| Background and Objective Air pollution is the largest environmental cause of disease and premature death in the world today.PM2.5 is particulate matter with aerodynamic diameter ≤2.5μm,which is the main component of air pollution.The composition of PM2.5 is relatively complex,mainly from human activities and natural environment.Its main toxic components include organic pollutants such as polycyclic aromatic hydrocarbons,heavy metal particulate matter and some biological pollutants.PM2.5 has been recognized as a key public health factor such as lung injury,cognitive impairment,and cardiac dysfunction.Lung cancer is one of the cancers with the highest morbidity and mortality in the world.It is a major public health problem and a huge social burden in my country.Lung cancer is a multistage,multifactorial disease with multiple histological subtypes and its causative factors,including smoking,air pollution,occupational exposure,and chronic lung disease.Therefore,research on the occurrence and development mechanism of lung cancer,screening of molecular targets for early diagnosis of lung cancer,and exploration of factors affecting the progression of lung cancer have become the focus and focus of research in recent years.However,the impact of air pollution on the progression of lung cancer and the related molecular mechanisms are still difficult points in the field of lung cancer prevention and treatment.Noncoding RNAs are RNA molecules that are transcribed from the genome but do not encode proteins.Including r RNA,t RNA and common Lnc RNA（long non-coding RNAs,lnc RNAs）,circ RNA（circular RNA）.Circ RNA is a class of nucleic acid transcripts with a closed-loop structure,without 5’ cap structure and 3’ poly（A）structure.Although circ RNAs are relatively rare in cells,they play key roles in a wide range of biological functions and influence cellular processes such as proliferation,differentiation,senescence,and maintenance of cellular pluripotency.Alterations of certain circ RNAs are associated with disease progression and patient prognosis,especially in lung cancer,and differentially expressed circ RNAs may serve as biomarkers and therapeutic targets for potential clinical interventions.Therefore,it is particularly important to elucidate the molecular mechanism of circ RNAs in lung cancer progression under PM2.5 exposure.RNA-binding proteins（RBPs）are among the most abundant proteins in cells and are often ubiquitously expressed.RBPs play a key role in the post-transcriptional processing of RNA,which can occur in various aspects of m RNA,and thus it plays an important role in different cellular processes.Currently,the roles of a large number of RBPs in a variety of human tumors have been identified,indicating the importance of RBPs in human biology and pathogenesis.Studies have shown that RBPs form RNARBP complexes with specific RNAs,thereby regulating the expression and function of their targets.Therefore,searching for the related RBPs in the progression of lung cancer under PM2.5 exposure and elucidating the RBPs that interact and interact with circ RNAs will help to explore the molecular mechanism of circ RNA/RBP binding affecting the progression of lung cancer under PM2.5 exposure.In this study,by screening the differentially expressed circ RNAs in clinical samples of lung cancer patients,through comparative screening,we obtained and identified the target circ RNAs and RNA-binding proteins specifically expressed in lung cancer cells exposed to PM2.5.Specific target proteins regulated by downstream RBPs.To verify the regulatory role and molecular mechanism of circ RNA/RNA-binding protein in the progression of PM2.5-induced lung cancer.This study re-explored the occurrence and development of lung cancer under PM2.5 exposure from the perspective of non-coding RNA and RBP,which is helpful to clarify the development mechanism of PM2.5-induced lung cancer.The scheme provides theoretical and experimental basis.Methods 1.The CCK8 assay was used to detect the PM2.5 dosage of lung cancer cell lines（H1299,A549,H460）.2.To analyze the circ RNA microarray data of clinical lung cancer,review the literature,and screen out three differentially expressed circ RNAs（circ CDR1 as,circ RPPHI,circ PRKCI）that are related to PM2.5-promoted lung cancer progression.The expression levels of circ CDR1 as,circ RPPHI and circ PRKCI in lung cancer cell lines（H1299,A549,H460）were detected by q RT-PCR experiments.3.Analyze the genomic location of circ CDR1as;design divergent primers and polymeric primers of circ CDR1 as,respectively,and detect the circular structure of circ CDR1 as by agarose electrophoresis;treat lung cancer cell lines with actinomycin D,and detect the circular structure of circ CDR1 as by q RT-PCR experiment structure.4.The specific location of circ CDR1 as in lung cancer cell lines was detected by fluorescence in situ hybridization（FISH）;the expression of circ CDR1 as in H1299 and A549 cells was down-regulated respectively,and the m RNA levels of circ CDR1 as and CDR1 after regulation were detected by q RT-PCR experiment to verify the transfection efficiency.5.The changes of cell activity of lung cancer cell lines after down-regulation of circ CDR1 as under PM2.5 exposure were detected by CCK8 assay;the changes of apoptosis level of lung cancer cell lines after down-regulation of circ CDR1 as under PM2.5 exposure were detected by flow cytometry experiments;Plate cloning assay and EDU assay were used to detect the changes of cell proliferation ability of lung cancer cell lines after down-regulation of circ CDR1 as under PM2.5 exposure;cell scratch assay was used to detect the changes of cell migration level of lung cancer cell lines after down-regulation of circ CDR1 as under PM2.5 exposure;Transwell assay detected the changes of cell invasion level of lung cancer cell lines after down-regulation of circ CDR1 as under PM2.5 exposure.6.The in vivo tumorigenicity changes of the lung cancer cell lines after down-regulation of circ CDR1 as were verified by the subcutaneous tumorigenesis experiment in nude mice in vivo;the lung cancer cell lines after down-regulation of circ CDR1 as were verified in PM2.5.Changes in lung metastases under exposure;hematoxylin-eosin staining（HE）was used to detect the pathological levels of corresponding tissue sections;immunohistochemical experiments were used to detect the expression levels of Caspase-3,E-cadherin and Ki-67 in each tissue section.7.RNA pull down assay was used to detect the protein expression of lung cancer cell lines combined with circ CDR1 as under PM2.5 exposure;mass spectrometry and Western Blot experiments confirmed SRSF1 protein and verified whether there is an interaction between SRSF1 and circ CDR1 as.8.Fluorescence in situ hybridization（FISH）was used to detect the expression intensities of circ CDR1 as and SRSF1 labeled with different probes in lung cancer cell lines,and to confirm whether there was a co-localization relationship between the two.Bioinformatics predicted the protein structure of SRSF1 and performed GO analysis.9.The lung cancer cell lines were treated with PM2.5 for different times,and the SRSF1 m RNA and protein expression levels were detected by q RT-PCR,Western Blot and cellular immunohistochemistry（ICC）experiments;the level of circ CDR1 as in lung cancer cell lines was down-regulated,and PM2.5 The m RNA and protein expression levels of SRSF1 after regulation were detected by q RT-PCR,Western Blot and cellular immunohistochemistry（ICC）experiments.10.Down-regulate the expression of circ CDR1 as in lung cancer cell lines,use MG-132 to act on PM2.5-treated cells,and detect the expression level of SRSF1 protein by Western Blot assay to verify the stability of SRSF1 protein;down-regulate the expression of circ CDR1 as in lung cancer cell lines,Using cycloheximide（CHX）to act on PM2.5-treated cells,Western Blot assay was used to detect the expression level of SRSF1 protein to verify the degradation level of SRSF1 protein.11.Phospho Site Plus bioinformatics predicted potential ubiquitination modification sites of SRSF1 protein;down-regulated the expression of circ CDR1 as in lung cancer cell lines,and detected the ubiquitination level of SRSF1 by co-immunoprecipitation assay（CO-IP）under PM2.5 exposure;CO-IP experiment confirmed the interaction between SRSF1 and PARK2 proteins in lung cancer cell lines;GST pull down experiment confirmed the interaction between SRSF1 and PARK2 proteins in lung cancer cell lines;CO-IP experiment detected whether circ CDR1 as could regulate the interaction between SRSF1 and PARK2 proteins;CO-IP experiments to verify whether PARK2 can regulate the ubiquitination level of K48 site of SRSF1 in lung cancer cell lines.12.Up-regulate and down-regulate the expression of SRSF1 in lung cancer cell lines,respectively.q RT-PCR and Western Blot experiments were used to detect the expression levels of SRSF1 m RNA and protein in lung cancer cell lines after regulation to verify the transfection efficiency.13.q RT-PCR experiments were used to detect the m RNA levels of VEGFAtot,VEGFAIso8 a and VEGFAIso8 b in lung cancer cell lines under PM2.5 exposure,circ CDR1 as and SRSF1 regulation respectively;agarose gel electrophoresis experiments were used to detect lung cancer cell lines in PM2.5 exposure,circ CDR1 as and the m RNA levels of VEGFAtot,VEGFAIso8 a and VEGFAIso8 b under the regulation of SRSF1 were tested to verify the effects of PM2.5 exposure,circ CDR1 as and SRSF1 on the expression of VEGFA.14.Co-regulate the expression of circ CDR1 as and SRSF1 in lung cancer cell lines,and detect the changes of cell viability under PM2.5 exposure by CCK8 assay;plate cloning assay,EDU assay to detect changes in cell proliferation ability under PM2.5 exposure;cell scratches The changes in the level of cell migration under PM2.5 exposure were detected by the experiment;the changes in the level of cell invasion under PM2.5 exposure were detected by transwell experiment.15.Down-regulate the expression of circ CDR1 as in lung cancer cell lines,and q RTPCR,Western Blot and cellular immunofluorescence（IF）experiments were used to verify whether the wnt/β-catenin signaling pathway was activated.Whether the key signaling molecules of β-catenin signaling pathway can be regulated by circ CDR1as/SRSF1.Results 1.According to the CCK8 test results,combined with the IC50 value,the PM2.5 exposure doses of different lung cancer cells were determined as: H1299: 300μg/m L;A549: 200μg/m L;H460: 300μg/m L.According to the data of GEO lung cancer circ RNA chip（GSE158695 and GSE152434）and the literature,the circ RNAs（circ CDR1 as,circ RPPHI,circ PRKCI）that may be related to PM2.5-induced lung cancer progression were screened.According to the results of q RT-PCR experiments,the expression of circ CDR1 as was stably and significantly up-regulated in H460,A549 and H1299 cells exposed to PM2.5.According to the results of agarose electrophoresis experiments,the polymer primers can amplify bands in both c DNA and g DNA,but the divergent primers can only amplify bands in c DNA.After treatment with actinomycin D,the expression of circ CDR1 as in lung cancer cell lines did not change significantly,while the level of CDR1 m RNA gradually decreased.Fluorescence in situ hybridization experiments confirmed that circ CDR1 as was mainly distributed in the cytoplasm and a small amount in the nucleus of lung cancer cells.The above results suggest that the abnormal expression of circ CDR1 as may be closely related to PM2.5 exposure-induced lung cancer progression.2.Circ CDR1 as can effectively regulate the malignant behavior of lung cancer cell lines exposed to PM2.5.The results of q RT-PCR experiments confirmed that both circ CDR1 as si RNA1 and si RNA2 could significantly down-regulate the expression of circ CDR1 as without affecting the expression level of CDR1 m RNA.CCK8 experiments confirmed that the survival rate of lung cancer cell lines was significantly reduced after PM2.5 exposure and down-regulation of circ CDR1as;flow cytometry experiments verified that under PM2.5 exposure,after down-regulation of circ CDR1 as,the apoptosis level of lung cancer cell lines was significantly increased;Cloning experiments and EDU experiments confirmed that the proliferation ability of lung cancer cell lines was significantly reduced after down-regulation of circ CDR1 as under PM2.5 exposure;cell scratch experiments showed that under PM2.5 exposure,after down-regulation of circ CDR1 as,the migration ability of lung cancer cell lines was significantly reduced;Transwell experiments showed that under PM2.5 exposure,the invasive ability of lung cancer cell lines was significantly reduced after downregulation of circ CDR1 as.Subcutaneous tumorigenesis experiments in nude mice showed that under PM2.5 exposure,down-regulation of circ CDR1 as inhibited the proliferation of lung cancer cells in vivo;the results of hematoxylin-eosin staining showed that under PM2.5 exposure,down-regulation of circ CDR1 as could effectively alleviate the malignancy of subcutaneous tumor tissues The results of hematoxylineosin staining in the lung metastasis experiment of nude mice showed that downregulation of circ CDR1 as inhibited the metastasis of lung cancer cells in lung tissue;immunohistochemical experiments of tissue sections showed that after exposure to PM2.5,Caspase-3 and E-cadherin The expression level of circ CDR1 as was significantly decreased,and the expression level of Ki-67 was significantly increased;after downregulation of circ CDR1 as,the expression levels of Caspase-3 and E-cadherin were increased,and the expression level of Ki-67 was significantly decreased.These results suggest that down-regulation of circ CDR1 as levels effectively inhibits the progression of malignant behavior of lung cancer cells in vitro and in vivo under PM2.5 exposure.3.To further explore the interaction between circ RNA and protein,RNA pull down experiment combined with mass spectrometry analysis confirmed the interaction between circ CDR1 as and SRSF1 protein.SRSF1 protein interaction relationship;fluorescence in situ hybridization（FISH）found that circ CDR1 as and SRSF1 protein had obvious co-localization relationship in the cytoplasm.q RT-PCR experiments showed that the expression level of SRSF1 m RNA in lung cancer cell lines significantly increased over time under PM2.5 exposure.It also increased significantly under exposure.After down-regulation of circ CDR1 as in lung cancer cell lines,both m RNA and protein levels of SRSF1 were significantly decreased under PM2.5 exposure.The above results suggest that there is an interaction between circ CDR1 as and SRSF1 proteins in lung cancer cell lines under PM2.5 exposure.4.Next,explore the regulatory mechanism between circ CDR1 as and SRSF1.Lung cancer cell lines were treated with MG-132,and Western Blot experiments confirmed that SRSF1 protein stability was significantly reduced after down-regulating the expression of circ CDR1 as under PM2.5 exposure,while pretreatment with MG-132 could reduce the degradation level of SRSF1 protein,suggesting that circ CDR1 as may play a regulatory role by inhibiting the degradation of SRSF1 under PM2.5 exposure.At the same time,the treatment of lung cancer cells with cycloheximide（CHX）found that after down-regulating the expression of circ CDR1 as,the half-life of SRSF1 protein was significantly reduced compared with the control group.It is suggested that circ CDR1 as may function by increasing the half-life of SRSF1.Phospho Site Plus bioinformatics prediction results showed that there are more ubiquitination sites in SRSF1 protein.The co-immunoprecipitation assay（CO-IP）confirmed that the ubiquitination modification level of SRSF1 protein was significantly increased after down-regulating the expression of circ CDR1 as in lung cancer cell lines under PM2.5 exposure.At the same time,COIP and GST pull down experiment confirmed that SRSF1 and E3 ubiquitin ligase PARK2 protein were co-expressed,and there was an interaction relationship.After down-regulating the expression of circ CDR1 as in lung cancer cell lines,the expression level of PARK2 was increased,suggesting that the ubiquitination modification of SRSF1 protein may be up-regulated.CO-IP assay confirmed that PARK2 could regulate the level of ubiquitination at the K48 locus of SRSF1 in lung cancer cell lines.The above results suggest that circ CDR1 as may participate in the regulation of SRSF1 protein by affecting the stability and degradation level of SRSF1 protein in lung cancer cell lines exposed to PM2.5.5.To further clarify the regulatory effects of circ CDR1 as and SRSF1 proteins on vascular endothelial growth factor A（VEGFA）alternative splicing in lung cancer cell lines under PM2.5 exposure.According to the results of q RT-PCR experiments,the expression levels of circ CDR1 as,VEGFA tot and SRSF1 all increased in lung cancer cell lines under the exposure of PM2.5;for the two types of VEGFA,the proportion of VEGFA Iso8 a was significant increased,while the proportion of VEGFA Iso8 b decreased.After up-regulating the expression level of SRSF1,the expression level of VEGFA Iso8 a in lung cancer cells was increased,and the expression level of VEGFA Iso8b was significantly decreased.On the contrary,after down-regulating the expression level of SRSF1,the expression level of VEGFA Iso8 a in lung cancer cells decreased,and the expression level of VEGFA Iso8 b was significantly increased.At the same time,after down-regulating the expression level of circ CDR1 as,the expression level of VEGFA Iso8 a in lung cancer cells decreased,and the expression level of VEGFA Iso8 b was significantly increased.The above results suggest that circ CDR1 as and SRSF1 proteins jointly regulate the alternative splicing level of VEGFA in lung cancer cells under PM2.5 exposure.6.CCK8 experiments confirmed that under PM2.5 exposure,after down-regulation of circ CDR1 as,up-regulation of SRSF1 increased the survival rate of lung cancer cell lines,while down-regulation of SRSF1 decreased the survival rate of lung cancer cell lines;plate cloning experiments,EDU experiments confirmed that under PM2.5 exposure,After down-regulation of circ CDR1 as,up-regulation of SRSF1 increased the proliferation ability of lung cancer cell lines,while down-regulation of SRSF1 decreased the proliferation ability of lung cancer cell lines;cell scratch experiments showed that under PM2.5 exposure,after down-regulation of circ CDR1 as,up-regulation of SRSF1 increased the migration ability of lung cancer cell lines.Down-regulation of SRSF1 reduced the migration ability of lung cancer cell lines;Transwell experiments showed that under PM2.5 exposure,after down-regulation of circ CDR1 as,up-regulation of SRSF1 increased the invasive ability of lung cancer cell lines,while down-regulation of SRSF1 decreased the invasive ability of lung cancer cell lines.The above results suggest that co-regulation of circ CDR1 as and SRSF1 in lung cancer cell lines significantly affects the malignant ability of lung cancer cell lines under PM2.5 exposure.7.The circ CDR1as/SRSF1 axis activates the wnt/β-catenin signaling pathway in lung cancer cell lines under the effect of PM2.5 exposure.Under PM2.5 exposure,the expression of circ CDR1 as in lung cancer cell lines was down-regulated,and q RT-PCR,Western Blot and cellular immunofluorescence（IF）experiments confirmed that the expression of molecules related to the wnt/β-catenin signaling pathway was significantly reduced;After the expression of circ CDR1 as and SRSF1 in lung cancer cell lines,it was confirmed that the key signaling molecules of the wnt/β-catenin signaling pathway can be affected by the circ CDR1as/SRSF1 axis.The above results suggest that the wnt/β-catenin signaling pathway mediated and activated by the circ CDR1as/SRSF1 axis may be involved in PM2.5-induced lung cancer progression.Conclusions 1.Circ CDR1 as is aberrant expressed in lung cancer cell lines exposed to PM2.5;2.Abnormal expression of circ CDR1 as could affect the malignant behavior of lung cancer cell lines exposed to PM2.5 in vitro and in vivo;3.After exposure to PM2.5,circ CDR1 as interacts with SRSF1 protein to regulate downstream molecular mechanisms,forming a circ CDR1as/SRSF1 regulatory axis;4.Under PM2.5 exposure,circ CDR1 as regulates the stability of SRSF1 protein by affecting the protein degradation and ubiquitination level of SRSF1;5.The circ CDR1as/SRSF1 regulatory axis affects the alternative splicing of VEGFA under PM2.5 exposure;6.The circ CDR1as/SRSF1 regulatory axis abnormally activates the wnt/β-catenin signaling pathway and affects the malignant behavior of lung cancer cell lines exposed to PM2.5;... |
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