PRMT5 Regulates RNA M6A Methylation For Doxorubicin Sensitivity In Breast Cancer

BackgroundBreast cancer is a malignant tumor with the highest incidence in female worldwide,and its mortality ranks second among female malignant tumors,severely threatening the life and health of women.Chemotherapy occupies an important position in the treatment of breast cancer,which includes anthracycline chemotherapeutic drugs represented by doxorubicin.It works mainly through damaging the double-stranded DNA structure of breast cancer cells.Recent studies found that,PRMT5,as an arginine methyltransferase,can improve the ability of breast cancer cells to repair DNA damage and reduce the sensitivity of breast cancer cells to chemotherapy.Our previous study showed that high level of PRMT5 in patients with breast cancer usually indicated a poor prognosis and promoted doxorubicin resistance in breast cancer.However,the molecular mechanism of regulation of DNA damage repair by PRMT5 is not fully clarified.N6-adenylate methylation（m6A）is the most common internal modification in RNA,which is a dynamic equilibrium process involving RNA methylase and demethylase in vivo.Currently,proven methylases include METTL3,METTL14,WTAP,KIAA1492,etc.,and demethylases include only ALKBH5 and FTO.A large number of studies have shown that abnormal expression of m6A modifier is closely related to tumorigenesis and development.In particular,RNA m6A levels were up-regulated after cells were subjected to UV-induced DNA damage.However,it is unknown whether the modification of RNA m6A is affected by chemotherapy drugs such as doxorubicin and whether it is involved in the regulation of doxorubicin sensitivity.Therefore,in the response of breast cancer cells to doxorubicin,we will clarify the relationship between PRMT5-mediated DNA damage repair and RNA m6A modification,analyze its regulatory mechanism,and reveal changes of cell biology and animal function involved.In addition,we also screened for new inhibitor of PRMT5,and determine its effect on doxorubicin sensitivity of breast cancer in vitro and in vivo.In conclusion,our study will be of great significance to the improvement of breast cancer cells’sensitivity to doxorubicin and to the better prognosis of breast cancer patients.ObjectiveExplore how PRMT5 modulates the change in the level of m6A induced by DNA-damage;analyze how PRMT5 regulates ALKBH7 methylation;clarify how ALKBH7 regulates the nuclear translocation of ALKBH5;reveal whether Tadalafil enhances the sensitivity of breast cancer doxorubicin treatment by inhibiting PRMT5activity.Methods And Results1.Doxorubicin was first demonstrated to up-regulate RNA m6A modification in breast cancer cellsFirstly,we extracted the RNA from the tissues of breast cancer patients with Trizol method,and after purification of m RNA,detected the m6A level in RNA with Dot blot method.It was found that the breast cancer patients who received neoadjuvant chemotherapy showed significantly higher m6A levels in their tissues than those patients without neoadjuvant chemotherapy.Then,we performed PDX model construction on two cases of triple-negative breast cancer,and treated model nude mouse with doxorubicin,after treatment we detected the m6A modification level of m RNA in tumor.The results showed that the m6A level in the group treated with doxorubicin was significantly higher compared to the group without doxorubicin treatment.Finally,we treated MDA-MB-231and T47-D breast cancer cells with different concentrations of doxorubicin and with different processing time.Dot blot revealed that doxorubicin increased the m6A modification levels in these two cell lines and this effect was both dose-dependent and processing-time-dependent.2.PRMT5 decreased the sensitivity of breast cancer cells to doxorubicin treatmentFirstly,we used lentiviral infection techniques to successfully construct the following three lines of breast cancer cells with PRMT5 over-expressed:MDA-MB-231,MCF-7 and T47-D,and treated these cells with different concentrations of doxorubicin and with different processing time.Western blot showed that PRMT5 significantly reduced theγH2AX levels in breast cancer cells.Then,bear breast cancer tumor in nude mouse and treated them with doxorubicin,we monitored the tumor volume at a fixed interval during the treatment and drew the tumor volume growth curve after treatment.It was found that PRMT5 can cause a reduction in the sensitivity of breast cancer cells to doxorubicin.Dot blot in PRMT5 over-expressed breast cancer cell lines showed that,PRMT5 can reduce the doxorubicin-induced m6A level in the m RNA of breast cancer cells.In the MEF cells knocked-down with prmt5,the MTT assay revealed a higher sensitivity to doxorubicin,Western blot assay showed a higher expression level of doxorubicin-mediatedγH2AX,and a higher level of doxorubicin-mediated m6A in the m RNA.3.PRMT5 regulates m6A methylation for DNA repairWe performed Me RIP-Seq on different groups of breast cancer cells.The results showed that m6A peaks were mainly located near the stop codon in 3’UTR regions and they were rich in 5’-RRACU-3’motif.In breast cancer cells,doxorubicin can promote the formation of m6A peaks,whereas PRMT5 can reduce the enrichment of doxorubicin-mediated m6A peaks.GO analysis found that,doxorubicin promoted the m6A modification of those transcripts that were related to DNA damage stimulus and DNA repair in breast cancer cells,whereas PRMT5 reduced the m6A methylation level of those transcripts.Among these,the change in m6A methylation level of BRCA1 signal was remarkable.Furthermore,RT-q PCR results showed that while doxorubicin reduced the stability of BRCA1 m RNA,on the contrary,PRMT5 increased this stability.RNA-immunoprecipitation-q RT-PCR results showed that,doxorubicin could promote the m6A modification on BRCA1 m RNA in the breast cancer cells,whereas PRMT5 can reduce its methylation level.Compared to wild MEF cells,the MEF cells with PRMT5knocked-down that were processed with doxorubicin had a higher m6A level in BRCA1m RNA.In addition,indirect immunofluorescence revealed that PRMT5 could promote the generation of RPA32 foci in the breast cancer cells,and comet assay showed that PRMT5could shorten the doxorubicin-mediated DNA tail length in the breast cancer cells.4.PRMT5 methylates ALKBH7 for m6A modificationBioinformatics data analysis combined with co-immunoprecipitation revealed that,PRMT5 could induce symmetrical methylation of ALKBH7 and they interacted with each other when doxorubicin existed.Both human and mouse ALKBH7 contain classical modification sites of PRMT5,that is RGRR.After mutation of RGRR in the ALKBH7 to KGKK or AGAA,the half-life period of protein in ALKBH7 was remarkably shortened and the level of symmetrical methylation in ALKBH7 was significantly reduced.After treating breast cancer cells using different types of protein degradation pathway inhibitors（MG132,leupeptin and MLN4924）,it was found that only MG132 can partly recover the expression of ALKBH7^3RK,suggesting the extremely important role of ubiquitin proteasome pathway in the modulation of the degradation of ALKBH7^3RK.Further analysis by combining bioinformatics analysis and protein stability analysis revealed that FBW7αmay be the E3 ubiquitin ligase to degrade ALKBH7,and that FBW7αcould promote the combination of ubiquitin molecules and ALKBH7^3RK.Moreover,it was found that knocking down ALKBH7 in the PRMT5 over-expressed cells,to some degree,could increase the sensitivity of breast cancer cells to doxorubicin,promote the expression ofγH2AX in the cells,prolong the DNA tail length and decrease the number of RPA foci.In the breast cancer cells,the over-expression of ALKBH7 could lower the level of m RNA m6A and the level of BRCA1 m6A,whereas ALKBH7^3RK could weaken this effect.In addition,it was found that knocking down ALKBH7 in the PRMT5 over-expressed cells made both the reduced m RNA m6A and BRCA1 m6A levels increased to a higher level,thereby lowering the stability and expression level of BRCA1 m RNA.5.PRMT5 promotes nuclear localization of ALKBH5Western blot showed that both PRMT5 and ALKBH7 were not able to change the expression level of m6A demethylase,that was ALKBH5 and FTO,in the cells,whereas the results of indirect immunofluorescence and nuclear plasma separation showed that under the influence of doxorubicin both PRMT5 and ALKBH7,but not ALKBH7^3RK,could promote ALKBH5 enter into the nuclear.Additionally,indirect immunofluorescence assay found the knock-down of ALKBH7 could significantly reduce the PRMT5-mediated ALKBH5 entering into the nuclear.While co-immunoprecipitation found ALKBH7 could co-work with ALKBH5,nuclear immunoprecipitation found that ALKBH7 and ALKBH5showed a stronger interaction in the nuclear with the treatment of doxorubicin.After knocking down ALKBH5 in the PRMT5 or ALKBH7 over-expressed cells,there was a remarkable increase in the m RNA m6A level and in the m6A modification level of BRCA1 in the cells,further lowering m RNA stability and expression of BRCA1.6.Approved drug Tadalafil is a novel PRMT5 inhibitorUsing virtual screening and molecular docking,it was found that Tadalafil may serve as a potential new inhibitor of PRMT5.Surface plasmon resonance experiments revealed that Tadalafil could specifically bind with PRMT5/MEP50 complex.Tadalafil could inhibit the symmetrical methylation of histone H4R3 and the expression of PRMT5another target SDMA,it could also decrease the methylation level and protein stability of ALKBH7.Tadalafil could significantly increase the sensitivity of breast cancer cells to doxorubicin.However,this effect was lost after knocking down PRMT5 in breast cancer cells with si RNA,suggesting that under current dose of doxorubicin,Tadalafil works mainly through targeting PRMT5.Nude mouse xenograft experiments and PDX model experiments found that,Tadalafil could dramatically enhance the response of tumor-bearing nude mouse to doxorubicin,which may be completed through inhibiting PRMT5 and DNA damage repair.In addition,MTT assay and tumor xenograft model experiments showed that Tadalafil could partially reverse the drug resistance of MDA-MB-231 against doxorubicin and make these drug-resistant cells more sensitive to doxorubicin.Tadalafil could also increase the m RNA m6A modification level in the cells and PDX model nude mouse,increase BRCA1 m RNA m6A modification level,improve the expression level of damage factorγH2AX,lower the stability and expression level of BRCA1 m RNA.Conclusion1.Doxorubicin-induced DNA damage was first demonstrated to up-regulate RNA m6A modification in breast cancer cells;2.PRMT5 might regulate doxorubicin sensitivity through inhibiting RNA m6A modification;3.PRMT5 promoted DNA damage repair through regulating BRCA1 m RNA m6A methylation;4.PRMT5 regulated RNA m6A methylation and DNA damage repair required for arginine methylation of the novel substrate ALKBH7;5.The RNA demethylase ALKBH5 was clarified to be expressed in the cytoplasm for the first time,ALKBH7 could be used as its chaperone to promote its m6A demethylase effect in the nucleus,and PRMT5 could promote ALKBH7-mediated nuclear translocation of ALKBH5;6.Tadalafil as a novel PRMT5 inhibitor was firstly demonstrated to increase doxorubicin sensitivity of breast cancer through regulating RNA m6A methylation.