A Study On Effects And Mechanisms Of ERCC1 Exon-skipping In Cisplatin-resistance Of Lung Cancer

Objective:The DNA damage response(DDR)is an evolutionarily conserved signaling cascade that senses and responds to endogenous sources(for example,ROS)and exogenous sources(like chemical mutagens and genotoxic chemotherapy drugs)of DNA-damaging agents.Nucleotide excision repair(NER)is a key component of the DNA damage response(DDR)and it is essential to safeguard genome integrity against genotoxic insults.NER with ERCC1 gene as the core is responsible for a plethora of DNA lesions induced by cisplatin and contributes to drug resistance.Consequently,ERCC1 was used as a predictor for chemotherapy responses of platinum drugs.However,it failed to reach a consistent conclusion.The proposal of alternative splicing may widen new field for the functional studies of ERCC1 gene.Numerous studies had emphasized the significance of alternative splicing modified by many compounds and oligonucleotides which can disrupt or manipulate spliceosome or splice site.Recent studies had shown that targeting the key factors of the DNA damage repair system might interfere with the DDR signal cascade and futher modulate the tumor microenvironment:DDR-mediated extracellular signals can transmit information to surrounding undamaged and damaged cells to initiate the local immune response of the tumor,which was conducive to the prognosis of the tumor.Among them,cGAS-STING as a cytoplasmic DNA sensor activated adaptive immune response and innate immune response pathways,and its clinical relevance to DNA damage therapy had become a new research hotspot.Therefore,this study aimed to explore the effect of alternative splicing of ERCC1 gene on cGAS-STING pathway.Expectantly,our study will contribute to fully understanding of the underlying mechanism of ERCC1 gene splicing and exploring effective strategies for immune activation.Furthermore,it is expected to provide an important the theoretical basis and scientific clues for finding novel and promising molecular targets in the treatment of lung cancer and prognosis biomarkers.Methods:1.Clinical samples combined with cisplatin-sensitive/resistant A549 cells(A549 and A549/DDP)was used to clarify the association between different splicing isoforms of the ERCC1 gene and cisplatin resistance:(1)evaluate the IC₅₀ of cisplatin by MTT assay and detect the expression of ERCC1 isoforms(ERCC1-E8,ERCC1-E8D,ERCC1-E3 and ERCC1-E3D)by QPCR in lung cancer tissues to clarify the association between IC₅₀ of cisplatin and the expression of ERCC1 isoforms,subsequently verify the above results in A549 and the cisplatin-resistant A549/DDP cells.(2)Evaluate cisplatin resistance by CCK8 assays and immunocytochemical analysis of ?H₂AX following overexpression of different isoforms in A549 and knockdown them in A549/DDP,respectively.(3)Analyse the ERCC1-XPA and ERCC1-XPF interaction by COIP in HEK293T cells transfected with different isoforms of ERCC1.(4)Elucidate the activity of the cGAS-STING pathway(cGAS,STING,IRF3,p IRF3 and IFN-?)by WB or ELISA in the above cells A549 and A549/DDP.(5)Clarify the interplay of the ERCC1 isoforms with cGAS-STING pathway by the above overexpression and knockdown of ERCC1 isoforms cell models.2.Identify the key regulatory factors responsible for the alternative splicing of ERCC1 gene:(1)Screen the key regulatory factors of ERCC1 gene alternative splicing via bioinformatics websites,and verify the binding of the candidate splicing factor PRPF8 to the pre-m RNA of ERCC1 gene via RIP experiments in A549 and A549/DDP cell.(2)Detect the isoforms expression of ERCC1 gene and futher clarify the changes of the downstream cGAS-STING pathway after inhibition of the expression of PRPF8 by si RNA.(3)CCK8 assays and immunocytochemical analysis of?H₂AX were used to evaluate the influence of PRPF8 down-regulation on cisplatin resistance.(4)Analyze the relation of cisplatin resistance(IC₅₀)to PRPF8 protein expression detected by immunohistochemical staining in lung cancer tissues.3.Determine whether?-elemene governs cisplatin resistance via the PRPF8-ERCC1-cGAS/STING signal axis.(1)Evaluate whether?-elemene alters cisplatin resistance by CCK8 assays and immunocytochemical analysis of?H₂AX.(2)Determine the effect of?-elemene treatment on the RNA binding protein PRPF8 and the alternative splicing of its target gene ERCC1.(3)Detect the activity of cGAS-STING pathway in A549/DDP cell treated by?-elemene and cisplatin.Results:1.The ERCC1 isoforms regulated cisplatin sensitivity in lung cancer via the cGAS-STING pathway:(1)ERCC1 gene exon 8 inclusion promoted cisplatin resistance,confirmed by the higher expression of ERCC1-E8 and PSI index(ratio of E8/E8D),the higher IC₅₀ of cisplatin(P<0.05);the exon 3 skipping of the ERCC1gene was negatively correlated with cisplatin resistance,but the ratio of ERCC1-E3and ERCC1-E3D was significantly positively correlated with the IC₅₀ of cisplatin in lung cancer tissues(P<0.05),namely,exon 3 skipping contibuted to patients'sensitivity to cisplatin chemotherapy.Meanwhile,the PSI index of exon 8 and exon 3 in A549 was significantly lower than that in A549/DDP.After cisplatin treatment,we observed the increased proportion in a dose-dependent manner.Similarly,the full-length ERCC1 protein in A549/DDP was significantly higher than that in A549.(2)The full-length isoform ERCC1-FL overexpression promotes cisplatin resistance,significantly up-regulated the IC₅₀ to cisplatin and alleviated DNA damage induced by cisplatin in A549,while exon 3 skipping isoform ERCC1-E3D and exon 8 skipping isoform ERCC1-E8D overexpression did not induced cisplatin resistance as ERCC1-FL.Subsequently,compared with NC group,we observed the significant down-regulation IC₅₀ and the severer DNA damage after the knockdown of isoforms ERCC1-E3 and ERCC1-E8 by si RNA targeting to exon 8 and exon 3 in A549/DDP.(3)COIP experiments confirmed that the isoforms ERCC1-E8D lost their ability to bind XPF resulting from exon 8 skipping;the deletion of exon 3 had no significant effect on the interaction of ERCC1 and XPA.(4)The cGAS-STING pathway was significantly activated in A549 exposured to cisplation,while was inhibited in A549/DDP exposured to the same concentration of cisplation.(5)The isoform ERCC1-FL overexpression significantly inhibits the cGAS-STING pathway activity,while ERCC1-E3D and ERCC1-E8D overexpression exhibited no effect on this pathway in A549 treated by 2?g/ml cisplatin.Then,we observed the knockdown of isoforms ERCC1-E3 and ERCC1-E8 by si RNA targeting to exon 3 and exon 8triggered the activation of the cGAS-STING pathway compared with the NC group in A549/DDP treated by 2?g/ml cisplatin.2.The RNA binding protein PRPF8 governed alternative splicing of ERCC1 to exacerbate cisplatin resistance:(1)Bioinformatics analysis indicated that PRPF8 was positively correlated with the expression of exon 8and exon 3 of the ERCC1 gene,and the RIP experiment provided the evidence that PRPF8 can bind to the pre-m RNA of ERCC1 gene and regulate its alternative splicing in A549 and A549/DDP.(2)The knockdown of PRPF8 by si RNA significantly reduced the proportion of ERCC1-E3 isoform,and activated the downstream cGAS-STING pathway to sensitize A549/DDP cell to cisplatin.(3)The high expressions of PRPF8 contibuted to poor prognosis of patients with lung carcinoma especially undergoing chemotherapy.3.?-elemene activated the cGAS-STING pathway to reverse cisplatin resistance via inhibition the splicing factor PRPF8-mediated the alternative splicing of ERCC1 in lung cancer:(1)A549/DDP cells was sensitized after?-elemene combined with cisplatin treatment,confirmed by the significantly reduced IC₅₀ and the severe DNA damage.(2)?-elemene inhibited the RNA binding protein PRPF8 and changes the alternative splicing of the target gene ERCC1.(3)?-elemene treatment rescued the inhibition of cGAS-STING activity caused by cisplatin.Conclusion:1.The ERCC1-FL isoform(exon 3 and exon 8 inclussion)was the sole one endowed with ERCC1 activity via interaction with XPA and XPF in DNA repair pathways to suppress the cGAS-STING pathway and further promote cisplatin resistance.2.RNA binding protein PRPF8 binded the pre-m RNA of ERCC1 gene to regulate its alternative splicing;inhibition of PRPF8 reduced the proportion of ERCC1-E3 isoform and futher activated cGAS-STING pathway to sensitize A549/DDP to cisplatin.3.?-elemene activated the cGAS-STING pathway via inhibition the splicing factor PRPF8-mediated the alternative splicing of ERCC1 to reverse cisplatin resistance in lung cancer.