| Backgrand:Lung cancer is the most common malignant tumor,which is seriously threatening to human health.It accounts for about 17% of all new cases in malignant tumors and about 23% of tumor-related deaths worldwide.At present,the 5-year survival rate is less than 10% in advanced lung cancer patients,so how to improve the long-term survival rate is the focus of clinical and mechanism research of tumors.The development of tumor cells,in the final analysis,results from the disorder of gene expression,especially the disorder expression related to apoptosis and proliferation.RNA splicing is a process in which genetic information from DNA forms m RNA in eukaryotic cells.In this process,introns are removed,and exons are connected,and m RNA molecules with continuous coding function are formed.Finally,two or more exon combinational subtypes can be formed.RNA splicing is an important mechanism in regulatting gene expression and forming proteome diversity.The splicing process of m RNA precursors is the most critical step in the expression of most genes.If the process of splicing cannot be carried out normally due to various reasons,and abnormal splicing is usually closely related to the occurrence and development of tumors.SRSF1 is one of the representative splicing members in selective family.Its main physiological functions include m RNA transport and processing,RNA precursor splicing,cell cycle and apoptosis regulation.However,the mechanism of SRSF1 in lung cancer remains unclear.In previous studies,we found that the apoptosis-related protein Anxa5 was low expressed in lung cancer,and the high expression of Anxa5 could inhibit the proliferation,migration and invasion of lung cancer cells,and affect the expression of key proteins in the EMT process.The protein expression profiles of the high-expressing Anxa5 cell line and the no-load cell line were analyzed by dimethyl stable isotope labeling and mass spectrometry,and the differences in SRSF1 expression were found in the two cell lines,which were verified by western blotting.In previous studies,we found that the apoptosisrelated protein Anxa5 was low expressed in lung cancer,and the high expression of Anxa5 could inhibit the proliferation,migration and invasion of lung cancer cells,and affect the expression of key proteins in the EMT process.The protein expression profiles of the high-expressing Anxa5 cell line and the no-load cell line were analyzed by dimethyl stable isotope labeling and mass spectrometry,and the differences in SRSF1 expression were found in the two cell lines,which were verified by western blotting.So what role does SRSF1 play in lung cancer? Is it also associated with apoptosis? Does it also play a role in the migration and invasion of lung cancer cells? What’s the mechanism then? We did some research on that.Objective:1.To determine the relationship between SRSF1 and the radiosensitivity,we detected the effect of SRSF1 gene expression on the radiation injury in lung cancer cells.2.To lay a foundation for further research,we analyze the splicing events of SRSF1 gene in lung cancer cells after radiotherapy.3.In order to provide new directions and new targets for future treatment of lung cancer,we explore the molecular mechanism of SRSF1 involvement in DNA damage and repair after radiotherapy for lung cancer.Methods:1.Bioinformatics analysis(1)Genes related to radiotherapy sensitivity were screened out from the CNKI gene database,and genes with FPKM value of >3 were selected.It was found that the anxa5-related SRSF1 protein was correlated with radiotherapy sensitivity,.(2)We used Western blot to verify the changes of SRSF1 protein after radiotherapy.2.Establishment of stable silenced SRSF1 lung cancer cell lines and related phenotypic studies:(1)Plasmid construction: Set up three stable cell lines with plasm PLKO.1,SRSF1-si RNA1 and SRSF1-si RNA2 in the lung cancer cell.(2)Cell line validation: Use the Western blot and q-PCR to detect SRSF1 gene expression.(3)phenotypic detection: Use the plate cloning experiment,CCK-8 cell growth experiment and apoptosis experiment to detect the malignant biological behavior changed when lung cancer cells with low SRSF1 expression after radiotherapy.(4)Verification of DNA damage after radiotherapy: Use immunofluorescence technology to detect γ-H2 AX,p-Chk2 localized expression in cells with and without radiotherapy,at the same time use the comet tail experimental detect DNA damage.Finally use the Western blot technique to detect intracellular γ-H2 AX,p-Chk2 change in protein level.3.Whole-genome RNA sequencing:(1)SRSF1 knocked down lung cancer cells after radiotherapy were sequenced by whole RNA sequencing.(2)Verify bioinformatics accuracy: SRSF1-related genes were screened by GO analysis and STRING analysis.Six genes were randomly selected use PT-PCR technology to verify the accuracy of bioinformatics analysis.(3)Verify the accuracy of splicing events: SRSF1-related genes were stratified and selected to participate in splicing events.Similarly,GO analysis and STRING analysis were used to screen out SRSF1-related splicing variation genes.Six genes were randomly selected and PT-PCR technology was used to verify the accuracy of bioinformatics analysis.4.DNA damage and its mechanism:(1)Plasmid construction: Construction eukaryotic expression plasmid of PCDH,SRSF1,plko-1(Ctl sh1),srsf1-sirna1(SRSF1 sh1),PTPMT1 B,PCDH+ p LKO.1(Ctl sh1+ Ctl),SRSF1-si RNA1+PCDH(SRSF1 sh1+Ctl),SRSF1-si RNA1(Ctl sh1+Ctl),SRSF1-si RNA1 +PCDH(SRSF1 sh1+Ctl),SRSF1-si RNA1+PTPMT1A(SRSF1sh1+PTPMT1A),and then transfection those corresponding plasmid with lentivirus.(2)Cell line validation: Detection the RNA levels in those stable transfected lung cancer cell lines and analysis the spliceosome changes of PTPMT1 A and PTPMT1 B respectively.(3)Effects of overexpression of PTPMT1 B spliceosome on lung cancer cell phenotype and DNA damage: Detecting malignant biological behaviors we used plate cloning and RTCA cell growth experiment.Then detecting the expression level and intracellular localization Change of γ-H2 AX in lung cancer cells after radiotherapy by immunofluorescence technology at the cell level.Meanwhile,DNA damage in cells after radiotherapy was detected by comet tail experiment.Finally,detecting the changes of protein levels of γ-H2 AX and p-CHK2 use Western blot in cells.(4)The phenotype and DNA damage effects of silencing SRSF1 and overexpression of PTPMT1 A spliceosome on lung cancer cell: We detected cell growth by plate clonal assay,nuclear γ-H2 AX use immunofluorescence,and the expression of γ-H2 AX by Western blot at the protein level after radiotherapy.(5)Molecular mechanism related to SRSF1 gene and radiotherapy: Using Western blot experiments to test AMPK,m TOR,p21 and p27 protein changes in SRSF1 sh1 +PTPMT1B spliceosome lung cancer cell line after radiation therapy in the m TOR and AMPK signaling.Results1.Effects of SRSF1 gene on radiation injury of lung cancer cells(1)Bioinformatics analysis results showed that there were 30 genes(FPKM value >3)related to radiotherapy sensitivity,and SRSF1 gene expression was most related to radiotherapy sensitivity.Western blot results showed that SRSF1 gene protein expression was significantly increased after radiotherapy.(2)Phenotypic experiments showed that the number of lung cancer cells with silenced SRSF1 genome was lower and the cell growth was slower than that of the control group,and the number of lung cancer cells with silenced SRSF1 genome and the cell growth rate were further reduced after radiotherapy than that of the control group.Apoptosis experiments showed that the proportion of apoptosis in SRSF1 knockdown group after radiotherapy was significantly higher than that in the non-radiotherapy group.(3)DNA damage test results: immunofluorescence test showed that the expression of γ-H2 AX in SRSF1 knockdown group was significantly higher than that in the control group after 2h of radiotherapy.Comet trail experiment showed that after 0.5h of radiotherapy,the trail length of SRSF1 knockdown group cells was significantly longer than that of the control group.Western Blot results showed that the expression of γ-H2 AX in SRSF1 knockdown group was significantly higher than that in the control group after radiotherapy.2.The splicing events induced by SRSF1 gene in lung cancer cells after radiotherapy(1)Bioinformatics analysis after radiotherapy: total RNA was extracted from SRSF1 knocked down lung cancer cells after radiotherapy for full RNA sequencing,Some SRSF1 related genes were screened by GO analysis.These genes are involved in cell cycle regulation,cell adhesion,metabolic process,the function such as phosphorylation,transcription,gene expression.STRING analysis showed that these genes involved in splicing events mainly involved in cell adhesion and ATP synthesis in the mitochondria,the splicing factor take part in the cell adhesion PTPMT1,PPLP1,RPS3 A,RPLP2,MYL6 etc,.(2)Splicing validation: six genes were randomly selected according to GO analysis and STRING analysis,and the results of PT-PCR showed that the changes in their expressions were consistent with the results of bioinformatics analysis.3.The molecular mechanism of SRSF1 involved in DNA damage and repair after radiotherapy in lung cancer(1)The expression level of SRSF1 can effect the spliceosome changes of PTPMT1:after SRSF1 knockdown,the spliceosome of PTPMT1 A decreased while the spliceosome of PTPMT1 B increased.After SRSF1 overexpression,the spliceosome of PTPMT1 A increased while the spliceosome of PTPMT1 B decreased.(2)The effects of PTPMT1 long spliceosome changes on cell phenotype: lung cancer cells with PTPMT1 B overexpression were irradiated by 0Gy,2Gy,4Gy and 6Gy,respectively.and the number of clones of lung cancer cells with PTPMT1 B overexpression was reduced and cell growth was slowed down.Lung cancer cells with PTPMT1 B overexpression grew the slowest after radiotherapy.(3)The effect of the short spliceosome of PTPMT1 changes on DNA injury:immunofluorescence assay showed that the expression of γ-H2 AX in the nucleus when overexpression of PTPMT1 B was increased after radiotherapy 2h compared with the control group.Comet tail experiment further showed that lung cancer cells with overexpression of PTPMT1 B showed significantly longer tail dragging phenomenon at0.5 h and 4 h after radiotherapy than the control group.(4)The effects of long spliceosome changes in PTPMT1 on cell phenotype: in the three cell lines of Ctl sh1+Ctl,SRSF1 sh1+Ctl,and SRSF1 sh1+PTPMT1A,plate clonal formation experiments showed that the number of cell clonal formation in the SRSF1sh1+PTPMT1A group was higher than that in the SRSF1 sh1+Ctl and Ctl sh1+Ctl groups after irradiation with different doses of radiation(0Gy,2Gy,4Gy).Immunofluorescence experiments showed that the expression levels of γ-H2 AX in Ctl sh1+Ctl and SRSF1sh1+PTPMT1A group were both lower than that of SRSF1 sh1+Ctl,suggesting that overexpression of PTPMT1 A after knockdown of SRSF1 can reduce DNA damage in the nucleus.Western blot showed that the expression of γ-H2 AX protein in Ctl sh1+Ctl and SRSF1 sh1+PTPMT1A cells was lower than that in SRSF1 sh1+Ctl.(5)the molecular mechanisms of DNA damage and repair: Western blot experiments show that the control of Ctl sh1 and SRSF1 sh1 lung cancer cells in radiotherapy 0 h,1 h,6 h,12 h,24 h,p-MAPK protein in control group after radiotherapy with the extension of time,the amount of protein expression showed a trend of decrease,in 24 h content is significantly less than SRSF1 sh1 group,SRSF1 sh1 after radiotherapy p-MAPK protein decrease trend obvious delays.Similarly,Western blot results showed that p21 and p27 protein expressions increased after PTPMT1 B overexpression,while pmapk and m TOR protein showed no significant changes.However,when PTPMT1 B overexpression of lung cancer cells was radiated,p-MAPK protein expression increased significantly,and p21 and p27 protein expression increased correspondingly,but m TOR protein decreased.Conclusion1.The down-regulated expression of SRSF1 gene can increase the radiation damage of lung cancer cells and increase the double-strand DNA breakage.2.SRSF1 gene is an important factor affecting the radiotherapy sensitivity of lung cancer,and may become one of the indicators for evaluating the radiotherapy efficacy and prognosis of lung cancer.3.SRSF1 gene is likely to participate in the radiotherapy resistance through selective splicing events in lung cancer cells.4.SRSF1-mediated selective splicing of PTPMT1(PTPMT1A pro-cancer splices and PTPMT1 B anti-cancer splices)to affect radiotherapy resistance of lung cancer cells is achieved by inhibiting AMPK/m TOR signaling pathway.5.The study of SRSF1 inhibitor or PTPMT1 A inhibitor will be conducive to finding drug action targets related to radiosensitivity of lung cancer,which is expected to provide new directions and new targets for the treatment of lung cancer. |
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