Profilin1 Induces Drug Resistance Through Autophagy In Multiple Myeloma

Background and objective:Multiple myeloma(MM)is a plasma derived hematological malignancy,the current incidence is next only to lymphoma.Recently,the approval of proteasome inhibitor Bortizomib(BTZ)made a great breakthrough in the diagnosis of MM,but the widely using of BTZ caused many patients with drug resistance who has poor prognosis.Profilin-1(PFN1)is a cytoskeleton protein which has the function of proliferation,survival,motility and membrane trafficking.In addition to the physiological function,PFN1 can also regulate tumor metastasis in breast cancer,but the other function is not clear in cancer.In this study,we used MM samples and MM cell lines to detect the function of PFN1 in MM,determine the relationship of PFN1 and drug resistance,and then reveal the possible mechanism of PFN1 related drug resistance,which may provide theoretical basis and clinical ideas for the therapy of MM patients with drug resistance.Methods:In this study,we analyzed the Gene Expression Profiles(GEP)data from MM patients to check the change of PFN1 m RNA expression level.The effect of different expression level of PFN1 m RNA on the survival time of patients with multiple myeloma,the difference of PFN1 m RNA expression between different MM subgroups,and the change of PFN1 m RNA expression in MM sequential patients were studied.The changes of PFN1 protein level in plasma cells of Health Donor and MM patients and the expression level of PFN1 protein in MM cell lines were detected.Secondly,PFN1 overexpression MM cell lines were constructed,and the expression of PFN1 was down-regulated by si RNA in MM cell lines.The proliferation of MM cells was detected by CCK8 proliferation assay,soft agar cloning assay and cell cycle assay.Thirdly,we detected the changes of autophagy-related molecule(Beclin1,LC3)in different MM cell lines by Western blotting,and examined autophagy level in PFN1 overexpression and knockdown cell lines using Western blotting,LC3 fluorescence transfection and electron microscopy.Then we explored the relationship of PFN1 and autophagy-related molecule Beclin1 by Co-immunoprecipitation.Finally,the expression of PFN1 in 8226 and R10 R cell lines was detected.After overexpression and knockdown of PFN1,we treated these cell lines with bortezomib,and cell proliferation rate was detected by CCK8,apoptosis was detected by flow cytometry,and cell clone formation rate was detected by soft agar clone formation assay.The autophagy process was blocked by 3-MA(Beclin1-complex inhibitor)or Beclin1 si RNA,flow cytometry and soft agar clone formation assays were used to investigate whether it could reverse the bortezomib resistance induced by PFN1.Results:1.GEP data analysis showed that the expression level of PFN1 m RNA in MM patients was significantly higher than that in Health Donor and MGUS patients(P < 0.05).Survival analysis indicated that the prognosis of patients with high PFN1 expression of MM was significantly worse than that of patients with low PFN1 expression.Further subgroup analysis showed that the expression of PFN1 m RNA in PR group and MF group was significantly higher than that in other groups(P < 0.05).Sequential analysis showed that the expression of PFN1 m RNA gradually increased with the development of MM.Immunofluorescence detection showed that the expression of PFN1 protein was up-regulated in CD138 + cells of MM patients.2.We constructed 8226 and 8226 R10 R PFN1 over-expressed cell lines and used PFN1 si RNA to down-regulate the expression of PFN1 in MM.1R and MM.1S cell lines.After overexpression of PFN1,we found that the cell viability of 8226 and 8226 R10 R increased significantly(P < 0.05),and the cloning rate of soft agar increased significantly(P < 0.05).Flow cytometry showed that the proportion of G1 phase cells decreased and that of S and G2 phase cells increased after PFN1 overexpression.After PFN1 knockdown,the cell viability of MM.1R and MM.1S decreased significantly(P < 0.05),and the cloning rate of soft agar also decreased significantly(P < 0.05).3.The expression of PFN1 and autophagy-related molecules Beclin1 and LC3 in MM cell lines were detected by Western blotting.It was found that the expression of PFN1 was positively correlated with that of Beclin1.The autophagy levels of 8226 and 8226 R10 R PFN1 overexpressed cell lines and MM.1R and MM.1S PFN1 knockdown cell lines were further examined.Western blotting assay showed that autophagy-related molecules Beclin1 and LC3 were significantly increased after overexpression of PFN1.After knocking down of PFN1,Beclin1 and LC3 were significantly decreased.After transfection into LC3-GFP adenovirus,the number of LC3 puncta was significantly increased after overexpression of PFN1,and the number of LC3 puncta was significantly decreased after knocking down of PFN1.Autophagic bodies increased significantly in overexpressed cell lines.Co-immunoprecipitation showed that there was a protein-protein interaction between PFN1 and Beclin1.4.The expression of PFN1 was detected in 8226 and 8226 R10 R cell lines.It was found that the expression of PFN1 was increased in 8226 R10 R resistant cell lines,and then bortezomib was treated in 8226 and 8226 R10 R PFN1 overexpressed cell lines and MM.1R and MM.1S PFN1 knockdown cell lines.It was found that after overexpression of PFN1,the drug resistance of 8226 and 8226 R10 R was significantly increased,apoptosis was significantly reduced,and the cloning rate of soft agar was significantly increased(P < 0.05).When PFN1 was knocked down,the drug resistance of MM.1R and MM.1S was significantly decreased,apoptosis was significantly increased,and the cloning rate of soft agar was significantly decreased(P < 0.05).5.After 3-MA(Beclin1 complex inhibitor)was added to 8226 PFN1 overexpressed cell lines,the expression of autophagy-associated molecule(Beclin1,LC3)was significantly inhibited and apoptosis-associated molecule PARP was significantly increased by Western blotting assay.In 8226 PFN1 overexpressed cell lines,the expression of Beclin1 was knocked down by Beclin1 si RNA,and then treated with bortezomib,the cloning rate of soft agar was significantly lower than that of NC group(P < 0.05),and the apoptosis rate was significantly higher than that of NC group.Conclusions:1.The expression of PFN1 in multiple myeloma cells was significantly increased,and the expression level was closely related to the progression and prognosis of the disease.2.In multiple myeloma,PFN1 can affect cell cycle,promote cell proliferation,and cell proliferation is significantly inhibited after down-regulation of PFN1.3.In multiple myeloma,PFN1 promotes autophagy by interacting with Beclin1,and down-regulation of PFN1 blocks the process of autophagy.4.In multiple myeloma,the up-regulation of PFN1 expression can promote cell proliferation,reduce cell apoptosis and induce cell bortezomib resistance.5.In multiple myeloma,the bortezomib resistance induced by PFN1 is caused by up-regulation of autophagy levels and blocking autophagy can reverse the bortezomib resistance induced by PFN1.