Berberinesuppresses bladdercancercell proliferation by inhibiting JAK1-STAT3 signaling via upregulation of miR-17-5p.

Hyperactivation of signal transducer and activator of transcription 3 (STAT3) is strongly associated with cancer initiation, progression, metastasis, chemoresistance, and immune evasion; thus, STAT3 has beenintenselystudiedasatherapeutictargetfor cancer treatment. Berberine (BBR), an active component extracted from Coptis chinensis, has shown anti-tumor effects in multiple tumors. However, its underlying mechanisms have not yet been fully elucidated. In this study, we investigated the effects and the underlying mechanisms of BBR on bladder cancer (BCa) cells. We found that BBR showed significant cytotoxic effects against BCa cell lines both in vivo and in vitro, with much lower cytotoxic effects on the human normal urothelial cell line SV-HUC-1. BBR treatment induced DNA replication defects and cell cycle arrest, resulting in apoptosis or cell senescence, depending on p53 status, in BCa cells. Mechanistically, BBR exerted anti-tumor effects on BCa cells by inhibiting Janus kinase 1 (JAK1)-STAT3 signaling through the upregulation of miR-17-5p, which directly binds to the 3'UTR of JAK1 and STAT3, downregulating their expressions. Collectively, our results demonstrate that BBR exerts anti-tumor effects by perturbing JAK1-STAT3 signaling through the upregulation of miR-17-5p in BCa cells, and that BBR may serve as a potential therapeutic option for BCa treatment.


Background
Bladder cancer (BCa) is the 10th most common malignancy globally, with an estimated 549,400 new cases and 199,900 cancer-related deaths in 2018 1 .The majority of BCa is pathologically classi ed as urothelial carcinomas 2 .Overall, about 75% of BC patients initially present with non-muscle invasive bladder cancer (NMIBC), and the primary choice for treatment is transurethral resection of bladder tumor (TURBT) followed by intravesical therapy 3 .Although under intensive management, approximately half of NMIBC cases recur and 10-15% would progress to muscle-invasive bladder cancer (MIBC) or metastasis within 5 years 2 .For those patients with MIBC, radical cystectomy with lymphadenectomy is still considered as standard treatment.Cisplatin-based chemotherapy is recommended in advanced patients.However, the 5-year survival rate is under 50% due to chemo-resistance and relapse.In recent years, checkpoint blockade immunotherapy (ICI) and FGFR2/3 target therapy have emerged as potential therapeutic strategies for BCa treatment 4 .But these medicines are applicable for certain patients and the objective response rate (ORR) is limited [5][6][7] .Thus, it is vital to explore novel therapeutic drugs for BCa.
Comprehensive genomic analysis reveals the genomic landscape of BCa and identi es a number of molecular targets 8 .Increasing evidence shows that signal transducer and activator of transcription 3 (STAT3) plays a vital role in tumorigenesis and development of BCa 9,10 .Generally, protein ligands in tumor microenvironment activate STAT3 via its phosphokinase-the Janus kinase (JAK), and phosphorylated STAT3 monomer is dimerized to translocate into nucleus and promote transcription of target genes.Phosphorylated STAT3 levels are shown to be upregulated in BCa tissues, and aberrant activation of STAT3 could promote proliferation, metastasis and acquired drug-resistance of BCa [9][10][11] .Transgenic mice overexpressing constitutively active STAT3 in bladder epithelium develop invasive bladder cancer 9 .Moreover, blockade of JAK-STAT3 pathway elicits signi cant anti-tumor effect and reverses resistance in BCa both in vitro and vivo [11][12][13] .Therefore, JAK-STAT3 pathway may be a potential target for BCa treatment 14 .Several selective inhibitors targeting STAT3 are undergoing preclinical and clinical researches, however, no one has been approved for clinical cancer treatment due to limited e cacy and unexpected side effects 15 .
Natural products, which are valuable sources in pharmaceutical development, have a reputation for strong potency with minimal side effects.Therefore, the molecular mechanisms underlying the effects of natural products are needed to extensively understand for further pharmaceutical development.Berberine (BBR), derived from Chinese medicinal plant Coptis chinensis, has a proven medical history as an Ayurvedic and Chinese medicine for its therapeutic effects on microbial-related or -independent gastrointestinal disorders 16 .As the most thoroughly studied natural plant alkaloid in the world, BBR has been reported to possess numerous pharmacological activities including antioxidant, anti-in ammatory, organ-protective activities 17 , and recent studies have discovered additional clinical indications.Oral administration of BBR is proved to display bene cial effects in treatment of patients with hypercholesterolemia and diabetics 17,18 .Notably, its antineoplastic effects have been investigated in a broad spectrum of cancer cells.Particularly, BBR has been shown to inhibit proliferation and migration of BCa cells, and induce DNA damage and apoptosis in BCa cells 19,20 .however, the underlying molecular mechanisms of BBR in attenuating BCa are still poorly understood.
In this study, we investigated the role and underlying molecular mechanisms of BBR in BCa cells.Our results demonstrated that BBR induced apoptosis in p53 de cient while caused senescence in p53 pro cient BCa cells via targeting JAK1-STAT3 signaling.We further showed that the inhibitory effect of BBR on JAK1-STAT3 pathway is through upregulating miR-17-5p.

Cell culture and manipulation
Human BCa cell lines T24, 5637 and immortalized human uroepithelial cell SV-HUC-1 were purchased from American type culture collection.Human BCa cell line 253J was purchased from Cell Bank of the Chinese Academy of Science.All cell lines were identi ed using short tandem repeat markers.Human BCa cell lines, including T24, 5637 and 253J were cultured in RPMI-1640 medium (#11875093, Gibco Life Technologies, Carlsbad, CA, USA,).SV-HUC-1 cell line was maintained in F12K medium (#CM10025, Macgene Technology Ltd, Beijing, China,).All medium was supplemented with 10% fetal bovine serum (#10270-106, Gibco), 100U/ml penicillin and 100μg/ml streptomycin.All cells were maintained at 37℃ in a humidi ed atmosphere with 5% CO 2 .

MTT, colony formation, and EdU incorporation assays
Cell proliferation and viability were measured by MTT, colony formation and EdU incorporation assays, which were performed as previously described 22 .

Wound-healing, transwell migration, and invasion assays
To detect the migration and invasion capacities of BCa cells, the wound-healing, transwell migration, and invasion assays were performed as previously described 21 .
Tunel assay was carried out using one-step Tunel Apoptosis Assay Kit (#C1089, Beyotime) and performed as previously described 22 .

ROS assay and alkaline comet assay
Intracellular ROS levels were measured with Reactive Oxygen Species Assay Kit (#S0033S, Beyotime).DNA damages were measured with alkaline comet assay using Single Cell Gel Electrophoresis Assay Kit (#4251-050-K, Trevigen, Gaithersburg, MD, USA).ROS assay and alkaline comet assay were performed as previously described 23 .

Apoptosis assay
Percentage of apoptotic cells was evaluated by Annexin V-FITC apoptosis Kit (#556547, BD Biosciences, Shanghai, China) according to the manufacturer's protocols.The apoptosis assay was performed as previously described 23 .

Protein extraction and western blot
Total proteins were extracted using RIPA lysis buffer containing proteinase and phosphatase inhibitors.Nuclear and cytoplasmic proteins were isolated with Nuclear and Cytoplasmic Protein Extraction Kit (#P0027, Beyotime) according to the manufacturer's instructions.Western blot analysis was performed as described previously 21 .The primary antibodies are listed in Supplementary Table 1.

RNA extraction and real-time quantitative PCR (qRT-PCR) assay
Isolation of total RNA and qRT-PCR assays were performed as described previously 21 .Primer sequences used for qRT-PCR were listed in Supplementary Table 2.All primers used for miRNA qRT-PCR were purchased from GeneCopoeia (Rockville, MD, USA).

Luciferase assay
The segments of JAK1 and STAT3 3'UTR containing wild-type and mutated miR-17-5p binding site were cloned into the pmir-GLO vectors (#E1330, Promega) which were purchased from BioSune (Shanghai, China).The luciferase assays were performed as previously described 21 .

In vivo tumor xenografts
For generation of BCa xenografts, 5×10 6 T24 cells suspended in 100 μl PBS were subcutaneously injected into the dorsal ank of female 5-to 6-week-old BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd, Beijing, China).One week later, the tumor-bearing mice were randomly divided into 2 groups and treated daily by oral gavage with DMSO and BBR (200 mg/kg body weight) respectively for two weeks.All mice were euthanized after 2 weeks and tumors were harvested.Tumors were measured using calipers, and tumor volume was calculated by the formula: Tumor volume (mm 3 ) = 0.5 ×tumor length (mm) × [tumor width (mm) 2 ].After weighing, tumor tissues were snap-frozen or embedded in para n for further analysis.The experimental protocols were approved by the Institutional Animal Care and Use Committee of Shandong University and all procedures were performed in compliance with the institutional guidelines.

Immunohistochemistry analysis
The tumor tissues were collected, fixed with formalin buffer, and embedded in paraffin.Immunohistochemical (IHC) staining was performed as previously described 21 .

Statistical analysis
All results were expressed as mean±SD.The statistical analysis and box plots were carried out using GraphPad Prism 6 software (La Jolla, CA, USA).The statistical comparisons were calculated by unpaired two-tailed Student's t-test and were considered signi cant if p<0.05.And levels of significance were presented at *P<0.05, **P<0.01,and ***P<0.001respectively.

BBR inhibits proliferation and induces cell cycle arrest in BCa cells.
We rst evaluated the anti-proliferation effects of BBR on BCa cells.MTT assays showed that BBR signi cantly decreased cell viability in a dose-dependent manner (Fig. 1a and Supplementary Fig. 1a).The results of colony formation assay also showed that BBR induced a concentration-dependent decrease in the number of cell colonies (Fig. 1b).Moreover, BCa cells treated with BBR exhibited much smaller colonies (Supplementary Fig. 1b).Next, EdU incorporation assays were utilized to detect the effects of BBR on DNA replication.Treatment of BBR signi cantly decreased the percentage of EdU positive cells in BCa cells (Fig. 1c and Supplementary Fig. 1c-e).Notably, the inhibitive effect of BBR on cell viability, colony formation and DNA replication were much less evident in the human urothelial cells (SV-HUC-1) (Fig. 1a-c and Supplementary Fig. 1a, 1f and 1 g), suggesting that the normal urothelial cells were less sensitive to BBR treatment than that of BCa cells.
Next, we investigated the underlying mechanisms of anti-proliferative effects of BBR in BCa cells.Cell cycle distribution analysis by ow cytometry showed that the effects of BBR on cell cycle varied in BCa cell lines.BBR treatment increased the proportion of cells in G0/G1 phase, accompanied by a decrease in S phase and G2/M in T24 cells (Fig. 1d).While, BBR elevated proportion of S phase in 5637 cells, along with a decline in proportion of G0/G1 and G2/M phase (Fig. 1d).Treatment of 253J cells with BBR resulted in an increase in proportion of G2/M phase (Fig. 1d).Western blots analysis on cell cycleassociated proteins revealed that the protein levels of CDK1, CDK2, CDK4, cyclin A, cyclin D1 and cyclin E were signi cantly decreased in BBR-treated T24, 5637 and 253J cells (Fig. 1e and Supplementary Fig. 1h).
We then established a xenograft model to further assess the effects of BBR on tumor growth in vivo.Applications of BBR signi cantly suppressed tumor growth of T24 cells (Fig. 1f).Consistently, the tumor volume and tumor weight were obviously reduced in BBR-treatment group (Fig. 1g).Moreover, immunohistochemistry (IHC) assays showed that BBR administration signi cantly decreased Ki-67 expression levels (Fig. 1h).Taken together, these results suggest that BBR inhibits the proliferation of BCa cells both in vivo and vitro.

Bbr Suppresses Migration And Invasion Of Bca Cells
We further determined the roles of BRR in migration and invasion of BCa cells.Wound healing assays showed that BCa cells treated with BBR exhibited a lower wound closure rate compared with that of cells treated with DMSO (Fig. 2a and Supplementary Fig. 2a).Transwell assays were further veri ed the decreased capabilities of migration in BBR-treated BCa cells (Fig. 2b and Supplementary Fig. 2b).
Matrigel invasion assays also showed BBR treatment resulted in a smaller number of invasive cells (Fig. 2c-e).These results indicated that BBR signi cantly attenuated the abilities of migration and invasion of BCa cells.Considering the vital role of Epithelial-Mesenchymal Transition (EMT) in regulating cancer cell migration and invasion, we next detected expression levels of several marker proteins involved in EMT.Western blot analysis revealed that BBR signi cantly down-regulated the expression of Snail, N-Cadherin and Vimentin in BCa cells (Fig. 2f-h), suggesting that BBR suppresses EMT in BCa cells.Besides, BBR treatment suppressed mRNA expression levels of MMP2 and MMP9 (Fig. 2i), two important genes for tumor metastasis.

BBR induces apoptosis or senescence in BCa cells in a p53-dependent way
Previous studies reported that BBR exerted anti-proliferative effects through apoptosis induction in various cancer cells.Thus, we next investigated whether BBR caused apoptosis in BCa cells.As shown in Fig. 3a-c, treatment of BBR signi cantly increased the percentage of apoptotic cells in T24 and 5637 cells, however, no signi cant changes in apoptotic cells percentage were observed in 253J cells.Consistently, the percentages of Tunel positive cells were signi cantly increased in BBR-treated T24 and 5637 cells (Fig. 3d and Supplementary Fig. 3a-b), but not in 253J cells (data not shown).Moreover, the levels of cleaved caspase-3, a marker of apoptosis, were up-regulated in T24 and 5637 cells with BBR treatment (Fig. 3e).In contrast, BBR treatment didn't affect the levels of cleaved caspase-3 in 253J cells (Supplementary Fig. 3c).
BBR was reported to induce cell apoptosis through accumulation of reactive oxygen species (ROS) and oxidative DNA damages.The comet assays showed that the mean olive tail moment values were signi cantly increased in T24 and 5637 cells treated with BBR (Fig. 3f-g).Moreover, the intracellular ROS levels were also drastically increased with increasing concentrations of BBR in these two cell lines (Fig. 3h), suggesting BBR caused ROS accumulation and DNA damage in T24 and 5637 cells.In contrast, BBR-treated 253J cells displayed similar olive tail moment values and ROS levels with that of DMSOtreated cells respectively (Supplementary Fig. 3d-e), indicating that the underlying mechanisms about proliferative inhibition of BBR might be different between 253J and the other two BCa cell lines.To investigate the mechanisms of BBR in cell proliferation regulation in 253J cells, we next performed SA-βgal activity assays to evaluate the effect of BBR on cell senescence.The results showed that BBR treatment signi cantly increased the number of SA-β-gal positive cells (Fig. 3i), substantiating that BBR induces cell senescence of 253J cells.
p53 is considered as one of the most critical tumor suppressors, and previous studies showed that cancer cells harboring wild-type or mutant-type p53 displayed different outcomes of drug-induced cell death 24 .The results that BBR induced apoptosis in p53 mutant T24 and 5637 cells, while induced cell senescence in p53 wild-type 253J cells prompted us to investigate whether the apoptosis induced by BBR in BCa cells was dependent on p53 status.253J cell line with stable p53 knock-out (253J p53-KO) was established (Fig. 3j-k).As shown in Fig. 3l, BBR treatment in 253J p53-KO cells caused a signi cant increase in apoptotic cells percentage compared with that of negative control (253J NC) cells.Besides, knockdown of p53 signi cantly attenuated the cell senescence caused by BBR in 253J cells (Fig. 3m), suggesting that the cell fate decisions, by BBR, depend on p53 function in BCa cells.
BBR down-regulates the expression and activity of STAT3 in BCa cells.
To further investigate the mechanism that BBR inhibits proliferation of BCa cells, the levels of phosphorylated ERK (p-ERK), phosphorylated AKT (p-AKT) and phosphorylated STAT3 (p-STAT3), which were known as important regulators in cancer progression, were examined.As shown in Fig. 4a, no signi cant changes in protein levels of p-ERK and p-AKT were observed in BBR treated T24 and 5637 cells.In contrast, both the p-STAT3 and total STAT3 protein levels were signi cantly decreased in BBR treated T24 and 5637 cells (Fig. 4b).Given that nuclear translocation of activated STAT3 was necessary for transcription of its target genes, the levels of cytoplasmic and nuclear STAT3 and p-STAT3 were detected.As shown in Fig. 4c, both cytoplasmic and nuclear STAT3 and p-STAT3 protein levels were down-regulated by BBR.Furthermore, luciferase analysis con rmed an obvious decrease in transcriptional activity of STAT3 in BBR-treated cells (Fig. 4d).Together, these results suggest that the activity of STAT3 was suppressed by BBR in BCa cells.
We next sought to evaluate whether STAT3 inhibition was responsible for BBR-mediated proliferation suppression in BCa cells.Stattic, a STAT3 inhibitor, signi cantly inhibited cell viability in a concentrationdependent manner (Fig. 4e).Importantly, similar with that of BBR, stattic treatment induced apoptosis in T24 cells, while induced cell senescence in 253J cells (Fig. 4f-4 g and Supplementary Fig. 4a-b).Moreover, stattic treatment in 253J p53-KO cells also signi cantly induced apoptosis accompanied by reduction of cell senescence (Fig. 4h-4i), resembling that of BBR treated cells.Altogether, these results suggest that the proliferation inhibition, apoptosis and senescence induction caused by BBR in BCa cells are potentially dependent on downregulation of STAT3.

Bbr Inhibits Stat3 Activation Through Suppressing Jak1
To further investigate whether the anti-tumor effect of BBR on BCa cells was mediated by STAT3 expression inhibition, we established STAT3 overexpressed T24 and 5637 cells (STAT3 OVE ) (Fig. 5a).Western blot analysis showed that overexpression of exogenous STAT3 signi cantly restored the reduced endogenous total STAT3 protein level caused by BBR (Fig. 5b).Of note, BBR also signi cantly decreased the exogenous STAT3 expression (Fig. 5b).MTT assays showed that expression of exogenous wild-type STAT3 didn't rescue BCa cells from proliferative inhibition caused by BBR (Fig. 5c).EdU incorporation assays showed that the number of EdU positive cells was statistically increased in STAT3 OVE cells than that of control cells under BBR treatment, however, it still signi cantly less than that of DMSO treated cells (Fig. 5d and Supplementary Fig. 5a), indicating the protective effect of introduction of exogenous STAT3 on DNA replication is not signi cantly effective to attenuate the e cacy of BBR in BCa cells.Consistently, overexpression of STAT3 didn't obviously restore p-STAT3 levels downregulated by BBR (Fig. 5e).Together, these results indicate that BBR-induced proliferation and STAT3 activity inhibition are not only mediated by decreasing STAT3 expression.
Given the critical role of Janus kinases (JAKs) in activating STAT3, we next examined the expression levels of JAK1 and JAK2.As shown in Fig. 5f and Supplementary Fig. 5b, BBR signi cantly decreased both the total and phosphorylated protein levels of JAK1 and JAK2 in T24 and 5637 cells.Importantly, overexpression of JAK1, but not JAK2, signi cantly restored p-STAT3 levels downregulated by BBR (Fig. 5g-i and Supplementary Fig. 5c), indicating that JAK1 is a key target through which BBR exerted antitumor effects on BCa cells.As expected, overexpression of JAK1 signi cantly attenuated the BBRinduced proliferation inhibition, cell cycle arrest and apoptosis (Fig. 5j-m and Supplementary Fig. 5d).Together, these results suggested that, BBR suppresses JAK1 expression to inhibit STAT3 activation in BCa cells.

Bbr Suppresses Signaling Upregulation Of Mir-17-5p
To elucidate the mechanisms that BBR downregulates JAK1 and STAT3 expression, we rst detected their mRNA levels.As shown in Fig. 6a and Supplementary Fig. 6a, BBR treatment did not decrease mRNA levels of JAK1 and STAT3 in 5637 and T24 cells.Moreover, the decreased protein levels of JAK1 and STAT3 caused by BBR treatment could not be restored by MG132 (Supplementary Fig. 6b).All these results suggest that downregulation of JAK1 and STAT3 expression induced by BBR was not mediated by regulating transcription or protein degradation.MicroRNAs (miRNAs) degrades or inhibits translation of downstream target genes via binding to complementary mRNAs.Accumulating evidence has proved that dysregulated miRNAs induced by drug treatment were important regulators of cell fate in cancer progression 25 .Thus, we then investigated whether BBR downregulated JAK1 and STAT3 via miRNAs.The online softwares-TargetScan and miR WALK were utilized to predict miRNAs targeting both JAK1 and STAT3, and 5 candidate miRNAs were identi ed (Fig. 6b).qRT-PCR assays showed that only miR-17-5p level was upregulated by BBR treatment (Fig. 6c).Further analysis revealed that BBR promoted expression of miR-17-5p in a concentrationdependent manner (Fig. 6d).Importantly, transfection of miR-17-5p mimics signi cantly decreased protein levels of JAK1 and STAT3 in T24 and 5637 cells, leading to downregulate p-STAT3 expression (Fig. 6e).Moreover, miR-17-5p mimics signi cantly inhibited cell proliferation (Fig. 6f) in 5637 cells, similar with that of BBR.To determine whether miR-17-5p directly binds to 3' UTR regions of JAK1 and STAT3 as in-silico prediction (Supplementary Fig. 6c), dual-reporter luciferase assays were performed.As shown in Fig. 6g, the activities of reporters containing wild-type JAK1 and STAT3 3'UTR, but not the miR-17-5p binding sites mutant 3'UTR, were decreased by transfection of miR-17-5p mimics.Collectively, these data indicate that miR-17-5p directly binds to 3' UTR of JAK1 and STAT3 and inhibits their expression in BCa cells.
Last, we examined JAK1 and STAT3 expression levels in xenograft tumors.The results showed that mRNA levels of JAK1 and STAT3 were not signi cantly altered by BBR treatment (Fig. 7a-b), however, miR-17-5p expression was obviously upregulated in BBR-treatment tumors (Fig. 7c).Moreover, the results of western blot and IHC analyses on xenograft tumors con rmed that BBR decreased JAK1, STAT3 and p-STAT3 protein levels in vivo (Fig. 7d-e).Collectively, these results suggest that BBR promotes miR-17-5p expression to suppress JAK1 and STAT3 both in vivo and vitro.

Discussion
great progresses in treatment of BCa, emerging medicines only provide therapeutic bene ts in certain applicable patients.Most patients ultimately undergo cancer progression, which is still a great challenge for BCa clinical management 4 .Therefore, novel drugs for BCa treatment are highly needed.Previous studies have reported that BBR inhibits proliferation, migration and induces apoptosis in T24 cells 19,20 .In this study, we demonstrated that BBR signi cantly inhibited proliferation, migration and invasion of BCa cells.Notably, BBR exhibited much less evident anti-proliferation effects on human normal urothelial cells (SV-HUC-1), suggesting the potential of BBR in BCa treatment.Moreover, we found that BBR induced apoptosis in p53 de cient while caused senescence in p53 pro cient BCa cells.Further analyses on the underlying mechanism revealed that BBR inhibited JAK1-STAT3 signaling pathway to exert its cytotoxic effects.
Hyperactivation of STAT3 occurs in over 70% of human malignant cancers, including bladder cancer, which usually results in poor patient outcomes 26 .Previous studies have reported the important role of STAT3 in BCa.Recruitment and activation of STAT3 induce epithelial-mesenchymal transition (EMT) to maintain metastatic potential of BCa 10,27 .STAT3 activation in urothelial stem cells leads to development of invasive BCa 9 , and activated STAT3 triggers self-renewal of bladder cancer stem cells, which plays a crucial role in tumor initiation, progression and drug resistance 11,28 .On the contrary, blocking STAT3 signaling impairs progressions of bladder cancer both in vitro and vivo 13,29 .Moreover, STAT3 inhibition sensitizes bladder cancer to anti-PD-L1 immune therapy and reverses cisplatin resistance [11][12][13] , suggesting that STAT3 may serve as an attractive target for BCa management.BBR was reported to inhibit STAT3 activation in several malignant tumors.BBR suppresses tumorigenesis and tumor growth via inhibiting STAT3 activation induced by cancer-associated broblasts (CAFs) in colorectal cancer and nasopharyngeal carcinoma 30,31 .Activation of STAT3 mediated by COX2-PGE2 pathway could be inhibited by BBR, which leads to inhibition of migration and invasion in colorectal cancer 32 .Besides, BBR is proved to enhance the antitumor effects of EGFR inhibitors in gastric cancer by inhibiting STAT3 activation 33 .Inhibition of STAT3 activation by BBR is also reported in autoimmune myocarditis, ulcerative colitis, ectopic stromal cells, podocytes and ocular Behcet's disease via down-regulating expression of IL-6, CXCL12, IL-17, etc [34][35][36][37][38] .All these results indicate that STAT3 might be a key target through which BBR exerts its effects.Notably, the majority of these studies showed that though activation of STAT3 was decreased by BBR treatment, total STAT3 protein levels were not affected.Here, we found that BBR downregulated expression of both total and phosphorylated STAT3 in BCa cells.Selectively blocking STAT3 inhibited cell viability and induced apoptosis or senescence in BCa cells, similar to that of BBR.While, overexpression of exogenous STAT3 didn't attenuate the effects of BBR or restore p-STAT3 levels downregulated by BBR in BCa cells, indicating that BBR exerted cytotoxic effects in BCa cells via suppressing both the total expression and activation of STAT3.
In tumor microenvironment, activation of STAT3 in response to cytokines is mediated by JAKs 39 .Suppression of JAKs results in abolishing STAT3 activation, which leads to repress progression and reverse chemoresistance in multiple cancers, including BCa.Importantly, several clinical studies have proved JAK inhibitors as an attractive candidate target for various malignancies 40 .BBR is reported to inhibit expression and phosphorylation of JAK1 and induces apoptosis in keratinocytes 23 , however, the underlying mechanism is poorly understood.Here we demonstrated that BBR inhibited expression of JAK1, and overexpression of JAK1 effectively restored STAT3 activation down-regulated by BBR, and attenuated BBR-induced proliferation inhibition, cell cycle arrest and apoptosis, indicating that JAK1 is the key target of BBR in BCa cells.Until now, a large number of studies have demonstrated the effect of BBR on repressing STAT3 activation through multiple ways, however, seldom research has discovered inhibition of JAK1-STAT3 signaling via suppressing both expression and activation of STAT3.In this study, we showed that BBR down-regulated expressions of both JAK1 and STAT3, which resulted in down-regulation of STAT3 activity.To our knowledge, it is the rst time that BBR is proved to inhibit both JAK1 and STAT3 total protein expression in neoplasm.Our results revealed further regulatory mechanism of BBR on JAK1-STAT3 axis, which would provide supporting evidence for potential clinical application.
MicroRNAs (miRNAs) are endogenously non-coding RNAs about 19-23 nucleotides in length.miRNAs bind with imperfect complementarity to mRNAs of their target genes and facilitate translational repression or mRNA degradation.Accumulating evidence suggests that aberrant expressions of miRNAs In summary, we demonstrated that BBR effectively inhibited proliferation, migration, invasion and induced apoptosis or senescence of BCa cells.Moreover, we showed that BBR exerted its anti-tumor effects via suppressing JAK1-STAT3 signaling pathway.BBR could promote expression of miR-17-5p and consequently down-regulates JAK1 and STAT3 expression, indicating miR-17-5p-JAK1-STAT3 axis is involved in the anti-tumor activity of BBR in BCa (Fig. 7f).As most available data has shown that BBR was relatively safe in the majority of laboratory and clinical situations 60 , our results provided supporting evidence for the clinical use of BBR in BCa treatment.