alternative splicingof PPP1R13L gene was selected and detected by bioinformatics and RACE experiment.Human lung cancer tissues and adjacent samples,cell models of BPDE induced malignant transformation and in vitro transfection were used to further explore the functional mechanism and characteristics of candidatealternative splicingof PPP1R13Lin the malignant transformationof lung cancer.In our study,it provides an important theoretical basis for the research on the specific alternative splicing mode of lung cancer and new ideas and targets of the prevention and diagnosis of tumors.Methods:In this study,we recruited 100 patients suffering from lung cancer from the First Affiliated Hospital of China Medical University and Liaoning Cancer Hospital and Institute.The cases were diagnosed from 2013 to 2018 by the local authorized diagnosing pathologists.Informed consent was obtained from each participant after a detailed explanation of the possible consequences of this study.In addition to the previous medical history,family history,lifestyle and other general demographic characteristics,the pathological characteristics of lung cancer such as tumorsizes,stages,pathological types and lymph node metastasis should be also recorded in detail and based on patient consultation and pathological report results.Firstly,according to the comprehensive analysis results of Affymetrix gene chips and bioinformatics software（UCSC,etc.）,PPP1R13L alternative splicing that may be related to the occurrence and development of lung cancer were predicted and screened.And candidate alternative splicing was conducted to verify by RACE test.mRNA expression levels of candidate alternative splicing and P53 gene were detected using realtime PCR.Protein expression of PPP1R13L transcripts was observed by Western blot.The correlation betweencandidate alternative splicing and clinical features is analyzed through realtime PCR and Western blot results.Secondly,16HBE was induced by low-dose BPDE in vitro to establish a cell malignant transformational model.The changes of malignant degree between differentpassage cells were observed through cell scratch,transwell chamber invasion experiment in vitro,soft AGAR cloning experiment and tumor formation in nude mice.HE staining and immunohistochemical analysis of the tumorigenic tissues were performed to identify the pathological types of the malignant transformed cells.And P53genotype was also analyzed by gene sequencing.During the process of malignant transformation,real-time PCR and Western blot were used to detect the changes in mRNA levels and protein expressions of twocandidate alternative splicings of PPP1R13L.Finally,two alternative splicingsof PPP1R13L gene overexpression plasmid or siRNA were designed and transfected into 293T and 16HBE cells respectively.Meanwhile,endogenous down-regulated expression cells of PPP1R13L-L were obtained by precise knockout of CRISPR-Cas9 system.After environmental carcinogen BPDE treatment,apoptosis of transfected cells in each group was dtected by flow cytometry,and the changes of nuclear expression of P53 was dtected byimmunofluorescence assay in transfected cells before and after BPDEexposing.Results:1.According to gene chip technology and UCSC database screening,RACE test confirmed that PPP1R13L-L and PPP1R13L-SV transcripts were expressed in both human populations and cell lines and may be associated with lung cancer.Excluding samples with incomplete patient information and poor quality of tissues,the final sample size was determined to be 98 cases.Among 98 patients with lung cancer,the mRNA expression level of PPP1R13L-SV in the cancer tissues was significantly higher than that in the paracancerous tissues（P<0.05）,while it was significantly increased in theparacancerous tissues ofP53 gene（P<0.05）.In the stratified analysis of clinicopathological features,it was found that the mRNA expression level of PPP1R13L-SV was significantly increased in patients older than60 years old（including 60 years old）,but the mRNA expression level of P53 gene was significantly decreased in cancer tissues（P<0.05）.In smoking patients,the mRNA expression level ofPPP1R13L-SVwas significant increasing than in the adjacent tissues（P<0.05）.In the patients of squamous cell carcinoma（LUSC）,mRNA expression level of PPP1R13L-SVwas increased,while it was decreased in P53 gene expression（P<0.05）,and the same trend was found in protein detection with comparative analysis.When tumor diameter≥3cm,the mRNA expression level of PPP1R13L-SV was significantly higher in LUSC（P<0.05）,but lower inP53 gene expression（P<0.05）;in cases with lymph node metastasis,PPP1R13L-SVwas high expression（P=0.054,bound value,considered statistically significant）;in the high malignant tumor staging group（Ⅲ/Ⅳstaging）,PPP1R13L-SV has high level expression（P<0.05）,while P53 gene was low level expression（P<0.05）.The mRNA or protein expression level of PPP1R13L-SVwas negatively correlated with that of P53 gene expression（P<0.05）.In carcinoma,para-carcinoma and normal tissues of 15patients with lung cancer,mRNA expression level of PPP1R13L-SVwas showed a downward trend（F=11.183,P<0.05）,but there was no significant difference in the expression of protein between para-carcinoma and normal tissues.2.The malignant transformation of 16HBE was induced by 1 M BPDE in vitro.With increasing algebra of malignant transformational cells,nuclear to plasma ratio was gradually increased from scattered growth to clustered growth state,and the cell scratch area ratio was gradually decreased（P<0.05）.Cell abilitiesof invasion and malignant proliferation were continuously enhanced,and theabilities of 16HBE-40T were almost the same as that of LK₂.In tumorigenic test of nude mice,tumorigenic histopathological sections was identified by HE staining and immunohistochemistry,and P53 exon5-8sequencing analysis of 16HBE-40T cells was performed.Their results showedlung squamous cell carcinoma with wild type of P53.In the process of cell malignant transformation,mRNA expression levels of PPP1R13L-L and PPP1R13L-SV showed a wave-like increase,while the levels of P53 showed a zigzag decline.In protein detection,the expression levels of PPP1R13L-SV and P53 showed the same trend as those of mRNA,while PPP1R13L-L protein were inconsistent with its mRNA expression.3.The overexpressed plasmid and siRNA of PPP1R13L-L and PPP1R13L-SV were designed and transfected into 293T and 16HBE cells in vitro,respectively.Green fluorescence was observed under a fluorescence microscope after48h.mRNA and protein expression levels of PPP1R13L-L or PPP1R13L-SV in the transfected cells were both higher than those in the cells transfected with EV plasmid.The cells transfected with siRNA showed no fluorescence expression,and the transfection efficiency was determined only by the changes of mRNA and protein expression levels.When theexpression levels of PPP1R13L-L or PPP1R13L-SV in the siRNA respective transfected cells were lower than those in the NC control group,it proved that cell transfection model wasconstructed successfully.After 24 hours of BPDE exposure,the apoptosis rate of each group increased.Compared with the NC group,the apoptosis of 293T and 16HBE cells transfected with siRNA was increased in the si PPP1R13L-L and siPPP1R13L-SV groups（P<0.05）,but after BPDE exposing,only siPPP1R13L-SV group was significant increase（P<0.01）.In 293T cells transfected with overexpressed plasmids,no significant changes of apoptosis were observed in OE PPP1R13L-Land OE PPP1R13L-SV groups when compared with EV group.However,after 24 hours of BPDE exposure,apoptosis in OE PPP1R13L-SV group was significantly reduced（P<0.05）.Apoptosis in 16HBE cells was basically consistent with that in 293T cells.Similarly,after 24 hours of BPDE exposure,we found that the fluorescence intensity in the nucleus of each group showed an increased trend.Compared with NC group,the nuclear fluorescence intensity in siPPP1R13L-L and siPPP1R13L-SVgroups was increased significantly（P<0.05）,but after BPDE exposing,it was increased only in the si PPP1R13L-SV group significantly（P<0.05）.The nuclear fluorescence intensity of the OE PPP1R13L-SV group was significantly lower than that in the EV group（P<0.05）.The results was basically consistent in the transfected 16HBE cells.Meanwhile,endogenous down-regulated expression cells of PPP1R13L-L was obtained by precise knockout with CRISPR-Cas9 system.Green fluorescence and proteinexpression wereboth confirmed the success of transfection,which were observed by fluorescence microscopes and western blot respectively.After 24 hours of BPDE treatment,the apoptosis rate of each group increased.There was no significant difference between the 16HBE Cas+kdL3 and the 16HBE-40T Cas+kdL3 group when the cells without BPDE.However,after BPDE treatment,apoptosis ratio in 16HBE Cas+kdL3 group was significantly higher than that in 16HBE-40TCas+kdL3 group（P<0.05）.No significant difference was observedbetween 16HBE-40T Cas+EV group and 16HBE-40T Cas+kdL3 group.The changes of fluorescence intensity in immunofluorescence experiments were were consistented with the results of apoptosis.Conclusion:1.In the research on human samples of lung cancer tissue,it implied that PPP1R13L-SV may play acrucialrole in the occurrence and development ofLUSC by affecting the expression of P53 serving as an effective biomarker to predict the malignant transformation of LUSC.2.In malignant transformed 16HBE cells,according to gene sequencing and pathological analysis,16HBE-40T was identified as lung squamous cell carcinoma cells with P53 wild-type,and PPP1R13L-SV was the specific alternative splicing during malignant transformation of 16HBE cells induced by BPDE.3.High expression of PPP1R13L-SV can inhibit pro-apoptotic function from P53regulating,and promote malignant transformation ofcells.PPP1R13L-SV acts as a specific alternative splicing mode of PPP1R13L gene in the malignant transformation of LUSC.