MiR-324-5p Inhibits Multiple Myeloma Cell Growth By Targeting The Hedgehog Pathway

Objective: To investigate whether mi R-324-5p exert an inhibitory effect on the growth of multiple myeloma cells by targeting the hedgehog pathway.Methods: Real time RT-PCR and western blotting analysis was used to analyze the expression level of mi R-324-5p and the protein SMO,GLI1 of Hedgehog pathway in multiple myeloma cell lines and bone marrow derived CD138+ cells from multiple myeloma patients with or without del(17p)and healthy individuals.Agomir-324-5p was designed and synthesized to activate endogenetic mi R-324-5p and transfected into JJN3 cells by the carrier of Hipfect,and set a negative control transfected group at the same time.Then real time PCR and western blotting were used to detect the expression level of SMO and GLI1 in protein and m RNA levels.And to detect the rate of MM cells' proliferation and apotosis under the treatment of bortezomib before and after being transfected through the methold of CCK8 and FITC/PI FCM.After constructing BMSC-MM coculture system,western blotting was used to analysis the expression of SMO and GLI1,FITC/PI FCM was used to detect the influence on MM cells' apotosis with bortezomib,before and after coculture.And agomir-324-5p was transfectd into MM cells in cocultural system,then to assess the rate of MM cells' viability with mi R-324-5p overexpression by CCK8.Finally,the establishment of subcutaneous xenografted NOD/SCID mice of MM cell line was used to evaluate the influence of overexpressing mi R-324-5p on the growth of MM cells in vivo,to analysis the expression level of mi R-324-5p in tumor cells by Taqman RT-PCR,and to detect tumor load and the related proteins in tumor tissue,including SMO,GLI1,Caspase3 and Ki67,with the methold of HE and immunohistochemistry,respectively.Result:Comparing with MM patients without del(17p),the bone marrow CD138+ cells derived from MM patients with del(17p)expressed higher level of mi R-324-5p,and the common MM patients expressed higher than the CD138~+ cells from normal control group.And the expression level of mi R-324-5p in MM cells line was negatively correlated with the protein expression of SMO and GLI1.Overexpression of mi R-324-5p in MM cells could downregulate the expression of SMO and GLI1 in the level of m RNA and protein,and could promote killing effect of bortezomib on MM cells.After coculturing with BMSC,the protein level of SMO and GLI1 in MM cells was higher than before,the apoptosis rate of bortezomib on MM cells decreased,and mi R-324-5p enhanced the anti-tumor effect on MM cells of bortezomib.With the treatment of agomir-324-5p,tumor growth in vivo could be effectively repressed.Correspondly,the reduced expression of SMO,GLI1,Ki67,and the increased level of Caspase3 were detected in agomir-324-5p treated tumor tissue.What's more,agomir-324-5p synergistical treatment with bortezomib could significantly repress turmor cell growth,and the protein expression of SMO,GLI1 and Ki67 decreased more,Caspase3 was more activated,consistently.Conclusion:mi R-324-5p could inhibit the growth of MM cells by targeting the Hedgehog pathway,and it can enhance the sensitivity of MM cells to bortezomib.In conclusion,mi R-324-5p may play a significant role in the therapeutic strategies of MM.