MiRNA-324-5p Suppresses The Migration And Invasion Of Multiple Myeloma Cells Through Inhibiting The SCF^?-TrCPE3 Ligase

Part 1 Expression of miR-324-5p in MM patients and cell lines and its influence on the ubiquitin-proteasome systemObjective:According to the RISS for MM,MM patients with del(17p)were stratified into high-risk group which implies poorer therapeutic effect and worse prognosis.However,the specific mechanisms of del(17p)leading to MM progression remains to be elucidated.It was demonstrated that miR-324-5p,located on chromosome 17p13.1,behaved as a tumor suppressor and its abnormalities were involved with the proliferation and differentiation of tumor cells,as well as their migration and invasion.Nevertheless,some other researches found that the elevated expression level of miR-324-5p was related to the genesis of cancer.The role of miR-324-5p in MM needs to be elucidated.UPS(ubiquitin-proteasome system)plays an important role in balancing the intracellular activities.What's more,both of proteasome inhibitors(PIs)and immunomodulatory drugs(IMiDs)which comprise the standard therapeutic regimens for MM exert their anticancer effect by disturbing the UPS.Therefore,this experiment was aimed to detect the expression status of miR-324-5p in MM patient samples and cell lines and predict its role in the regulation of UPS.Methods:Primary plasma cells were purified from bone marrow aspirates of 26 newly diagnosed MM(NDMM)patients and 2 healthy volunteers from Wuhan Union Hospital with CD138 magnetic beads(Miltenyi Biotec).Purity of plasma cells was assessed by flow cytometry analysis and was found to be>95%.Risk stratifications of all the MM patients was defined according to the RISS for MM.Six MM cell lines(NCI-H929,OPM-2,U266,RPMI 8226,MM.1R,and MM.1S)were cultured in RPMI1640 with 10%FBS.Then the total RNAs of all the extracted plasma cells and MM cell lines were isolated with TRIzol method and the expression levels of miR-324-5p were determined with TaqMan RT-PCR.We analyzed the correlation between miR-324-5p and the risk stratifications of MM patients with GraphPad Prism 5.The expression of miR-324-5p in MM cell lines was compared with that in healthy controls.The MM cell line MM.1R with quite low miR-324-5p expression was chose for further transfection experiments.MM:1R cells were transfected with miR-324-5p mimic and mimic negative control(mimic-NC)respectively.After cultured for 48h,these cells were collected for the analysis of ubiquitinated proteins levels with Western Blot and Ubiquitination Pathway PCR Array assay in the Yingbio Company in Shanghai.By means of bioinformatics,the diverse results varied between the two groups of MM JR were analysed and the target gene of miR-324-5p would be predicted.Results:The expression levels of miR-324-5p in plasma cells from bone marrow aspirates of NDMM patients was downregulated during the disease progression and its expression in RISS stage III patients(P<0.05)was remarkably lower than that in patients of stage I.Compared to healthy volunteers,miR-324-5p was expressed at lower levels in all the six MM cell lines.The expression levels of ubiquitinated proteins in MM.1R was slightly decreased with the overexpression of miR-324-5p.Among the 89 genes evaluated with Ubiquitination Pathway PCR Array,5 genes(BTRC,DAZIP3,HECW2,UBR2,and VHL)were down-regulated and only one(CBL)was up-regulated.Specifically,BTRC was most significantly decreased with a fold of-2.42Conclusion:The abnormal expression of miR-324-5p in MM cells could exert its role in the progression of MM through regulating the expression of related genes in Ubiquitination Pathway.Part 2 MircoRNA-324-5p suppresses the migration and invasion of MM cells through inhibiting the SCF ?-TrCP E3 ligaseObjective:?-TrCP serves as the substrate-recognition domain of SCF E3 ligases complex and selectively recognize target proteins for the subsequent ubiquitination process.Then,the ubiquitinated proteins can be presented to 26S proteasome and be degraded so that many kinds of signaling pathways and physiological activities can be regulated.The murine 5TGM1 myeloma cells with ?-TrCP mutated attenuated the cell growth and PDTC—inhibitor of ?-TrCP could reduce the tumor burden of mice bearing the 5TGM1 myeloma cells.Therefore,it was postulated that ?-TrCP also take part in the development of MM in humans.Some researches suggest that the conserved phospho-degron DSGxxS/T in target proteins is the recognition site of ?-TrCP for ubiquitination.MTSS1(metastasis suppressor 1),the degron-containing protein,is the substrate of(3-TrCP and associated with tumor invasion and metastasis.The role of ?-TrCP and MTSS1 in MM still remains to be elucidated.On the basis of the conclusion in Part 1,we know that miR-324-5p miR-324-5p in all the six MM cell lines was expressed at lower levels than that in healthy controls and BTRC was the most significant one among those genes downregulated by the overexpression of miR-324-5p.This part of experiment was designed to verify the relation between miR-324-5p and ?-TrCP/MTSS1 and explore the impact of miR-324-5p to the cellular activities of MM.Methods:Total RNAs and proteins were extracted from six MM cells cultured with RPMI1640(10%FBS)in vitro.Then,the expression level of BTRC mRNA was examined by SYBR Green RT-PCR and the related protein ?-TrCP was detected by Western Blot(WB).Meanwhile,the plasma cells from the two healthy volunteers were taken as the negative control.The correlations of the expressions of miR-324-5p,BTRC and MTSS1 were assessed by means of GraphPad Prism 5.0.MM.1R and H929 cells were transfected with miR-324-5p mimics and then cultured for further 48-96h.Finally,these cells were collected and used to determine the expression variations of(3-TrCP and MTSS1.At the meantime,cells transfected with miR-324-5p mimic negative control(NC)was prepared as control.What' more,MM.1R cells transfected with miR-324-5p mimic and NC were gathered for proliferation test and Transwell assay to find out the influence of overexpressed miR-324-5p to the proliferation ability and motility of MM cells.Besides,the proteasome inhibitor MG132 was used to treat MM.1R and H929 cell lines for detecting the expression variations of ?-TrCP and MTSS.Results:Compared to healthy volunteers,the expression levels of P-TrCP were significantly increased in all the six MM cell lines.While,the opposite is that the expression of MTSS 1 was decreased in different degrees.Correlation analysis by software GraphPad Prism 5.0 showed that the expression levels of miR-324-5p and BTRC mRNA were negatively correlated(Pearson correlation coefficient =-0 374).Meanwhile,the expression of MTSS 1,either on mRNAs or proteins levels,were negatively correlated to the expression of BTRC(Pearson corrplation coefficient =-0.538,Pearson correlation coefficient =-0.678).In MM.1R and H929 with high expression of miR-324-5p,the expressions of BTRC was significantly reduced(P=0.0153;P =0.0111)but the expressions of MTSS1 was prominently enhanced(P=0.0026;P =0.0386).Besides,the proliferation and migration ability of MM.1R with overexpressed miR-324-5p were both suppressed(P=0.0172;P=0.0356).Conclusion:MiR-324-5p may act as a tumor suppressor by impairing the motility of MM cells through suppressing the ubiquitination pathway.