Mechanisms Of Niraparib Inhibiting Cell Proliferation And Migration Via Regulating 6mA Modification In BRCAwt Ovarian Cancer

Objective : Ovarian cancer is the most lethal gynecologic cancer,and the maintenance treatment of niraparib significantly improves the prognosis of patients with ovarian cancer,but the mechanism of its effect on prolonging progression-free survival of BRCA wild-type ovarian cancer patients is still unknown.6mA modification,a DNA modification that can regulate gene expression,has been confirmed to play an important role in the development of cancer.Therefore,this study was designed to investigate whether niraparib could affect the tumor development by modulating 6mA modification in a BRCAwt ovarian cancer model.Methods:The level of 6mA modification was determined by Dot Blot,ELISA and 6mA-IP-seq.The effect of niraparib on methylase was analyzed using RT-q PCR and bioinformatics.The effect of niraparib and methylase on m RNA expression and 6mA modification level of the TM4SF1 was determined using RNA-seq,6mA-IP-seq,6mA-IP-q PCR and RT-q PCR.Following the interference of TM4SF1 expression by RNAi technology,CCK8 assay,scratch assay and Transwell assay and subcutaneous transplantation tumor experiment were performed to explore its effect on the proliferation and migration ability of BCRA wild-type ovarian cancer cells.Results: Compared with the DMSO-treated group,all the Dot Blot and ELISA results confirmed that the level of 6mA modification was significantly decreased in the SKOV3 and OVCAR8 cells after niraparib treatment(p<0.001),meanwhile the 6mA-IP-seq showed that the decreased 6mA modification was mainly enriched in the transcription start regions(TSSs).RT-q PCR results revealed that the m RNA expression of ALKBH1 was significantly upregulated in the two BRCAwt ovarian cancer cells after niraparib treatment(p<0.01),meanwhile the m RNA expression of other three methylase N6AMT1,METTL4 and ALKBH4 was not significantly altered(p>0.05).Database analysis revealed that ALKBH1 expression was reduced in ovarian cancer tissues(p<0.001),and survival analysis was performed to find that low expression of ALKBH1 is positively associated with the poor prognosis of ovarian cancer(GEO database: p<0.012;TCGA database: p<0.023).After niraparib treatment on SKOV3,6mA-IP-seq was paired with the differential genes obtained by RNA-seq,and 10 intersecting genes were found;only 3 of the 10 intersecting genes were verified to be differentially expressed after niraparib treatment in OVCAR8 cell line.The RT-q PCR results showed that only TM4SF1 and SCL26A7 were differentially expressed among the three genes after interfering with the expression of ALKBH1(p<0.001),while 6mA-IP-q PCR revealed that ALKBH1 could bind to the transcriptional start region of TM4SF1 and regulate the level of 6mA modifications in this region.Database analysis revealed a significant increase in m RNA expression of TM4SF1 in ovarian cancer tissues(p<2.2e-16),which was further confirmed at the protein level by immunohistochemistry,and a trend of correlation between TM4SF1 expression and prognosis was observed in survival analysis(p=0.068).After interfering with the expression of TM4SF1,CCK8 assay,scratch assay and Transwell assay confirmed that TM4SF1 could promote the proliferation,invasion and migration ability of BRCAwt ovarian cancer cells.And the in-vivo experiments also revealed a significant decrease in the ability of the SKOV3 cell line that interfered with TM4SF1 to develop tumors in BALB/c mice(p<0.001).Conclusion:Through a series of studies,we finally found that in BRCA wild-type ovarian cancer cells,niraparib could reduce the 6mA modification level and expression of TM4SF1 by upregulating the expression of demethylase ALKBH1,and thus inhibit the proliferation and migration capability of BRCA wild-type ovarian cancer cells.