| Objective:In recent years,lung cancer has become one of the malignant tumors with the highest morbidity and mortality in the world,seriously endangering people’s health.Therefore,finding effective biological markers in the occurrence and development of lung cancer,realizing accurate early prevention and reasonable treatment are of great significance for reducing the incidence and mortality of lung cancer.Studies have shown that smoking is the main risk factor of lung cancer Cigarette contains many carcinogens,among which nicotine has been shown to be closely related to lung cancer.Nicotine as the main alkaloid in tobacco plants,although it cannot cause cancer in humans,which can participate in multiple biological processes such as cell proliferation,apoptosis inhibition,cell migration and angiogenesis,increasing tumor migration and invasion capabilities,thereby affecting the epithelial mesenchymal transition(EMT)of many tumors including NSCLC.This study used the method of combining lung cancer clinical case studies with in vitro cell experiments to explore whether the PPP1R13L splicing isomers and negative feedback loop of PPP1R13L-SP1 can play a certain regulatory role in the EMT process caused by nicotine,thus exerting a vital influence on the metastasis and invasion of lung cancer cells and other biological processes.Methods:1.Bioinformatics analysis(1)The expression levels of PPP1R13L and SP1 genes in NSCLC and their correlation with clinicopathological features were analyzed by Xena and TCGA databases;(2)The expression levels of PPP1R13L-L and PPP1R13L-SV genes in NSCLC were analyzed by TSV db database.(3)The SP1 binding sites that regulate the expression of PPP1R13L-L and PPP1R13L-SV genes were predicted by UCSC and TRANSFAC databases.2.Clinical case study of lung cancer(1)After signing the informed consent,50 cases of lung cancer tumor tissues were collected during surgery,and the mRNA expression levels of PPP1R13L-L,PPP1R13L-SV and SP1 were detected by qRT-PCR.3.In vitro cell experiment research(1)Human lung adenocarcinoma cell line A549 and squamous cell carcinoma cell line LK2 were treated with different concentrations of nicotine for 24 hours to detect cell migration and invasion ability,and detect PPP1R13L-L,PPP1R13L-SV,SP1 and EMT related indicators mRNA and protein expression,analyze the expression regulation mode of PPP1R13L-L,PPP1R13L-SV and SP1 when EMT occurs in lung cancer cells;COIP assay was used to analyze whether SP1 protein could bind to PPP1R13L-L and PPP1R13L-SV proteins;Chip was used to analyze whether SP1 as a transcription factor could regulate the expression of PPP1R13L-L gene.(2)A549 cells were used to construct a si-SP1 transfected cell model to analyze the effects on cell migration and invasion ability,as well as the effects on the mRNA and protein expression of PPP1R13L-L,PPP1R13L-SV,SP1 and EMT related indicators.(3)A549 cells were used to construct si-PPP1R13L-L and si-PPP1R13L-SV transfected cell models to analyze the effects on cell migration and invasion ability,as well as the mRNA and protein expression of PPP1R13L-L,PPP1R13L-SV,SP1 and EMT related indexes.Results:1.In NSCLC,the expression of PPP1R13L gene(PPP1R13L,PPP1R13L-L and PPP1R13L-SV)was different in carcinomas and paracancerous tissues,and the correlation between the expression and pathological characteristics was found to be significantly correlated with smoking and tumor stage of lung cancer patients(P<0.05);The expression of SP1 and PPP1R13L were both low in paracancerous tissues and high in cancer,showing a positive correlation;PPP1R13L-L and PPPIR13L-SV have SP1 binding sites near the transcription start sites.2.The results of lung cancer case study showed that PPP1R13L-L was significantly higher in men,lung cancer patients with smoking history,LUSC or≥3 cm tumor(P<0.05).PPP1R13L-SV in male,history of smoking or tumor III/IV crowd significantly high expression(P<0.05);SP1 mRNA expression level was decreased in LUSC(P<0.05).The mRNA of SP1 was positively correlated with PPP1R13L-L and PPP1R13L-SV(P<0.05).3.Compared with the control group,the migration ability of A549 and LK2 cells treated with 0.1～10 μM nicotine was significantly improved(P<0.05);1 μM nicotine treatment of cells for 24 h obviously improve the invasion ability(P<0.05);in A549 and LK2 cells,the protein expression levels of PPP1R13L-L and PPP1R13L-SV increased in the nicotine-treated groups,the mRNA and protein expression levels of N-cadherin and β-catenin increased,while the mRNA and protein expression levels of SP1 and E-cadherin decreased(P<0.05);it was found that compared with the control group,the nicotine-treated group had obvious nuclear metastasis when cells were treated with 1 μM nicotine.Sp1 protein could bind to PPP1R13L-L and PPP1R13L-SV proteins.As a transcription factor,SP1 can regulate the gene transcription of PPP1R13L-L.4.When the expression of SP1 in A549 cells was reduced,the migration and invasion ability of the cells are apparently enhanced(P<0.05);the mRNA and protein expression of PPP1R13L-L,PPP1R13L-SV and E-cadherin are reduced(P<0.05),N-cadherin and β-catenin were improved(P<0.05);β-catenin protein has obvious nuclear transfer.5.In A549 cells,compared with the empty plasmid group,cell migration and invasion were significantly reduced in the si-PPP1R13L-L and si-PPP1R13L-SV groups(P<0.05);the mRNA and protein expression of SP1 and E-cadherin increased(P<0.05),while N-cadherin and β-catenin decreased(P<0.05);there was no nuclear transfer occurred in β-catenin protein.Conclution:1.The lung cancer cell lines(A549,LK2)were treated with nicotine,and it was found that the high expression of PPP1R13L-L and PPP1R13L-SV was closely related to the occurrence of EMT in lung cancer cells.2.PPP1R13L-L and PPP1R13L-SV alternative splicing isomers could regulate the expression of SP1 and indirectly affect the EMT process of lung cancer cells.PPP1R13L may be an effective biomarker for predicting metastasis of smoking lung cancer.3.SP1 acted as a transcription factor to promote gene transcription of PPP1R13L-L,which could form a negative feedback loop with PPP1R13L-L and played an important regulatory role in the EMT process of lung cancer cells. |
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