| BACKGROUNDMultiple myeloma(MM)is the second most frequent hematological malignancy.Introduction of novel therapeutics such as proteasome inhibitor bortezomib have greatly improved overall survival in MM patients.However,relapse and drug resistance remain inevitable.Hypoxic conditions of the bone marrow microenvironment were closely related to the progression,chemotherapy resistance and poor prognosis of MM.Thus,it is urgent to find a new therapeutic drug that can efficiently kill hypoxic myeloma cells for MM patients,especially for drug-resistant or relapsed MM patients.miR-21 is an oncogene in almost all tumors,and there is a functional link between miR-21 expression and hypoxia in multiple types of tumors.The expression of miR-21 has been found closely associated with invasion,metastasis,and drug resistance of MM cells.Therefore,inhibition of miR-21 could induce the apoptosis of myeloma cells and enhance the sensitivity of myeloma cells to chemotherapy drugs.Gambogenic acid(GNA),one of the major active ingredients of traditional Chinese medicine gamboge,has been shown to possess antitumor effect against a variety of tumors with high-efficiency,low-toxicity,and multiple targets.Recently,studies have shown that miRNA is involved in the antitumor activity of traditional Chinese medicine active ingredients.Collectively,this study aimed to clarify whether GNA exerts antitumor effect in hypoxic MM cells via regulation of miR-21 and the underlying mechanisms,providing the framework for its clinical development.METHODSThe effect of GNA on proliferation of MM cell line U266 under normoxia was evaluated by the CCK8 assay.After treated with different concentrations of GNA under normoxia or hypoxia for indicated time,cell proliferation and apoptosis were evaluated by the CCK8 assay and AnnexinV-FITC/PI double staining,respectively.After incubation under hypoxia for different periods of time,the levels of miR-21 expression were detected by quantitative real-time PCR(qRT-PCR)and the expression of PTEN was detected by Western Blot.Furthermore,the expression of miR-21/PTEN in U266 cells was detected after treatment with GNA under hypoxia.After transfection with miR-21 inhibitor,the apoptosis rate of hypoxic MM cells was detected by flow cytometry(FCM)and the expression of miR-21/PTEN was detected.The effects of GNA on the STAT3,p-STAT3 and HIF-1? proteins in hypoxic MM cells were detected,and the effects of p-STAT3 inhibitors and HIF-1? inhibitors on the expression of miR-21/PTEN were further detectedMyeloma xenografts established by subcutaneous injection of U266 were used to evaluate the tumor inhibitory rate of GNA(5 mg/kg per 2 days)in vivo.The mice body weight and volume of tumor growth were observed during treatment.After intravenous treatment for two weeks,the tumors were dissected and photographed.Heart,liver,spleen,lung,kidney and tumor tissues were isolated from each group and the internal structure of the organization was examined by Hematoxylin-eosin(HE)staining Moreover,the expression of miR-21 in tumor was detected by qRT-PCR and that of Ki67 and PTEN was determined by immunohistochemistry stainingRESULTSThe results of CCK-8 assay showed that exposure to GNA resulted in a dose-dependent inhibition of cell proliferation under normoxia or hypoxia.Under normoxia,the apoptosis rate was 4.98±0.34%,9.63±0.99%,19.95±1.39%for 0,0.8 and 1.6?M GNA-treated group,respectively.Additionally,under hypoxia,the apoptosis rate was 4.49 ± 0.70%,13.01± 2.04%,27.50 ± 2.56%for 0,0.8 and 1.6 ?M GNA-treated group,respectivelyAfter incubation under hypoxia for 3,6,12 and 24 hours,the expression of miR-21 in U266 cells was 1.71 ± 0.14,2.20 ± 0.28,2.26 ± 0.22,and 2.73 ± 0.23 folds compared with normoxic group(p<0.05),respectively.Western Blot demonstrated that expression of the miR-21 target,PTEN,was decreased after the induction of hypoxia when compared with normoxic group(p<0.05)After treatment with different concentrations of GNA(0,0.1,0.4,and 1.6 ?M)under normoxia or hypoxia for 24 h,the expression of miR-21 in hypoxic U266 cells was 0.61 ± 0.10,0.42± 0.06,and 0.22± 0.04 folds for 0.1,0.4,and 1.6 ±M GNA compared with 0 ±M GNA,respectively.And in normoxic U266 cells,the expression of miR-21 was 0.82±0.04 and 0.44±0.12 folds for 0.4 and 1.6 ?M GNA compared with 0 ?M GNA,while there was no significant effect for 0.1 ?M GNA.In addition,hypoxia downregulated the expression of PTEN in U266 cells compared with normoxic group,and GNA treatment could upregulate that in a dose-dependent manner(p<0.05)To verify whether decreased miR-21 mediates cell apoptotic activity of GNA,U266 cells were transfected with miR-21 inhibitor.The results of FCM showed that the apoptotic rate of U266 cells was 14.93±3.74%under normoxia and 17.81±2.32%under hypoxia after treatment with miR-21 inhibitor.Additionally,the results obtained from Western Blot demonstrated that inhibition of miR-21 increased PTEN expression in normoxic U266 cells and attenuated reductions in PTEN protein levels under hypoxia(p<0.05)As compared with normoxia,hypoxia increased the level of hypoxia induced factor-1?(HIF-1?)and phospho-signal transducer and activator of transcription 3(p-STAT3)proteins.Under hypoxia,expression levels of HIF-1? and p-STAT3 were significantly reduced after treated with GNA.Notably,S31-201,an inhibitor of p-STAT3,and 2-ME,an inhibitor of HIF-1?,could decrease the expression of the miR-21 and in turn significantly increase the expression of PTEN when compared with normoxic group,and there were significant differences between them(p<0.05)Furthermore,in vivo study revealed that GNA could significantly suppress tumor growth compared with control group(p<0.05),and it did not obviously affect the body weight of mice(p>0.05).HE staining showed that there were no significant pathological changes in the main organs of each group,including heart,liver,spleen,lung and kidney.In addition,qRT-PCR results showed that the expression of miR-21 in the tumor tissue significantly decreased in the GNA treated group compared with the control group.Meanwhile,immunohistochemistry results showed that PTEN,target of miR-21,was increased after GNA treatment.Besides,a pronounced decrease in the expression of ki67,the marker of tumor proliferation,was noted in GNA-treated group.CONCLUSIONSHypoxia could induce an increase of miR-21 expression in U266 cells and trigger a suppression of its down-stream target PTEN.GNA exerts antitumor activity in hypoxic MM cells by regulation of miR-21/PTEN via reducing HIF-1? accumulation and STAT3 phosphorylation.All our results provide evidences that GNA can affect malignant properties of U266 cells even under hypoxia both in vitro and in vivo.Thus,GNA appears to be a new potent therapeutic agent against human MM. |
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