PCBP2 Stabilizes The Degradation Of PARP1 By Binding To PARP1 MRNA And Participates In Olaparib Resistance

【Background】Cancer is often described as the disease of genome through the accumulation of DNA mutations and genome instability.Global cancer statistics reveals that breast cancer still the leading cause of deaths among women worldwide.Triple-negative breast cancer estimated about 15%of all breast cancer Patients.There is no target standard treatment due to the lack of endocrine and anti-HER2 therapeutic targets.Therefore,it is Possible to figure out the genes related to triple-negative breast cancer occurrence and discover therapeutic target drugs for breast cancer Patients to Provide a novel study Plan.The PARP1 is a key enzyme in DNA repair,and TNBC cells with BRCA1 gene depletion are sensitive to PARP1 inhibitors.Moreover PARP1 inhibitors are widely used to the treatment of ovarian cancer Patients with BRCA gene depletion but are still in Phase III clinical trials for breast cancer.PCBP2(Poly(r C)-binding Protein 2)belong to RNA-binding Protein family,it can specifically bind the target transcript and Play a role in Post-transcriptional regulation.There were examine 331 breast cancer Patients by immunohistochemistry and results showed that PCBP2 was highly expressed in the nucleus of breast cancer tissue cells and was associated with Poor Prognosis of the Patients,while PARP1 was Positively correlated with the nuclear expression level of PCBP2.We speculate that this may be related to the Post-transcriptional regulatory function of PCBP2.【Objectives】1.To explore the regulatory network between PCBP2 and PARP1;2.To investigate,whether PCBP2 affects the Process of PARP1 inhibitor resistance.【Methods】(1)The expression of PCBP2 and PARP1 in the continuous sections of47 breast cancer Patients were detected and correlation between these were determined by the chi-square test analysis method according to the staining score.(2)PCBP2 Protein immunoprecipitation reaction was Performed,Furthermore,the Precipitated interacting Proteins were silver-stained and sent to mass spectrometry for identification.(3)PCBP2knockout cell line PCBP2-/-SUM149PT was constructed.(4)RNA and Protein expression of PARP1 were detected in wild-type and PCBP2 knockout cell lines.(5)The localization and expression of PARP1 m RNA in wild-type and PCBP2 knockout cell lines were identified by RNA immunofluorescence and RNA nucleocytoplasmic separation techniques.(6)RNA Binding Protein Immunoprecipitation test was used to identify PCBP2 binding sites in PARP1m RNA.(7)The degradation rate of PARP1 RNA was detected by RT-qPCR at different times in wild-type and PCBP2 knockout cell lines after treatment with transcription inhibitors.(8)The sequence interaction between PARP1 and PCBP2 was identified by co-transfection of PARP1 double luciferase reporter gene Plasmid containing the full length of PARP1 3’UTR and Plasmid containing the sequence of Possible binding sites of PCBP2.(9)Olaparib was added to cell lines with or without PCBP2 knockout to determine the sensitivity of cells to drugs.(10)Two Olaparib resistant cells were successfully induced by increasing concentration induction:HCC1937 OR(RI=4.69±0.67)and SUM149PT OR(RI=10.45±1.02);(11)Expression of PARP1 and PCBP2 in drug-resistant strains and Parental cells was detected by Fluorescence in Situ Hybridization and Western Blot.【Results】(1)There was a Positive correlation between the expression of PARP1 and PCBP2 in the tissues of breast cancer Patients(χ~2=7.875,P=0.005).The disease-free survival(P=0.047)and overall survival(P=0.01)of the Patients with high expression of PARP1 were shorter than those with low expression.(2)We analyzed the Precipitated Proteins by mass spectrometry and found 518 Proteins interacting with PCBP2.In this study we found that these Proteins were mainly involved in RNA catabolic Process and translation.(3)Moreover,Western Blot,RT-qPCR,RNA FISH,and RIP showed that PCBP2 could affect the Protein and m RNA expression of PARP1 m RNA,but did not affect the localization.(4)Furthermore,to inhibit the transcription Process of DNA,it was found that the degradation rate of PARP1 was accelerated after the knockout of PCBP2.(5)The dual-luciferase reporter gene results showed that PCBP2 could bind to the CU-rich elements of PARP1 3’UTR to stabilize RNA degradation.(6)Breast cancer cells lost their sensitivity to Olaparib after PCBP2 knockout detected by CCK8 assay.(7)FISH and Western blot results showed that,after resistance to Olaparib,PARP1 Protein expression was increased and localization was still in the nucleus.However,the intracellular DNA damage,together with the Protein levels of PCBP2 were reduced,which indicates that drug-resistant cells could recover the DNA damage repair function,and PCBP2 was involved in the cellular Process.【Conclusions】(1)PCBP2 Played an important role to stabilizing RNA degradation by binding to CU-rich element in PARP1 3’UTR.(2)The resistance to Olaparib increased after PCBP2 knockout.(3)DNA damage repair function of breast cancer cells recovered after Olaparib resistance.