Figures & data
Table 1. The primer sequences for qRT-PCR assay in this study
Table 2. Correlation of hsa_circ_0007841 expression with multiple myeloma patients’ clinicopathological characteristics
Figure 1. Up-regulation and stability of circ_0007841 in MM. (a) QRT-PCR assay for the relative expression of circ_0007841 in bone marrow aspirates from MM patients (MM) and donors (Control). (b) Kaplan-Meier analysis for the overall survival of MM patients with high or low expression level of circ_0007841. (c) QRT-PCR assay for the relative expression of circ_0007841 in nPCs, NCI-H929, OPM2, U266 and JJN3 cells. (d-e) QRT-PCR assay for the relative expression of circ_0007841 and SEC61A1 mRNA in NCI-H929 and OPM2 cells treated with RNase R or not. (f-g) QRT-PCR assay for the relative expression of circ_0007841 and SEC61A1 mRNA in NCI-H929 and OPM2 cells treated with Actinomycin D or dimethyl sulfoxide (DMSO). *P < 0.05
Figure 2. Depletion of circ_0007841 hindered MM cell proliferation and metastasis, while promoted apoptosis. NCI-H929 and OPM2 cells were transfected with si-NC, si-circ_0007841#1, si-circ_0007841#2 and si-circ_0007841#3. (a-b) QRT-PCR assay for the relative expression of circ_0007841 in transfected cells. (c) Colony formation assay for the colony formation ability of transfected cells. (d) Flow cytometry for the apoptotic rate of transfected cells. (e-g) Western blot assay for the protein levels of Bax, Bcl-2 and c-caspase 3 in transfected cells. (h) Wound healing assay for the cell migration of transfected cells. (i) Transwell assay for the cell invasion of transfected cells. *P < 0.05
Figure 3. Depletion of circ_0007841 reduced the BTZ resistance of MM cells and MM growth in vivo. (a-b) MTT assay for the cell viability and IC50 value of NCI-H929 and OPM2 cells disposed with BTZ at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM and 400 nM) and transfected with si-NC, si-circ_0007841#1 and si-circ_0007841#2. (c-e) Nude mice were inoculated with NCI-H929 cells stably transfected with sh-NC or sh-circ_0007841 and injected with BTZ or not. (c-d) Tumor volume (c) and weight (d) of formed tumors. (e) QRT-PCR assay for the relative expression of circ_0007841 in formed tumors. *P < 0.05
Figure 4. Circ_0007841 could sponge miR-129-5p. (a) The predicted binding sites between circ_0007841 and miR-129-5p, as well as the mutant. (b-c) Dual-luciferase reporter assay for the luciferase activity of wt-circ_0007841 and mut-circ_0007841 in NCI-H929 and OPM2 cells co-transfected with NC mimics or miR-129-5p mimics. (d-e) RIP and RT-qPCR assays for the binding efficiency of circ_0007841 and miR-129-5p to Ago2 protein in NCI-H929 and OPM2 cells. (f) RNA pull-down assay for confirming the binding ability of circ_0007841 and miR-129-5p in NCI-H929 and OPM2 cells. (g-j) QRT-PCR assay for the relative expression of miR-129-5p in nPCs, NCI-H929, OPM2, U266 and JJN3 cells (g), in NCI-H929 and OPM2 cells transfected with pCD-ciR or circ_0007841 (h), in NCI-H929 and OPM2 cells transfected with si-NC, si-circ_0007841#1 and si-circ_0007841#2 (i), as well as in bone marrow aspirates from MM patients (MM) and donors (Control) (j). (k) Spearman’s correlation analysis for the expression levels of circ_0007841 and miR-129-5p in bone marrow aspirates from MM patients. *P < 0.05
Figure 5. MiR-129-5p inhibition could relieve silencing of circ_0007841-mediated repressed impact on cell proliferation, metastasis and chemoresistance of MM cells. (a-i) NCI-H929 and OPM2 cells were transfected with si-NC, si-circ_0007841#1, si-circ_0007841#1+ NC inhibitor or si-circ_0007841#1+ miR-129-5p inhibitor. (a) QRT-PCR assay for the relative expression of miR-129-5p in transfected cells. (b) Colony formation assay for the colony formation ability of transfected cells. (c) Flow cytometry for the apoptotic rate of transfected cells. (d-g) Western blot assay for the protein levels of Bax, Bcl-2 and c-caspase 3 in transfected cells. (h) Wound healing assay for the cell migration of transfected cells. (i) Transwell assay for the cell invasion of transfected cells. (j-m) MTT assay for the cell viability and IC50 value of NCI-H929 and OPM2 cells disposed with BTZ at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM and 400 nM) and transfected with si-NC, si-circ_0007841#1, si-circ_0007841#1+ NC inhibitor or si-circ_0007841#1+ miR-129-5p inhibitor. *P < 0.05
Figure 6. JAG1 was a target of miR-129-5p. (a) The predicted binding site between miR-129-5p and JAG1 mRNA, as well as the mutant. (b-c) Dual-luciferase reporter assay for the luciferase activity ofwt-JAG1 3ʹUTR and mut-JAG1 3ʹUTR in NCI-H929 and OPM2 cells co-transfected with NC mimics or miR-129-5p mimics. (d) RNA pull-down assay for validating the binding capacity of miR-129-5p and JAG1 in NCI-H929 and OPM2 cells. (e-l) QRT-PCR assay and western blot analysis for the mRNA and protein levels of JAG1 in nPCs, NCI-H929, OPM2, U266 and JJN3 cells (e-f), in NCI-H929 and OPM2 cells transfected with NC mimics, miR-129-5p mimic, NC inhibitor or miR-129-5p inhibitor (g-j), as well as in in bone marrow aspirates from MM patients (MM) and donors (Control) (k-l). (m) Spearman’s correlation analysis for the expression levels of JAG1 mRNA and miR-129-5p in bone marrow aspirates from MM patients. *P < 0.05
Figure 7. Up-regulation of miR-129-5p repressed MM cell proliferation, metastasis and chemoresistance by reducing JAG1 expression. (a-j) NCI-H929 and OPM2 cells were transfected with NC mimics, miR-129-5p mimic, miR-129-5p mimic+pcDNA or miR-129-5p mimic+JAG1. (a-b) QRT-PCR assay and western blot analysis for the mRNA and protein levels of JAG1 in transfected cells. (c) Colony formation assay for the colony formation ability of transfected cells. (d) Flow cytometry for the apoptotic rate of transfected cells. (e-h) Western blot assay for the protein levels of Bax, Bcl-2 and c-caspase 3 in transfected cells. (i) Wound healing assay for the cell migration of transfected cells. (j) Transwell assay for the cell invasion of transfected cells. (k-n) MTT assay for the cell viability and IC50 value of NCI-H929 and OPM2 cells disposed with BTZ at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM and 400 nM) and transfected with NC mimics, miR-129-5p mimic, miR-129-5p mimic+pcDNA or miR-129-5p mimic+JAG1. *P < 0.05
Figure 8. Circ_0007841 positively regulated JAG1 by absorbing miR-129-5p. (a-b) QRT-PCR assay and western blot analysis for the mRNA and protein levels of JAG1 in NCI-H929 and OPM2 cells transfected with si-NC, si-circ_0007841#1, si-circ_0007841#1+ NC inhibitor, si-circ_0007841#1+ miR-129-5p inhibitor. (c) Spearman’s correlation analysis for the expression levels of JAG1 mRNA and circ_0007841 in bone marrow aspirates from MM patients. *P < 0.05
