Inhibitory Effect And Mechanism Of RIB On Gastric Cancer Through PRMT5

Objective: To study the epigenetic mechanism of RIB inhibiting the growth of gastric cancer by PRMT5-mediated arginine methylation.Methods: MTT [3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide] colorimetry was used to investigate the effect of RIB on the growth of MFC and SGC-7901 cells.Plate cloning assay was used to detect the effect of RIB on the colony formation of MFC and SGC-7901 cells.Flow cytometry was used to detect the effect of RIB on cell cycle and apoptosis of MFC and SGC-7901 cells.Western blot was used to detect the expression of related proteins and study their anti-swelling effect in vitro.MTT assay was used to investigate the effect of RIB combined with chemotherapeutics on gastric cancer cells and the anti-tumor mechanism of RIB combined with chemotherapeutics in vitro.Results: 1.RIB can inhibit the proliferation of mouse gastric cancer cell MFC and human gastric cancer cell SGC-7901 in a time-and dose-dependent manner.The IC50 values of MFC cells treated with RIB for 48 h,72 h and 96 h were 13.70±1.52 ?g/mL,10.97±0.59 ?g/mL and 5.73±0.21 ?g/mL,respectively.The IC50 values of SGC-7901 cells treated with RIB for 48 h,72 h and 96 h were 34.93±1.44 ?g/mL,28.56±1.79 ?g/mL and 24.73±1.69 ?g/mL,respectively.2.Plate cloning experiment showed that RIB could inhibit colony formation of MFC and SGC-7901 cells.3.Flow cytometry was used to detect the effect of RIB on apoptosis of MFC and SGC-7901 cells.The results showed that after 72 hours of RIB treatment,MFC cells could be induced to apoptosis in a dose-dependent manner.However,RIB did not induce apoptosis of SGC-7901 cells.4.Flow cytometry was used to detect the effect of RIB on the cell cycle of MFC and SGC-7901.The results showed that RIB reduced the number of MFC and SGC-7901 cells in G0/G1 phase and increased S phase.Therefore,RIB blocked MFC and SGC-7901 cells in S phase,inhibited DNA replication and thus inhibited cell proliferation.5.Western blot results showed that PRMT5 and eIF4 E protein were low expressed in RIB-treated MFC and SGC-7901 cells,that is,RIB might inhibit the growth of tumor cells by down-regulating the expression of PRMT5 and eIF4 E.6.The combination of 25 ?g/mL RIB and 10~20 ?g/mL fluorouracil(5-FU)could inhibit the proliferation of MFC cells,but it could inhibit the proliferation of SGC-7901 cells.The combination of 25 ?g/mL RIB and 0.0625~0.125 ?g/mL adriamycin(Adriamycin or Doxorubicin,ADM or DOX)could inhibit the proliferation of MFC and SGC-7901 cells,and the concentration of 25 ?g/mL RIB was 0.0625~0.125 ?g/mL.The combination of 0625~0.125 ?g/mL of cisplatin(DDP)has a positive effect on inhibiting the proliferation of MFC and SGC-7901 cells.Conclusion: 1.RIB can inhibit the proliferation of MFC and SGC-7901 cells in a time-and dose-dependent manner in a certain concentration range.2.RIB can inhibit colony formation of MFC and SGC-7901 cells.3.RIB treatment of MFC cells for 72 hours could induce apoptosis of MFC cells,but had no effect on SGC-7901 cells.4.RIB can block MFC and SGC-7901 cells in S phase and block DNA replication,thus inhibiting cell proliferation.5.RIB may play an anti-tumor role by down-regulating the expression of PRMT5 and eIF4 E proteins.6.The combination of 25 ? g/mL RIB and 5-FU(10~20 ?g/mL)had a positive effect on the proliferation of MFC cells,but had an antagonistic effect on the proliferation of SGC-7901 cells.The combination of RIB and adriamycin(<0.125 ?g/mL)or cisplatin had a positive effect on the proliferation of MFC and SGC-7901 cells.