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Prostate cancer (PCa) is a common malignant tumor of the male urinary system and ranks the second in the causes of tumor-related deaths. Differential display code 3 (DD3) is a noncoding gene that is specifically expressed in PCa. High expression of sperm-associated antigen 9 (SPAG9) is closely related to tumorigenesis of PCa, and SPAG9 is a therapeutic target for PCa. In this study, a new oncolytic adenovirus DD3-ZD55-SPAG9 was constructed by using DD3 promoter to enhance the efficacy and safety of adenovirus. The combined use of DD3-ZD55-SPAG9 and docetaxel showed that DD3-ZD55-SPAG9 significantly improved the anti-tumor efficacy of docetaxel in PCa both in vitro and in vivo. The mechanism was related to the induction of tumor cell apoptosis and the inhibition of tumor cell invasion. In conclusion, DD3-ZD55-SPAG9 combined with docetaxel is an effective strategy for PCa therapy.

Supplementary Figure Legends

Quantitation for Western Blot data. A. Quantitation for Fig. 1C/D. B. Quantitation for Fig. 2A/D. C. Quantitation for Fig. 3D. D. Quantitation for Fig. 5A.

30 metastasis-related genes used for screening.

Morphology change of C4-2 S/P cells transfected with TR4 shRNA. C4-2 CD133+ S/P cells with TR4 knock down (B) showed morphology change to more differentiated PCa cells than control group (A).

The testicular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily that mediates various biologic functions with key impacts on metabolic disorders and tumor progression. Here, we demonstrate that TR4 may play a positive role in prostate cancer CD133+ stem/progenitor (S/P) cell invasion. Targeting TR4 with lentiviral silencing RNA significantly suppressed prostate cancer CD133+ S/P cell invasion both in vitro and in vivo. Mechanism dissection found that TR4 transcriptionally regulates the oncogene EZH2 via binding to its 5′ promoter region. The consequences of targeting TR4 to suppress EZH2 expression may then suppress the expression of its downstream key metastasis-related genes, including NOTCH1, TGFβ1, SLUG, and MMP9. Rescue approaches via adding the EZH2 reversed the TR4-mediated prostate cancer S/P cell invasion. Together, these results suggest that the TR4→EZH2 signaling may play a critical role in the prostate cancer S/P cell invasion and may allow us to develop a better therapy to battle the prostate cancer metastasis. Mol Cancer Ther; 14(6); 1445–53. ©2015 AACR.

Background Segmental testicular infarction is a rare condition that often occurs in the upper pole of the left testicle and usually presents with acute onset of scrotal pain. Contrast-enhanced ultrasound and MR are essential for diagnosing and differentiating segmental testicular infarction in clinical practice, and conservative treatment can only be adopted after a definitive diagnosis. In the present case, after conservative treatment, the infarct volume was reduced, the blood flow around the infarct was increased, and blood flow signals appeared in the infarct. We performed a correlation analysis to investigate the causes of these changes. Case presentation A 33-year-old male, without any specific disease history, was admitted to the hospital with a 5-day history of left testicular pain, and the imaging showed focal necrosis of the left testicle with hemorrhage. He was diagnosed with segmental testicular infarction after differentiating and excluding it from malignant tumors. Conservative medical treatment was given, and the symptoms of testicular pain were relieved after treatment. After discharge, regular reexamination at follow-ups showed that the infarct’s size was reduced, the blood flow around the infarct was increased, and blood flow signals appeared in the infarct. Conclusion Conservative treatment has become the standard treatment currently adopted after confirming the diagnosis of segmental testicular infarction through contrast-enhanced ultrasound and MR. The blood flow changes in and around the focus of testicular infarction can be related to various factors. At present, relevant conclusions of the underlying mechanisms were mainly deduced from infarction studies of other related organs such as the heart and brain; thus, the specific pathological mechanism needs further experimental verification.

While testicular nuclear receptor 4 (TR4) may promote prostate cancer (PCa) metastasis, its role in the clear cell renal cell carcinoma (ccRCC) remains unclear. Here we found a higher expression of TR4 in ccRCC tumors from patients with distant metastases than those from metastasis-free patients, suggesting TR4 may play positive roles in the ccRCC metastasis. Results from multiple in vitro ccRCC cell lines also confirmed TR4's positive roles in promoting ccRCC cell invasion/migration via altering the microRNA (miR-32-5p)/TR4/HGF/Met/MMP2-MMP9 signaling. Mechanism dissection revealed that miR-32-5p could suppress TR4 protein expression levels via direct binding to the 3'UTR of TR4 mRNA, and TR4 might then alter the HGF/Met signaling at the transcriptional level via direct binding to the TR4-response-elements (TR4RE) on the HGF promoter. Then the in vitro data also demonstrated the efficacy of Sunitinib, a currently used drug to treat ccRCC, could be increased after targeting this newly identified miR-32-5p/TR4/HGF/Met signaling. The preclinical study using the in vivo mouse model with xenografted ccRCC cells confirmed the in vitro cell lines data. Together, these findings suggest that TR4 is a key player to promote ccRCC metastasis and targeting this miR-32-5p/TR4/HGF/Met signaling with small molecules including TR4-shRNA or miR-32-5p may help to develop a new therapy to better suppress the ccRCC metastasis.

Castration‑resistant prostate cancer (CRPC) is difficult to treat in current clinical practice. Hypoxia is an important feature of the CRPC microenvironment and is closely associated with the progress of CRPC invasion. However, no research has been performed on the immune escape of CRPC from NK cells. The present study focused on this subject. Firstly, when the CRPC cell lines C4‑2 and CWR22Rv1 were induced by hypoxia, the expression of the UL16 binding protein (ULBP) ligand family of natural killer (NK) group 2D (NKG2D; ULBP‑1, ULBP‑2 and ULBP‑3) and MHC class I chain‑related proteins A and B (MICA/MICB) decreased. NKG2D is the main activating receptor of NK cells. Tumor cells were then co‑cultured with NK cells to conduct NK cell‑mediated cytotoxicity experiments, which revealed the decreased immune cytolytic activity of NK cells on hypoxia‑induced CRPC cells. In exploring the mechanism behind this observation, an increase in programmed death‑ligand 1 (PD‑L1) expression in CRPC cells induced by hypoxia was observed, while the addition of PD‑L1 antibody effectively reversed the expression of NKG2D ligand and enhanced the cytotoxic effect of NK cells on CRPC cells. In the process of exploring the upstream regulatory factors of PD‑L1, inhibition of the Janus kinase (JAK)1,2/signal transducer and activator of transcription 3 (Stat3) signaling pathway decreased the expression of PD‑L1 in CRPC cells. Finally, it was observed that combined inhibition of JAK1,2/PD‑L1 or Stat3/PD‑L1 was more effective than inhibition of a single pathway in enhancing the immune cytolytic activity of NK cells. Taking these results together, it is thought that combined inhibition of the JAK1,2/PD‑L1 and Stat3/PD‑L1 signaling pathways may enhance the immune cytolytic activity of NK cells toward hypoxia‑induced CRPC cells, which is expected to provide novel ideas and targets for the immunotherapy of CRPC.

Previous reports, including our experimental results, showed that macrophages migrate to prostate cancer (PCa) cells. We tested whether the migrated macrophages affect the susceptibility of castration-resistant PCa (CRPC) cells to cytotoxic actions of natural killer (NK) cells. We found treatment of tumor cells with the conditioned media (CM) of the PMA/IL-4 treated THP-1 cells (M2 type macrophages) (THP-1 CM) decreased the susceptibility of tumor cells to NK cell cytotoxicity, as a result of increased programmed death receptor ligand 1 (PD-L1) and decreased NK group 2D (NKG2D) ligands in CRPC cells. Meanwhile, the decreased susceptibility of tumor cells was also detected when NK cells were treated with THP-1 CM and used in NK cell cytotoxicity tests. Therefore, we observed higher resistance of CRPC cells when both tumor and NK cells were treated with THP-1 CM than when tumor cells or NK cells were individually treated. We further discovered that the PMA/IL-4 treated THP-1 cells secrete a high level of IL-6, so blocking the IL-6 action significantly decreased the PD-L1 level while recovering the NKG2D ligands, thus increasing the susceptibility of CRPC cells to NK cell action. Moreover, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling in triggering the THP-1 CM effect. Consequently, we found the susceptibility of CRPC cells to NK cells was increased when either JAK or Stat 3 inhibitor was added when tumor cells were treated with THP-1 CM, and that the best effect was observed when the JAK inhibitor and PD-L1 Ab were added together.

Castration-resistant prostate cancer (CRPC), also known as androgen-independent prostate cancer, frequently develops local and distant metastases, the underlying mechanisms of which remain undetermined. In the present study, surgical specimens obtained from patients with clinical prostate cancer were investigated, and it was revealed that the expression levels of ataxia telangiectasia mutated kinase (ATM) were significantly enhanced in prostate cancer tissues isolated from patients with CRPC compared with from patients with hormone‑dependent prostate cancer. CRPC C4‑2 and CWR22Rv1 cells lines were subsequently selected to establish prostate cancer models, and ATM knockout cells were established via lentivirus infection. The results of the present study demonstrated that the migration and epithelial‑mesenchymal transition (EMT) of ATM knockout cells were significantly decreased, which suggested that ATM is closely associated with CRPC cell migration and EMT. To further investigate the mechanisms underlying this process, programmed cell death 1 ligand 1 (PD‑L1) expression was investigated in ATM knockout cells. In addition, inhibitors of Janus kinase (JAK) and signal transducer and activator of transcription 3 (STAT3; Stattic) were added to C4‑2‑Sc and CWR22Rv1‑Sc cells, and the results demonstrated that PD‑L1 expression was significantly decreased following the addition of JAK inhibitor 1; however, no significant change was observed following the addition of Stattic. Furthermore, a PD‑L1 antibody and JAK inhibitor 1 were added to C4‑2‑Sc and CWR22Rv1‑Sc cells, and it was revealed that cell migration ability was significantly decreased and the expression of EMT‑associated markers was effectively reversed. The results of the present study suggested that via inhibition of the ATM‑JAK‑PD‑L1 signaling pathway, EMT, metastasis and progression of CRPC may be effectively suppressed, which may represent a novel therapeutic approach for targeted therapy for patients with CRPC.

Background Obesity affects prostate cancer (PCa) progression, and the periprostatic adipose tissue adjacent to the prostate is considered a driving force of disease progression. Adipocytes are the main cell population in adipose tissues and their paracrine role contributes to PCa progression, however its implication in modulating immune reactions remains largely unknown. We investigated the adipocyte role in controlling the susceptibility of castration-resistant PCa (CRPC) cells to the cytotoxic action of natural killer (NK) cells. Methods Using primary NK cells as the NK cell source, NK cell cytotoxicities to CRPC cells, either control media treated or adipocyte-conditioned media (CM) treated, were tested in lactate dehydrogenase (LDH) release-based assays. The levels of programmed death receptor ligand (PD-L1) and NK group 2D (NKG2D) ligands in adipocyte CM-treated CRPC cells were analyzed in qPCR analyses. Effects of blocking adipocyte action on altering PD-L1/NKG2D ligand levels and the susceptibility of CRPC cells to NK cell cytotoxicity were investigated. Results We found NK cell cytotoxicity to CRPC cells decreases when tumor cells are treated with adipocyte CM associated with PD-L1 and NKG2D ligand level alterations. Further, we discovered that the JAK/Stat3 signaling pathway was responsible for the adipocyte CM effect. Two adipokine molecules, IL-6 and leptin, were shown to be important in activation of the JAK/Stat3 signaling in CRPC cells to modulate the PD-L1/NKG2D ligand level alteration. Adding the inhibitors of JAK/Stat3 signaling or neutralizing antibodies of IL-6 or leptin increased the susceptibility of CRPC cells to NK cell action. Conclusions Blocking the adipocyte effect by inhibiting the IL-6/leptin-JAK/Stat3 signaling axis may enhance NK cell mediated immunity to CRPC cells and this strategy may help to develop future therapeutics to treat obese PCa patients.

To investigate whether IL‐6 signaling affects the susceptibility of castration‐resistant prostate cancer (CRPC) cells to cytotoxic action of natural killer (NK) cells, CRPC cell lines (having different IL‐6 levels) were developed by lentiviral transduction. While observing no secreted IL‐6 level in parental C4‐2 and CWR22Rv1 cells, we found the IL‐6 expression/secretion in these cells was induced after the transduction process and the IL‐6 level difference in C4‐2siIL‐6/sc and CWR22siIL‐6/sc cell CRPC cell sets could be detected. We then found that IL‐6‐knockdown cells were more susceptible to NK cell cytotoxicity than control cells due to lowered programmed death receptor ligand 1 (PD‐L1) and increased NK group 2D (NKG2D) ligand levels. In animal studies, to concur with the in vitro results, we found that IL‐6‐expressing cell‐derived tumors were more resistant to NK cell action than the tumors of IL‐6‐knockdown cells. Further, we discovered that JAK‐Stat3 is the most critical IL‐6 downstream signaling that modulates PD‐L1/NKG2D ligand levels in CRPC cells. Furthermore, inhibition of the JAK or Stat3 signaling effectively increased the susceptibility of C4‐2sc and CWRsc cells to NK cell cytotoxicity. We observed the most effective cytotoxicity when the PD‐L1 Ab and JAK inhibitor (or Stat 3 inhibitor) were used together. These results suggest that the strategy of targeting IL‐6 signaling (or its downstream signaling) may enhance the NK cell‐mediated immune action to CRPC tumors, thus yielding clinical implications in developing future immunotherapeutics of exploiting this strategy to treat patients with CRPC.

To evaluate the effectiveness and safety of low-concentration hydrogen peroxide solution (HPS) for continuous bladder irrigation after transurethral resection of the prostate (TURP).We retrospectively analyzed the clinical data about 148 cases of benign prostatic hyperplasia (BPH) treated by TURP from January 2013 to January 2016. Seventy-six of the patients received postoperative continuous bladder irrigation with 0.15% HPS (group A) and the other 72 with normal saline (group B). We compared the two groups of patients in their postoperative hemoglobin (Hb) levels, duration of bladder irrigation, frequency of catheter blockage, time of catheterization, and length of hospital stay.There were no statistically significant differences between the two groups of patients preoperatively in the prostate volume, International Prostate Symptoms Score, maximum urinary flow rate, postvoid residual urine, or levels of serum PSA and Hb (P0.05). At 48 hours after operation, a significantly less reduction was observed in the Hb level in group A than in group B (［3.38 ± 2.56］ vs ［7.29 ± 6.58］ g/L, P0.01). The patients of group A, in comparison with those of group B, also showed remarkably shorter duration of postoperative bladder irrigation (［32.57 ± 5.99］ vs ［46.10 ± 8.79］ h, P0.01), lower rate of catheter blockage (3.3% vs 11.8%, P0.01), shorter time of catheterization (［3.74 ± 0.79］ vs ［4.79 ± 0.93］ d, P0.01), and fewer days of postoperative hospital stay (［4.22 ± 0.81］ vs ［4.67 ± 0.88］ d, P0.01).Low-concentration HPS for continuous bladder irrigation after TURP can reduce blood loss, catheter blockage, bladder irrigation duration, catheterization time, and hospital stay, and therefore deserves a wide clinical application.目的： 评价低浓度过氧化氢溶液在经尿道前列腺电切术（TURP）后持续膀胱冲洗中有效性和安全性。方法： 回顾性分析2013年1月至2017年1月期间共148例良性前列腺增生（BPH）行TURP手术治疗的患者临床资料。根据患者术后持续膀胱冲洗液的不同分为A、B两组，A组76例接受0.15%过氧化氢溶液行膀胱冲洗；B组72例接受生理盐水行膀胱冲洗。记录两组患者术后血红蛋白浓度值下降情况，膀胱持续冲洗时间，导尿管堵塞次数、留置导尿管时间，术后住院天数。结果： 术前两组患者前列腺体积、国际前列腺症状评分（IPSS）、最大尿流率（Qmax）、残余尿（PVR）、PSA及血红蛋白值相比差异无统计学意义（P0.05）。术后48 h内平均血红蛋白（Hb）下降水平：A组Hb下降了（3.38±2.56） g/L，B组Hb下降了（7.29±6.58）g/L；持续膀胱冲洗时间：A组为（32.57±5.99）h，B组为（46.10±8.79） h；两组相比差异均有统计学意义（P0.01）。A组患者术后尿管堵塞率为3.3%、留置尿管时间为（3.74±0.79）d、住院天数为（4.22±0.81） d；B组术后尿管堵塞率为11.8%、留置尿管时间为（4.79±0.93）d、住院天数为（4.67±0.88）d；两组相比差异均有统计学意义（P0.01）。结论： 前列腺电切术后应用低浓度过氧化氢溶液行膀胱持续冲洗可以减少出血，降低导尿管堵塞发生率，缩短膀胱冲洗时间、术后导尿管留置时间和住院天数，值得在临床中进行推广。.

The insulin sensitizers, thiazolidinediones (TZDs), have been used as anti-diabetic drugs since the discovery of their ability to alter insulin resistance through transactivation of peroxisome proliferator-activated receptors (PPARs). However, their side effects in hepatitis, cardiovascular diseases, and bladder cancer resulted in some selling restrictions in the USA and Europe. Here, we found that the potential impact of TZDs on the prostate cancer (PCa) progression might be linked to the TR4 nuclear receptor expression. Clinical surveys found that 9% of PCa patients had one allele TR4 deletion in their tumors. TZD increased cell growth and invasion in PCa cells when TR4 was knocked down. In contrast, TZD decreased PCa progression in PCa cells with wild type TR4. Mechanism dissection found that the Harvey Rat Sarcoma (HRAS) oncogene increased on TZD treatment of the TR4 knocked-down CWR22Rv1 and C4-2 cells, and interruption with HRAS inhibitor resulted in reversal of TZD-induced PCa progression. Together, these results suggest that TZD treatment may promote PCa progression depending on the TR4 expression status that may be clinically relevant since extra caution may be needed for those diabetic PCa patients receiving TZD treatment who have one allele TR4 deletion.

A recent report indicated that the TR4 nuclear receptor might suppress the prostate cancer (PCa) initiation via modulating the DNA damage/repair system. Knocking-out peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor that shares similar ligands/activators with TR4, promoted PCa initiation. Here we found 9% of PCa patients have one allele of PPARG deletion. Results from in vitro cell lines and in vivo mouse model indicated that during PCa initiation TR4 roles might switch from suppressor to enhancer in prostate cells when PPARG was deleted or suppressed (by antagonist GW9662). Mechanism dissection found targeting TR4 in the absence of PPARG might alter the stem cell population and epithelial-mesenchymal transition (EMT) signals. Together, these results suggest that whether TR4 can enhance or suppress PCa initiation may depend on the availability of PPARG and future potential therapy via targeting PPARG to battle PPARG-related diseases may need to consider the potential side effects of TR4 switched roles during the PCa initiation.

A poly(2,2,6,6-tetramethylpiperidin-1-oxy-4-yl methacrylate)/carbon-nanotube-array (PTMA/CNT-array) electrode was used as a cathode to improve the high-rate charge/discharge performance in organic radical batteries. Scanning electron microscopy observations showed that the PTMA/CNT-array electrode provides continuous conduction paths for electrons, and its electrochemical behaviours were investigated using cyclic voltammetry, charge/discharge tests, and AC impedance measurements. The results indicated that the PTMA/CNT-array electrode exhibits a lower electron-transfer resistance between CNTs and either the current collector or CNTs compared with conventional PTMA/suspended-CNT composite electrodes, enhancing the C-rate performance of batteries.

While testicular nuclear receptor 4 (TR4) may promote prostate cancer (PCa) metastasis, its role in the clear cell renal cell carcinoma (ccRCC) remains unclear. Here we found a higher expression of TR4 in ccRCC tumors from patients with distant metastases than those from metastasis-free patients, suggesting TR4 may play positive roles in the ccRCC metastasis. Results from multiple in vitro ccRCC cell lines also confirmed TR4's positive roles in promoting ccRCC cell invasion/migration via altering the microRNA (miR-32-5p)/TR4/HGF/Met/MMP2-MMP9 signaling. Mechanism dissection revealed that miR-32-5p could suppress TR4 protein expression levels via direct binding to the 3'UTR of TR4 mRNA, and TR4 might then alter the HGF/Met signaling at the transcriptional level via direct binding to the TR4-response-elements (TR4RE) on the HGF promoter. Then the in vitro data also demonstrated the efficacy of Sunitinib, a currently used drug to treat ccRCC, could be increased after targeting this newly identified miR-32-5p/TR4/HGF/Met signaling. The preclinical study using the in vivo mouse model with xenografted ccRCC cells confirmed the in vitro cell lines data. Together, these findings suggest that TR4 is a key player to promote ccRCC metastasis and targeting this miR-32-5p/TR4/HGF/Met signaling with small molecules including TR4-shRNA or miR-32-5p may help to develop a new therapy to better suppress the ccRCC metastasis.

Testicular nuclear receptors 2 and 4 (TR2, TR4), also known as NR2C1 and NR2C2, belong to the nuclear receptor superfamily and were first cloned in 1989 and 1994, respectively. Although classified as orphan receptors, several natural molecules, their metabolites, and synthetic compounds including polyunsaturated fatty acids (PUFAs), PUFA metabolites 13-hydroxyoctadecadienoic acid, 15-hydroxyeicosatetraenoic acid, and the antidiabetic drug thiazolidinediones can transactivate TR4. Importantly, many of these ligands/activators can also transactivate peroxisome proliferator-activated receptor gamma (PPARγ), also known as NR1C3 nuclear receptor. Both TR4 and PPARγ can bind to similar hormone response elements (HREs) located in the promoter of their common downstream target genes. However, these two nuclear receptors, even with shared ligands/activators and shared binding ability for similar HREs, have some distinct functions in many diseases they influence. In cancer, PPARγ inhibits thyroid, lung, colon, and prostate cancers but enhances bladder cancer. In contrast, TR4 inhibits liver and prostate cancer initiation but enhances pituitary corticotroph, liver, and prostate cancer progression. In type 2 diabetes, PPARγ increases insulin sensitivity but TR4 decreases insulin sensitivity. In cardiovascular disease, PPARγ inhibits atherosclerosis but TR4 enhances atherosclerosis through increasing foam cell formation. In bone physiology, PPARγ inhibits bone formation but TR4 increases bone formation. Together, the contrasting impact of TR4 and PPARγ on different diseases may raise a critical issue about drug used to target any one of these nuclear receptors.

Testicular nuclear receptor 4 (TR4) plays protective roles against oxidative stress and DNA damage and might contribute to aging. Our recent clinical tumor tissue staining results showed higher expression of TR4 in prostate cancer (PCa) patients with high Gleason scores compared to the tissues with the low Gleason scores. In vitro migration/invasion assays after manipulation of the TR4 expression in PCa cells showed that TR4 promoted PCa cells migration/invasion. Mechanism dissection found that the CCL2/CCR2 signal plays the key role in the mediation of TR4-promoted PCa cells migration/invasion. Chromatin immunoprecipitation and Luciferase assays further confirmed TR4 modulation of CCL2 at the transcriptional level and addition of the CCR2 antagonist led to interruption of the TR4-enhanced PCa cells migration/invasion. Finally, the orthotopic xenografted mice studies using the luciferase expressing CWR22Rv1 cells found that TR4 enhanced PCa metastasis and this increased metastasis was reversed when the CCR2 antagonist was injected into the mice. Together, these in vitro and in vivo results revealed a positive role of TR4 in PCa metastasis and demonstrated CCL2/CCR2 signaling as an important mediator in exerting TR4 action. This finding suggests that TR4 may represent a biomarker related to PCa metastasis and targeting the TR4-CCL2/CCR2 axis may become a new therapeutic approach to battle PCa metastasis.

Testicular nuclear receptor 4 (TR4), a member of the nuclear receptor superfamily, plays important roles in metabolism, fertility and aging. The linkage of TR4 functions in cancer progression, however, remains unclear. Using three different mouse models, we found TR4 could prevent or delay prostate cancer (PCa)/prostatic intraepithelial neoplasia development. Knocking down TR4 in human RWPE1 and mouse mPrE normal prostate cells promoted tumorigenesis under carcinogen challenge, suggesting TR4 may play a suppressor role in PCa initiation. Mechanism dissection in both in vitro cell lines and in vivo mice studies found that knocking down TR4 led to increased DNA damage with altered DNA repair system that involved the modulation of ATM expression at the transcriptional level, and addition of ATM partially interrupted the TR4 small interfering RNA-induced tumorigenesis in cell transformation assays. Immunohistochemical staining in human PCa tissue microarrays revealed ATM expression is highly correlated with TR4 expression. Together, these results suggest TR4 may function as a tumor suppressor to prevent or delay prostate tumorigenesis via regulating ATM expression at the transcriptional level.

Peroxisome proliferator-activated receptor γ (PPARγ, NR1C3) and testicular receptor 4 nuclear receptor (TR4, NR2C2) are two members of the nuclear receptor (NR) superfamily that can be activated by several similar ligands/activators including polyunsaturated fatty acid metabolites, such as 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, as well as some anti-diabetic drugs such as thiazolidinediones (TZDs). However, the consequences of the transactivation of these ligands/activators via these two NRs are different, with at least three distinct phenotypes. First, activation of PPARγ increases insulin sensitivity yet activation of TR4 decreases insulin sensitivity. Second, PPARγ attenuates atherosclerosis but TR4 might increase the risk of atherosclerosis. Third, PPARγ suppresses prostate cancer (PCa) development and TR4 suppresses prostate carcinogenesis yet promotes PCa metastasis. Importantly, the deregulation of either PPARγ or TR4 in PCa alone might then alter the other receptor's influences on PCa progression. Knocking out PPARγ altered the ability of TR4 to promote prostate carcinogenesis and knocking down TR4 also resulted in TZD treatment promoting PCa development, indicating that both PPARγ and TR4 might coordinate with each other to regulate PCa initiation, and the loss of either one of them might switch the other one from a tumor suppressor to a tumor promoter. These results indicate that further and detailed studies of both receptors at the same time in the same cells/organs may help us to better dissect their distinct physiological roles and develop better drug(s) with fewer side effects to battle PPARγ- and TR4-related diseases including tumor and cardiovascular diseases as well as metabolic disorders.

Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133(+) stem/progenitor cells compared with CD133(-) non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133(+) population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC50 values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.

Testicular nuclear receptors 2 and 4 (TR2, TR4), also known as NR2C1 and NR2C2, belong to the nuclear receptor superfamily and were first cloned in 1989 and 1994, respectively. Although classified as orphan receptors, several natural molecules, their metabolites, and synthetic compounds including polyunsaturated fatty acids (PUFAs), PUFA metabolites 13-hydroxyoctadecadienoic acid, 15-hydroxyeicosatetraenoic acid, and the antidiabetic drug thiazolidinediones can transactivate TR4. Importantly, many of these ligands/activators can also transactivate peroxisome proliferator-activated receptor gamma (PPARγ), also known as NR1C3 nuclear receptor. Both TR4 and PPARγ can bind to similar hormone response elements (HREs) located in the promoter of their common downstream target genes. However, these two nuclear receptors, even with shared ligands/activators and shared binding ability for similar HREs, have some distinct functions in many diseases they influence. In cancer, PPARγ inhibits thyroid, lung, colon, and prostate cancers but enhances bladder cancer. In contrast, TR4 inhibits liver and prostate cancer initiation but enhances pituitary corticotroph, liver, and prostate cancer progression. In type 2 diabetes, PPARγ increases insulin sensitivity but TR4 decreases insulin sensitivity. In cardiovascular disease, PPARγ inhibits atherosclerosis but TR4 enhances atherosclerosis through increasing foam cell formation. In bone physiology, PPARγ inhibits bone formation but TR4 increases bone formation. Together, the contrasting impact of TR4 and PPARγ on different diseases may raise a critical issue about drug used to target any one of these nuclear receptors.

Background: One-third of US population is obese. The mortality of obese prostate cancer (PCa) patients is higher than non-obese patients, thus studying molecular mechanism of how obesity affects PCa progression has clinical relevance. The periprostatic adipose tissue adjacent to the prostate is considered a driving force of disease progression and adipocytes are the main cell population in adipose tissues. The paracrine role of adipocytes in PCa progression has been emphasized, but its implication in modulating immune reactions remains unknown. We investigated the role of adipocytes in the surveillance of castration-resistant PCa (CRPC) cells to immune actions. Especially we investigated the role of adipocytes in affecting tumor cell susceptibility to cytotoxic action of natural killer (NK) cells, as the strategy of improving the NK cell-mediated immunity is emerging as an immunotherapeutic option. Methods: Using primary NK cells as the NK cell source, NK cell cytotoxicities to CRPC cells, either control media treated or adipocyte-conditioned media (CM) treated, were tested in lactate dehydrogenase (LDH) release-based assays. The levels of programmed death receptor ligand (PD-L1) and NK group 2D (NKG2D) ligands in adipocyte CM-treated CRPC cells were analyzed in qPCR analyses. Effects of blocking adipocyte action on altering PD-L1/NKG2D ligand levels and the susceptibility of CRPC cells to NK cell cytotoxicity were investigated. Results: We found NK cell cytotoxicity to CRPC cells decreased when tumor cells were treated with adipocyte CM, and was associated with PD-L1 (up-regulation) and NKG2D ligand level (down-regulation) alterations. Further, we discovered that the JAK/Stat3 signaling pathway was activated by adipocyte CM. Two adipokine molecules, IL-6 and leptin, were shown to be important in the activation of the JAK/Stat3 signaling to modulate the PD-L1/NKG2D ligand level alterations in CRPC cells. Consequently, adding inhibitors of JAK/Stat3 signaling or neutralizing antibodies of IL-6 or leptin increased the susceptibility of CRPC cells to NK cell cytotoxic action. Conclusions and Impact: Results from our study showed the influence of adipocytes on tumor cells and resulted in reducing the susceptibility to NK cell-mediated antitumor immune function. This may be one of ways that adipocytes trigger CRPC cells to be more aggressive and out of immune surveillance control. Our study results showed that blocking the adipocyte effect by inhibiting the IL-6/leptin-JAK/Stat3 signaling axis enhanced the NK cell mediated immunity to CRPC cells. We suggest that this strategy can be applied to the development of future immune therapeutics to treat obese PCa patients. Citation Format: Lijun Xu, Mingjing Shen, Xiaodong Chen, Rongying Zhu, Dong-rong Yang, Ying Tsai, Peter Keng, Yuhchyau Chen, Soo Ok Lee. Adipocytes affect castration-resistant prostate cancer cells in resistance to cytotoxic action of NK cells through alterations of pd-l1/nkg2d ligand levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3147.

The focus of the present study was to evaluate transrectal real-time tissue elastography (RTE)-targeted two-core biopsy coupled with peak strain index for the detection of prostate cancer (PCa) and to compare this method with 10-core systematic biopsy. A total of 141 patients were enrolled for evaluation. The diagnostic value of peak strain index was assessed using a receiver operating characteristic curve. The cancer detection rates of the two approaches and corresponding positive cores and Gleason score were compared. The cancer detection rate per core in the RTE-targeted biopsy (44%) was higher compared with that in systematic biopsy (30%). The peak strain index value of PCa was higher compared with that of the benign lesion. PCa was detected with the highest sensitivity (87.5%) and specificity (85.5%) using the threshold value of a peak strain index of ≥5.97 with an area under the curve value of 0.95. When the Gleason score was ≥7, RTE-targeted biopsy coupled with peak strain index detected 95.6% of PCa cases, but 84.4% were detected using systematic biopsy. Peak strain index as a quantitative parameter may improve the differentiation of PCa from benign lesions in the prostate peripheral zone. Transrectal RTE-targeted biopsy coupled with peak strain index may enhance the detection of clinically significant PCa, particularly when combined with systematic biopsy.

Background TR4 nuclear receptor 4 (TR4) plays an important role in macrophages-associated foam cell formation of cardiovascular diseases and infiltrating macrophages are critical for prostate cancer (PCa) progression. However, the linkage of macrophages and TR4 and their impacts on PCa metastasis remains unclear.

The testicular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily that mediates various biologic functions with key impacts on metabolic disorders and tumor progression. Here, we demonstrate that TR4 may play a positive role in prostate cancer CD133+ stem/progenitor (S/P) cell invasion. Targeting TR4 with lentiviral silencing RNA significantly suppressed prostate cancer CD133+ S/P cell invasion both in vitro and in vivo. Mechanism dissection found that TR4 transcriptionally regulates the oncogene EZH2 via binding to its 5′ promoter region. The consequences of targeting TR4 to suppress EZH2 expression may then suppress the expression of its downstream key metastasis-related genes, including NOTCH1, TGFβ1, SLUG, and MMP9. Rescue approaches via adding the EZH2 reversed the TR4-mediated prostate cancer S/P cell invasion. Together, these results suggest that the TR4→EZH2 signaling may play a critical role in the prostate cancer S/P cell invasion and may allow us to develop a better therapy to battle the prostate cancer metastasis. Mol Cancer Ther; 14(6); 1445–53. ©2015 AACR.

To explore the effects of stromal interaction molecule 1 (STIM1) on the expression of apoptosis-related proteins in prostate cancer PC-3 cells.We transfected the lentivirus vector STIM1-pGCSIL-GFP carrying STIM shRNA into human hormone-independent prostate cancer PC-3 cells, and 3 days later observed the transfection efficiency by fluorescence microscopy. At 7 days after transfection, we determined the expression of STIM1 in the PC-3 cells by RT-PCR and Western blot and those of apoptosis-related proteins Bcl-2, Bax, survivin and activated Caspase-3 by Western blot.At 3 days, inverted microscopy revealed a transfection efficiency of80%. At 7 days, the STIM1 expression was significantly inhibited at both mRNA and protein levels. The Bcl-2/Bax rate was remarkably decreased as compared with that of the control group (0. 31 vs 1.24 ) , and the survivin expression was markedly reduced, 0. 14 times that of the relative expression in the control. However, the Caspase-3 cleavage was significantly activated, 1.52 times that of the control (P0.05).STIM1 can be regarded as an oncogene in prostate cancer PC-3 cells. Inhibition of its expression can induce PC-3 cell apoptosis by reducing the Bcl-2/Bax rate, decreasing the survivin expression, and activating the Caspase-3 pathway.

Testicular nuclear receptor 4 (TR4) plays protective roles against oxidative stress and DNA damage and might contribute to aging. Our recent clinical tumor tissue staining results showed higher expression of TR4 in prostate cancer (PCa) patients with high Gleason scores compared to the tissues with the low Gleason scores. In vitro migration/invasion assays after manipulation of the TR4 expression in PCa cells showed that TR4 promoted PCa cells migration/invasion. Mechanism dissection found that the CCL2/CCR2 signal plays the key role in the mediation of TR4-promoted PCa cells migration/invasion. Chromatin immunoprecipitation and Luciferase assays further confirmed TR4 modulation of CCL2 at the transcriptional level and addition of the CCR2 antagonist led to interruption of the TR4-enhanced PCa cells migration/invasion. Finally, the orthotopic xenografted mice studies using the luciferase expressing CWR22Rv1 cells found that TR4 enhanced PCa metastasis and this increased metastasis was reversed when the CCR2 antagonist was injected into the mice. Together, these in vitro and in vivo results revealed a positive role of TR4 in PCa metastasis and demonstrated CCL2/CCR2 signaling as an important mediator in exerting TR4 action. This finding suggests that TR4 may represent a biomarker related to PCa metastasis and targeting the TR4-CCL2/CCR2 axis may become a new therapeutic approach to battle PCa metastasis.

Testicular nuclear receptor 4 (TR4), also known as NR2C2, belongs to the nuclear receptor superfamily and shares high homology with the testicular nuclear receptor 2. The natural ligands of TR4 remained unclear until the recent discoveries of several energy/lipid sensors including the polyunsaturated fatty acid metabolites, 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, and their synthetic ligands, thiazolidinediones, used for treatment of diabetes. TR4 is widely expressed throughout the body and particularly concentrated in the testis, prostate, cerebellum, and hippocampus. It has been shown to play important roles in cerebellar development, forebrain myelination, folliculogenesis, gluconeogenesis, lipogenesis, muscle development, bone development, and prostate cancer progression. Here we provide a comprehensive summary of TR4 signaling including its upstream ligands/activators/suppressors, transcriptional coactivators/repressors, downstream targets, and their in vivo functions with potential impacts on TR4-related diseases. Importantly, TR4 shares similar ligands/activators with another key nuclear receptor, peroxisome proliferator-activated receptor γ, which raised several interesting questions about how these 2 nuclear receptors may collaborate with or counteract each other's function in their related diseases. Clear dissection of such molecular mechanisms and their differential roles in various diseases may help researchers to design new potential drugs with better efficacy and fewer side effects to battle TR4 and peroxisome proliferator-activated receptor γ involved diseases.

Bladder urothelial carcinoma (BUC) accounts for ∼90% of all cases of bladder cancer. Reduced expression of TGFBR3 has been frequently observed in several types of human cancers. However, little is known about whether expression of TGFBR3 reduced in BUC and the underlying mechanisms. In the present study, we performed quantitative real-time PCR to examine the mRNA expression of TGFBR3 and GATA3, and bisulfite genomic sequencing to evaluate the methylation status in TGFBR3 and GATA3 promoter regions in fresh tumor and the corresponding paracarcinoma tissues from 29 patients with BUC. As a result, the expression of TGFBR3 and GATA3, a transcriptional factor of the TGFBR3 gene, were found to be co-downregulated in BUC. Moreover, our findings indicated that GATA3 promoter methylation was one of the reasons for silencing of GATA3 and TGFBR3 in BUC, albeit TGFBR3 methylation and mutation were not associated with reduced expression of TGFBR3 mRNA in BUC. In summary, our findings suggest that methylation in the GATA3 promoter region may inhibit the expression of GATA3 mRNA, which leads to the reduced expression of TGFBR3 mRNA in BUC.

Objective To investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) in bladder cancer (BCa) progression. Materials and Methods The gene copy number of PPARγ in human BCa tissue samples was analyzed by fluorescence in situ hybridization. The migration and invasive ability of human BCa cell lines with different PPARγ expression levels or treated with thiazolidinedione-rosiglitazone, a PPARγ agonist and an antidiabetic drug, were investigated. Results PPARγ amplification was increased dramatically in BCa tissue compared with normal urothelium (38.1% vs 4.3%, P = .0082) and in tumors with lymph node metastasis compared with those without metastasis (75.0% vs 15.4%, P = .0176). The human BCa cell line 5637 with strong PPARγ expression demonstrated a greater ability for cell migration and invasion than the UMUC-3 cell line with weak PPARγ expression. Knocking down PPARγ in BCa 5637 cells led to decreased cell migration, and activation of PPARγ with thiazolidinedione-rosiglitazone promoted their migration and invasive ability. Conclusion PPARγ amplification in BCa could play an important role in BCa cell migration and invasion. Alteration of PPARγ expression by PPARγ-small interfering ribonucleic acid or activation by its agonist rosiglitazone, a diabetic thiazolidinedione drug, could lead to alternation of BCa cell migration and invasion.

To investigate and compare the effectiveness and safety of 80-W GreenLight laser vaporization and GreenLight high-performance system (HPS) 120-W laser vaporization for the treatment of benign prostatic hyperplasia (BPH) in high-risk patients.We allocated 290 high-risk patients with BPH to two groups to receive 80-W (n = 220) and HPS 120-W GreenLight laser vaporization (n = 70). We recorded and compared the pre-, intra- and post-operative clinical data of the two groups.The operations were successful in both of the groups. There were statistically significant differences in the prostate volume, IPSS, Qmax and PVR before and after surgery (P0.01), but not between the two groups (P0.05). The operation time, lasing time and energy consumption were (56.5 +/- 22.6) min, (31.2 +/- 10.3) min and (159.8 +/- 29.0) kJ in the 80-W group, as compared with (45.1 +/- 20.4) min, (24.6 +/- 8.3) min and (134.2 +/- 23.3) kJ in the 120 W group, with significant differences between the two (P0.01).GreenLight laser vaporization of the prostate is a safe and effective procedure for the treatment of BPH, and the new HPS 120-W laser therapy, with its advantages of easier operation and shorter surgical time, is an even better minimally invasive option for elderly high-risk patients.

To report the initial urological applications of single trocar laparoscopic surgery.From April to December 2010, a total of 45 urologic patients underwent single trocar laparoscopic surgery. The procedures included high ligations of spermatics vessels (n = 17), unroofing of renal cysts (n = 24) and ureterolithotomy (n = 4). Transperitoneal approach: A 15 mm incision was made in umbilicus and CO(2) pneumoperitoneum created. A 10-mm trocar was inserted into enterocoelia. After the installation of single punch laparoscope (STROZ), the operating instruments were guided through integrated working channel to complete operative procedures. Retroperitoneal approach: A 15 mm incision was made above the diseased-side crista iliaca for around 4 cm. Retroperitoneal cavity was prepared for retroperitoneoscopy with the IUPU (Institute of Urology, Peking University) technique. In the same way, a 10-mm trocar was inserted. And the single punch laparoscope was installed to complete operative procedures.The procedures were successful in 41 cases. But the others (renal cysts, n = 2; ureteral calculus, n = 2) were successfully managed by adding a 5 mm trocar. Operation time: transperitoneal approach, 23 - 70 min; retroperitoneal approach, 45 - 175 min. The intra-operative volume of blood loss was minimal. And the hospital stay was 1 - 5 days. No significant postoperative complication occurred.For some relatively simple procedures, a new and more minimally invasive approach of single trocar laparoscopic surgery may be attempted.

To detect the expressions of transforming growth factor-beta1 (TGF-beta1), Desmin and CD34 in the penile corpus cavernosum of SD rats in different age groups.We randomly selected 10 SD rats in each of the 2-, 5- and 20-month age groups, harvested their penile corpus cavernosum tissues under ether anesthesia, and detected the mRNA and protein expressions of TGF-beta1, Desmin and CD34 by RT-PCR and immunohistochemistry.The results of RT-PCR showed the mRNA expressions of TGF-beta1, Desmin and CD34 in the corpus cavernosum tissues, with significant differences between every two groups (P0.01). The TGF-beta1 protein was mainly expressed in the trabeculae and around the arteries of the corpus cavernosum for membrane and cytoplasm staining, the Desmin protein mainly in the membrane and cytoplasm for muscle tissue staining; and the CD34 protein mainly in the vascular and sinusoidal endothelia. The mRNA expression of TGF-beta1 was correlated positively (r = 0.944, P0.01) while those of Desmin and CD34 negatively with the age of the rats (r = -0.947, P0.01; r = -0.934, P0.01). And the mRNA expressions of both Desmin and CD34 had a significant correlation with that of TGF-beta1 (r = -0.888, P0.01; r = -0.887, P0.01).With the increase of age, the expression of TGF-beta1 is significantly up-regulated, while those of Desmin and CD34 significantly down-regulated in the corpus cavernosum tissues, and it is negatively correlated with the latter two. TGF-beta1 is an important influencing factor on ED.

To construct a recombinant adenovirus vector bearing dual-survivin short hairpin RNA (shRNA).Dual-survivin shRNA was designed and synthesized respectively, both inserted into adenovirus DNA. The recombinant adenovirus vector was confirmed via both sequencing and restriction digestion analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenoviruses.The recombinant adenovirus vector was constructed and the target sequence was obtained.The construction of the recombinant adenovirus vector provides a basis for the research of potential gene therapy for prostate cancer.

To construct and identify the eukaryotic expression plasmids encoding two short hairpin RNA (shRNA) of survivin for the purpose of paving the way for the studies of targeted gene therapy for prostatic carcinoma (PCa).Two shRNA of survivin were designed and synthesized respectively, and then both were cloned into plasmids. Finally, the recombinant plasmids were confirmed by sequencing and agarose gel electrophoresis after restriction digestion.The recombinant plasmids encoding two survivin shRNA were constructed and the aim sequence obtained.Successful construction of the recombinant provides a sound basis for the research of targeted gene therapy for PCa.

To classify the Chinese isolates of Coltiviruses.Three sets of primers were selected among them two were specific to the 9th and 12th segments of subgroup B2, and one was for the 12th segment of subgroup B1-All the Chinese isolates of Coltivirus selected in the experiment were classified according to the lengths of different amplicons of the reverse transcriptase-polymerase Chain reaction (RT-PCR). The homogenicity of the nucleic acids of the isolates BJ95-75 and YN-6 was also compared with other Coltivirus strains belonging to subgroup B2.With the primers 12-854-S/12-B2-R, which were specific to the 12th segment of Coltivirus subgroup B2-850 bp amplicons were obtained from Beijing isolate BJ95-75 and all the Yunnan isolates such as YN-6, -67-1, -68-1, -69, -70-1, -70-2, -90, -92-2, -93 of Coltivirus 492 bp DNA fragments were also amplified from all of them with the segment 9th specific primers 9-JKT-S/9-JKT-R. However no positive results were obtained from Northeast isolates NE97-12, NE97-31 and control viruses YN-99(Orbivirus),YN-151-1(JEV) with the same two sets of primers. With 12-B1-S/12-B1R primers specific to the 12th segment of subgroup B1, no amplicons of right length were obtained from any of the Chinese isolates of Coltivirus and the control viruses. When compared the nucleic acid sequences of BJ95-75 and YN-6 with other Coltivirus strains such as Bannavirus, JKT6423, JKT6969, JKT7043, the amplicons from segment 12th of these two strains had more than 89.4% homology with the other strains, especially to the earlier Chinese isolate Bannavirus, the homolog was more then 98.9%. Nearly 96.5% and 99.2% of the nucleic acids of the amplicons from segment 9th of the two strains were being homologous to Bannavirus and about 84.0% to JKT6423, which had been classified into type B2a. But the maximal homogenicity was about 53% when compared with the other two coltivirus strains. JKT6969 and JKT7043 which had been classified into type B2b.Genotyping the recent Chinese isolates of coltivirus for the first time in our country. Most of the Chinese isolates belong to subgroup B2, more exactly type B2a. The Northeast isolates NE97-12 and NE97-31 were not correctly grouped with the available primers.

To investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.Mosquitoes were collected from Lancang river downstream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation. New isolates were identified by neutralization test(NT), ELISA, immunofluorescence assay(IFA) and polyacrylamid gel electrophoresis(PAGE).Totally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4%. Ten strains were resistant to both ether and 5 prime-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PAGE and PAGE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5 prime-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and ElISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87 nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down-stream area in Yunnan province. Among them ten, nine, one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.