Hepatocellular carcinoma-derived exosomal miRNA-761 regulates the tumor microenvironment by targeting the SOCS2/JAK2/STAT3 pathway

BACKGROUND:Exosomes and exosomal microRNAs have been implicated in tumor occurrence and metastasis.Our previous study showed that microRNA-761(miR-761)is overexpressed in hepatocellular carcinoma(HCC)tissues and that its inhibition affects mitochondrial function and inhibits HCC metastasis.The mechanism by which exosomal miR-761 modulates the tumor microenvironment has not been elucidated.METHODS:Exosomal miR-761 was detected in six cell lines.Cell counting kit-8(CCK-8)and transwell migration assays were performed to determine the function of exosomal miR-761 in HCC cells.The luciferase reporter assay was used to analyze miR-761 target genes in normal fi broblasts(NFs).The inhibitors AZD1480 and C188-9 were employed to determine the role of the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in the transformation of cancer-associated fi broblasts(CAFs).RESULTS:In this study,we characterized the mechanism by which miR-761 reprogrammed the tumor microenvironment.We found that HCC-derived exosomal miR-761 was taken up by NFs.Moreover,HCC exosomes aff ected the tumor microenvironment by activating NFs via suppressor of cytokine signaling 2(SOCS2)and the JAK2/STAT3 signaling pathway.CONCLUSIONS:These results demonstrated that exosomal miR-761 modulated the tumor microenvironment via SOCS2/JAK2/STAT3 pathway-dependent activation of CAFs.Our fi ndings may inspire new strategies for HCC prevention and therapy.

Correlation of STAT3, CEA in lung adenocarcinoma cell A549

Objective: The purpose of this study was to analyze the relationship between signal transducer and activator of transcription 3 (STAT3) and carcinoembryonic antigen (CEA) in lung adenocarcinoma cell A549, and to explore the value of STAT3 on early diagnosis of lung adenocarcinoma. Methods: The expression of CEA, STAT3 mRNA and it's protein in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5 were tested by immunohistochemistry staining (PV) and quantitative real time fluorescent PCR. The correlation between STAT3 and CEA in human lung adenocarcinoma cell A549 was analyzed. Results: The protein and mRNA levels of STAT3, CEA in lung adenocarcinoma cell A549 were apparently higher than those in normal human lung cells MRC-5. The levels of STAT3 mRNA and it's protein were positively correlated with CEA in lung adenocarcinoma cell A549. Conclusion: STAT3 have the same value in diagnosis of lung adenocarcinoma.

Knockdown of STAT3 by iRNA Inhibiting Migration and Invasion of Epithelial Ovarian Cancer Cells

Signal transducer and activator of transcription 3（STAT3） is a dual functional transcription factor with the functions of signal transduction and transcription regulation. It is reported that the expression of STAT3 in ovarian cancer is significantly higher and STAT3 can facilitate ovarian cancer growth and metastasis. To clarify the definite effect and molecular mechanism of STAT3 involved in ovarian cancer growth and metastasis, STAT3 expression was significantly downregulated by transfecting ovarian cancer model SK-OV-3 cells with the plasmid vector which express specific RNAi that targets human STAT3. The downregulated STAT3 not only decreased the invasion and migration but also inhibited the proliferation of SK-OV-3 cells. Western blot assay shows that the expression of vascular endothelial growth factor（VEGF） and that of Survivin were reduced in the cells with the plasma vector expressing specific RNAi that targets human STAT3. These results demonstrate that STAT3 involved in the invasion and migration of SK-OV-3 regulates the expression of VEGF and Survivin. In addition, VEGF and Survivin could play an important role in ovarian cancer growth and metastasis.

Regulation Mechanism of TFP on TGF-β1/STAT3 Signaling Pathway in Immune-mediated Liver Injury in Mice

[Objectives]To study the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP)on immune-mediated liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide(BCG+LPS)in mice,and to explore its action mechanism.[Methods]60 Kunming mice were divided into normal group,model group,control group(bifendate)and TFP low,medium and high dose groups according to random number table method,with 10 mice in each group.On the first day of modeling,mice were injected with 0.2 mL of BCG solution(12.5 mg/mL)through the tail vein,and on the eleventh day,0.2 mL of LPS(37.5μg/mL)were injected into the tail vein to prepare a mouse model of immune-mediated liver injury;from the first day of modeling,the normal group and the model group were administered intragastrically with the corresponding volume of distilled water,and the bifendate group and the TFP high,medium,and low dose groups were administered intragastrically with the corresponding doses once a day for 11 d.After the last time administration,fasting but giving water for 16 h,took blood from eyes,then collected the liver tissue.The levels of alanine transaminase(ALT)and aspartate transaminase(AST)in serum were detected by biochemical method;transforming growth factor-β1(TGF-β1),intercellular adhesion molecule-1(ICAM-1),interleukin-6(IL-6)and interleukin-1β(IL-1β)expression levels in liver tissue were detected by enzyme-linked immunosorbent assay(ELISA);phosphorylated protein tyrosine kinase JAK-2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)protein expression levels were detected by Western Blot method;the degree of liver tissue lesions was detected by HE staining.[Results]Compared with the model group,the levels of ALT and AST in the serum of mice in each dose group of TFP(high dose 600 mg/kg,medium dose 400 mg/kg,and low dose 200 mg/kg)were reduced,and the activities of T-SOD and GSH-Px were increased;the content or expression ofβ1,ICAM-1,IL-6,IL-1βdecreased,and the expression of p-JAK2 and p-STAT3 protein decreased;pathological sections showed that the degree of inflammatory necrosis and the degree of lesions in the liver tissues of each dose group of TFP were reduced by varying degrees.[Conclusions]TFP has a protective effect on BCG+LPS-induced immune-mediated liver injury in mice.The mechanism may be related to regulating the phosphorylation level of JAK2 and inhibiting the inflammatory reaction,thereby regulating the TGF-β1/STAT3 signaling pathway and improving the immune-mediated liver injury.

Identification of marine natural product Pretrichodermamide B as a STAT3 inhibitor for efficient anticancer therapy

The Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT3)regulates the expression of various critical mediators of cancer and is considered as one of the central communication nodes in cell growth and survival.Marine natural products(MNP)represent great resources for discovery of bioactive lead compounds,especially anti-cancer agents.Through the medium-throughput screening of our in-house MNP library,Pretrichodermamide B,an epidithiodiketopiperazine,was identified as a JAK/STAT3 signaling inhibitor.Further studies identified that Pretrichodermamide B directly binds to STAT3,preventing phosphorylation and thus inhibiting JAK/STAT3 signaling.Moreover,it suppressed cancer cell growth,in vitro,at low micromolar concentrations and demonstrated efficacy in vivo by decreasing tumor growth in a xenograft mouse model.In addition,it was shown that Pretrichodermamide B was able to induce cell cycle arrest and promote cell apoptosis.This study demonstrated that Pretrichodermamide B is a novel STAT3 inhibitor,which should be considered for further exploration as a promising anti-cancer therapy.

Myeloid peroxisome proliferator-activated receptor α deficiency accelerates liver regeneration via IL-6/STAT3 pathway after 2/3 partial hepatectomy in mice

Background:Liver regeneration is a fundamental process for sustained body homeostasis and liver function recovery after injury.Emerging evidence demonstrates that myeloid cells play a critical role in liver regeneration by secreting cytokines and growth factors.Peroxisome proliferator-activated receptorα(PPARα),the target of clinical lipid-lowering fibrate drugs,regulates cell metabolism,proliferation,and survival.However,the role of myeloid PPARαin partial hepatectomy(PHx)-induced liver regeneration remains unknown.Methods:Myeloid-specific PPARa-deficient(Ppara^(Mye−/−))mice and the littermate controls(Ppara^(fl/fl))were subjected to sham or 2/3 PHx to induce liver regeneration.Hepatocyte proliferation and mitosis were assessed by immunohistochemical(IHC)staining for 5-bromo-2'-deoxyuridine(BrdU)and Ki67 as well as hematoxylin and eosin(H&E)staining.Macrophage and neutrophil infiltration into livers were reflected by IHC staining for galectin-3 and myeloperoxidase(MPO)as well as flow cytometry analysis.Macrophage migration ability was evaluated by transwell assay.The mRNA levels for cell cycle or inflammation-related genes were measured by quantitative real-time RT-PCR(qPCR).The protein levels of cell proliferation related protein and phosphorylated signal transducer and activator of transcription 3(STAT3)were detected by Western blotting.Results:Ppara^(Mye−/−)mice showed enhanced hepatocyte proliferation and mitosis at 32 h after PHx compared with Ppara^(fl/fl)mice,which was consistent with increased proliferating cell nuclear antigen(Pcna)mRNA and cyclinD1(CYCD1)protein levels in Ppara^(Mye−/−)mice at 32 h after PHx,indicating an accelerated liver regeneration in Ppara^(Mye−/−)mice.IHC staining showed that macrophages and neutrophils were increased in Ppara^(Mye−/−)liver at 32 h after PHx.Livers of Ppara^(Mye−/−)mice also showed an enhanced infiltration of M1 macrophages at 32 h after PHx.In vitro,Ppara-deficient bone marrow-derived macrophages(BMDMs)exhibited markedly enhanced migratory capacity and upregulated M1 genes Il6 and Tnfa but downregulated M2 gene Arg1 expressions.Furthermore,the phosphorylation of STAT3,a key transcript factor mediating IL6-promoted hepatocyte survival and proliferation,was reinforced in the liver of Ppara^(Mye−/−)mice after PHx.Conclusions:This study provides evidence that myeloid PPARαdeficiency accelerates PHx-induced liver regeneration via macrophage polarization and consequent IL-6/STAT3 activation,thus providing a potential target for manipulating liver regeneration.

Regulatory Effect of JAK2/STAT3 on the Immune Function of Endotoxin-tolerant Dendritic Cells and its Involvement in Acute Liver Failure

Background and Aims:Acute liver failure(ALF)is a potentially fatal clinical syndrome with no effective treatment.This study aimed to explore the role of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)pathway in modulating the phenotype and immune function of endotoxin-tolerant dendritic cells(ETDCs).In addition,we explored the use of EDTCs in an experimental model of ALF and investigated the associated mechanisms.Methods:In the in vitro experiment,ETDCs were transfected with adenovirus to induce SOCS1^(+/+)ETDCs and SOCS1^(−/−)ETDCs.Thereafter,costimulatory molecules and mixed lymphocyte reaction were assessed.Experimental mice were randomly divided into normal control,ALF,ALF+mock-ETDCs,ALF+SOCS1^(+/+)ETDCs,ALF+AG490,and ALF+AG490+SOCS1^(+/+)ETDCs groups.We examined the therapeutic effect of adoptive cellular immunotherapy by tail-vein injection of target ETDCs 12 h before ALF modeling.AG490,a JAK2/STAT3 inhibitor,was used in the in vivo experiment to further explore the protective mechanism of SOCS1^(+/+)ETDCs.Results:Compared with control ETDCs,SOCS1^(+/+)ETDCs had lower expression of costimulatory molecules,weaker allostimulatory ability,lower levels of IL-6 and TNF-αexpression and higher IL-10 secretion.SOCS1^(−/−)ETDCs showed the opposite results.In the in vivo experiments,the ALF+SOCS1^(+/+)ETDCs and ALF+AG490+SOCS1^(+/+)ETDCs groups showed less pathological damage and suppressed activation of JAK2/STAT3 pathway.The changes were more pronounced in the ALF+AG490+SOCS1^(+/+)ETDCs group.Infusion of SOCS1^(+/+)ETDCs had a protective effect against ALF possibly via inhibition of JAK2 and STAT3 phosphorylation.Conclusions:The SOCS1 gene had an important role in induction of endotoxin tolerance.SOCS1^(+/+)ETDCs alleviated lipopolysaccharide/D-galactosamine-induced ALF by downregulating the JAK2/STAT3 signaling pathway.

Effect of electroacupuncture on JAK2/STAT3 pathway in synovial tissues of rats with rheumatoid arthritis

Objective: To observe the effect of electroacupuncture (EA) on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in knee joint synovial tissues of rats with rheumatoid arthritis (RA) and to explore the action mechanism of EA on RA. Methods: Twelve of the 48 SPF male Sprague-Dawley (SD) rats were assigned to a normal group by the random number table method. The remaining 36 rats were subjected to RA model preparation by intradermal injection of the Freund's complete adjuvant into the right hind foot pad of each rat under sterile conditions. After the model was successfully prepared, rats were then divided into a model group, a drug group and an EA group according to a random number table method (n=12). Rats in the drug group were treated with 2 mL aqueous solution of tripterygium glycosides [8.1 mg/(kg?bw)];rats in the EA group were treated with EA at bilateral Yanglingquan (GB 34) and Zusanli (ST 36), for 30 min each time;rats in the normal group and the model group were placed in a special rat fixation tank for 30 min each time, and received the same dose of normal saline as those in the drug group. Rats in all groups received intervention once a day for 4 weeks. Diameter of rat ankle joint and rat arthritis index were measured before and after the intervention. At the end of the experiment, the expressions of phospho-JAK2 and phospho-STAT3 were determined by immunohistochemistry. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect JAK2 and STAT3 mRNAs expressions. Results: After the model was produced, the arthritis index >2 was considered successful in model preparation. Compared with the model group, the ankle joint diameters and arthritis indexes of rats in the drug group and the EA group were significantly lower (all P<0.01);immunohistochemical staining cells with phospho-JAK2 and phospho-STAT3 were significantly decreased (all P<0.01);the expression levels of JAK2 and STAT3 mRNAs were decreased with statistical differences (all P<0.01). There were no significant differences between the EA group and the drug group (all P>0.05). Conclusion: EA can alleviate the inflammatory response of RA rats, improve their pathological conditions, reduce the expressions of phospho-JAK2 and phospho-STAT3 in the synovial tissue of knee joint, and decrease the expressions of JAK2 and STAT3 mRNAs. The therapeutic effect of EA is comparable to that of the tripterygium glycosides. The mechanism of EA treatment may be related to the inactivation of the JAK2/STAT3 pathway.

STAT3 signal that mediates the neural plasticity is involved in willed-movement training in focal ischemic rats

Willed-movement training has been demonstrated to be a promising approach to increase motor per- formance and neural plasticity in ischemic rats. However, little is known regarding the molecular signals that are in- volved in neural plasticity following willed-movement training. To investigate the potential signals related to neural plasticity following willed-movement training, littermate rats were randomly assigned into three groups： middle cerebral artery occlusion, environmental modification, and willed-movement training. The infarct volume was measured 18 d after occlusion of the right middle cerebral artery. Reverse transcription-polymerase chain reaction （PCR） and im- munofluorescence staining were used to detect the changes in the signal transducer and activator of transcription 3 （STAT3） mRNA and protein, respectively. A chromatin immunoprecipitation was used to investigate whether STAT3 bound to plasticity-related genes, such as brain-derived neurotrophic factor （BDNF）, synaptophysin, and protein in- teracting with C kinase 1 （PICK1）. In this study, we demonstrated that STAT3 mRNA and protein were markedly increased following 15-d willed-movement training in the ischemic hemispheres of the treated rats. STAT3 bound to BDNF, PICK1, and synaptophysin promoters in the neocortical cells of rats. These data suggest that the increased STAT3 levels after willed-movement training might play critical roles in the neural plasticity by directly regulating plasticity-related genes.

Protein tyrosine phosphatase 1B expression contributes to the development of breast cancer

The protein tyrosine phosphatase 1B（PTP1B）is an important regulator of metabolism.The relationship between PTP1B and tumors is quite complex.The purpose of this study is to explore the expression pattern and role of PTP1B in breast cancer.The expression of PTP1B was detected in 67 samples of breast cancer tissue by Western blot.Cell growth assay,Transwell migration assay,and Scratch motility assay were used to examine the proliferation and migration of MCF-7 with and without PTP1B.The total levels and phosphorylated levels of signal transduction and activator of transcription 3（STAT3）and the expression of C-C motif chemokine ligand 5（CCL5）were also examined by Western blot.PTP1B was overexpressed in over 70%of breast cancer tissues,correlating with patients with estrogen receptor（ER）-negative,progesterone receptor（PR）-negative,and human epidermal growth factor receptor 2（HER2）-positive tumors.The data also showed that both tumor size and lymph node metastasis were significantly higher in patients with a higher level of PTP1B.The proliferation and migration of MCF-7 cells were found to be inhibited after knocking down the gene of PTP1B.Our data also showed that PTP1B could up-regulate the dephosphorylated level of STAT3,which could increase the expression of CCL5.These phenomena indicated that PTP1B may play a crucial role in the development of breast cancer.

Novel thioredoxin reductase inhibitor butaselen inhibits tumorigenesis by down-regulating programmed death-ligand 1 expression

The thioredoxin system plays a role in a variety of physiological functions, including cell growth, differenti- ation, apoptosis, tumorigenesis, and immunity. We previously confirmed that butaselen （BS）, a novel thioredoxin reductase inhibitor, can inhibit the growth of various human cancer cell lines, yet the underlying mechanism remains elusive. In this study, we investigated the anti-tumor effect of BS in vivo through regulating the immune system of KM mice. We found that BS inhibits tumor proliferation by promoting the activation of splenic lymphocytes in mice. BS can elevate the percentage of CD^4-CD8^＋ T lymphocytes and the secretion of downstream cytokines in mice via downregulating the expression of programmed death-ligand 1 （PD-L1） on the tumor cells＇ surface in vivo. Further study in HepG2 and BEL-7402 cells showed that decrease of PD-L1 level after BS treatment was achieved by inhibiting signal transducer and activator of transcription 3 （STAT3） phosphorylation. Taken together, our results suggest that BS has a role in promoting the immune response by reducing PD-L1 expression via the STAT3 pathway, and subsequently suppresses tumorigenesis.

Interleukin-22 in the pathogenesis and potential treatment of liver diseases

Interleukin(IL)-22,a member of the IL-10 cytokine family,plays critical roles in tissue repair and host defense.IL-22 binds to its hetereodimeric receptor(R)composed of IL-22R1 and IL-10R2 and activates downstream signaling,including Signal transducer and activator of transcription 3(STAT3)pathways.IL-22 promotes the expression of survival genes in a STAT3-dependent manner.IL-22R1 expression is restricted mainly in epithelial cells while IL-10R2 is ubiquitously expressed in almost all cell types.In the liver,IL-22R1 is expressed in hepatocytes,liver progenitor cells,hepatic stellate cells and liver cancer cells.IL-22 protects the liver in various liver damage models and promotes liver regeneration after liver injury.IL-22 also helps to resolve liver fibrosis by inducing hepatic stellate cell senescence.IL-22 is considered a pro-inflammatory cytokine in viral hepatitis although it does not directly act on immune cells.IL-22 is reported to be involved in the development of liver cancer and in regulating energy metabolism.Studies on IL-22 in liver inflammation,injury and repair will provide valuable information to clarify IL-22 as a potential candidate for treating liver injury and fibrosis.

Targeting the complement system in pancreatic cancer drug resistance:a novel therapeutic approach

Pancreatic cancer is ranked as the fourth leading cause of cancer-related mortality and is predicted to become the second leading cause of cancer-related death by 2030.The cause of this high mortality rate is due to pancreatic ductal adenocarcinoma’s rapid progression and metastasis,and development of drug resistance.Today,cancer immunotherapy is becoming a strong candidate to not only treat various cancers but also to combat against chemoresistance.Studies have suggested that complement system pathways play an important role in cancer progression and chemoresistance,especially in pancreatic cancer.A recent report also suggested that several signaling pathways play an important role in causing chemoresistance in pancreatic cancer,major ones including nuclear factor kappa B,signal transducer and activator of transcription 3,c-mesenchymal-epithelial transition factor,and phosphoinositide-3-kinase/protein kinase B.In addition,it has also been proven that the complement system has a very active role in establishing the tumor microenvironment,which would aid in promoting tumorigenesis,progression,metastasis,and recurrence.Interestingly,it has been shown that the downstream products of the complement system directly upregulate inflammatory mediators,which in turn activate these chemo-resistant pathways.Therefore,targeting complement pathways could be an innovative approach to combat against pancreatic cancer drugs resistance.In this review,we have discussed the role of complement system pathways in pancreatic cancer drug resistance and a special focus on the complement as a therapeutic target in pancreatic cancer.