Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Oncogenic role of MIR516A in human bladder cancer was mediated by its attenuating PHLPP2 expression and BECN1-dependent autophagy.

Although MIR516A has been reported to be downregulated and act as a tumor suppressor in multiple cancers, its expression and potential contribution to human bladder cancer (BC) remain unexplored. Unexpectedly, we showed here that MIR516A was markedly upregulated in human BC tissues and cell lines, while inhibition of MIR516A expression attenuated BC cell monolayer growth in vitro and xenograft tumor growth in vivo, accompanied with increased expression of PHLPP2. Further studies showed that MIR516A was able to directly bind to the 3'-untranslated region of PHLPP2 mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of PHLPP2 expression in MIR516A-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a MIR516A downstream mediator responsible for MIR516A oncogenic effect. PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly, therefore promoting BECN1-dependent macroautophagy/autophagy, and inhibiting BC tumor cell growth. In addition, our results indicated that the increased autophagy by attenuating MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo. Collectively, our results reveal that MIR516A has a novel oncogenic function in BC growth by directing binding to PHLPP2 3'-UTR and inhibiting PHLPP2 expression, in turn at least partly promoting CUL4A-mediated BECN1 protein degradation, thereby attenuating autophagy and promoting BC growth, which is a distinct function of MIR516A identified in other cancers.Abbreviation: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BAF: bafilomycin A1; BC: bladder cancer; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CUL3: cullin 3; CUL4A: cullin 4A; CUL4B: cullin 4B; IF: immunofluorescence: IHC-p: immunohistochemistry-paraffin; MIR516A: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.

Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

Because bladder cancer (BC) is one of the most common malignant cancers of the urinary system, identification of BC cell growth-associated effectors is of great significance. Cyclin-dependent kinase (CDK)6 is a member of the CDK family of cell cycle-related proteins and plays an important role in cancer cell growth. This is borne out by the fact that a CDK6 inhibitor had been approved to treat several types of cancers. Nevertheless, underlying molecular mechanisms concerning how to regulate CDK6 expression in BC remains unclear. In the present study, it was observed that miR-934 was much higher in human BCs and human BC cell lines as well. The results also revealed that miR-934 inhibition dramatically decreased human BC cell monolayer growth in vitro and xenograft tumor growth in vivo; the outcomes were accompanied by CDK6 protein down-regulation and G0-G1 cell cycle arrest. Moreover, overexpression of CDK6 reversed the inhibition of BC cell growth induced by miR-934. Further studies showed that miR-934 binds to a 3'-UTR of ubiquitin-conjugating enzyme 2N (ube2n) mRNA, down-regulated UBE2N protein expression; this, in turn, attenuated CDK6 protein degradation and led to CDK6 protein accumulation as well as the promotion of BC tumor growth. Collectively, this study not only establishes a novel regulatory axis of miR-934/UBE2N of CDK6 but also provides data suggesting that miR-934 and UBE2N may be potentially promising targets for therapeutic strategies against BC.-Yan, H., Ren, S., Lin, Q., Yu, Y., Chen, C., Hua, X., Jin, H., Lu, Y., Zhang, H., Xie, Q., Huang, C., Huang, H. Inhibition of UBE2N-dependent CDK6 protein degradation by miR-934 promotes human bladder cancer cell growth.

miR-3687 Overexpression Promotes Bladder Cancer Cell Growth by Inhibiting the Negative Effect of FOXP1 on Cyclin E2 Transcription.

Cyclin E2, a member of the cyclin family, is a key cell cycle-related protein. This protein plays essential roles in cancer progression, and, as such, an inhibitor of cyclin E2 has been approved to treat several types of cancers. Even so, mechanisms underlying how to regulate cyclin E2 expression in cancer remain largely unknown. In the current study, miR-3687 was upregulated in clinical bladder cancer (BC) tumor tissues, The Cancer Genome Atlas (TCGA) database, and human BC cell lines. Inhibition of miR-3687 expression significantly reduced human BC cell proliferation in vitro and tumor growth in vivo, which coincided with the induction of G0/G1 cell cycle arrest and downregulation of cyclin E2 protein expression. Interestingly, overexpression of cyclin E2 reversed the inhibition of BC proliferation induced by miR-3687. Mechanistic studies suggested that miR-3687 binds to the 3' UTR of foxp1 mRNA, downregulates FOXP1 protein expression, and in turn promotes the transcription of cyclin E2, thereby promoting the growth of BC cells. Collectively, the current study not only establishes a novel regulatory axis of miR-3687/FOXP1 regarding regulation of cyclin E2 expression in BC cells, but also provides strong suggestive evidence that miR-3687 and FOXP1 may be promising targets in therapeutic strategies for human BC.

MicroRNA-411 Downregulation Enhances Tumor Growth by Upregulating MLLT11 Expression in Human Bladder Cancer.

Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in bladder cancer growth are not explored yet at present. Here, we screened 1,900 known miRNAs and first discovered that miR-411 was one of the major miRNAs, which was down-regulated in n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCs. This miR-411 down-regulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA (The Cancer Genome Atlas) database indicated that miR-411 was positively correlated with DFS (disease-free survival). Our studies also showed that miR-411 inhibited tumor growth of human BC cells in a xenograft animal model. Mechanistic studies revealed that overexpression of miR-411 repressed the expression of ALL1-fused gene from the chromosome 1q (AF1q) (MLLT11) by binding to the 3' untranslated region (UTR) of mllt11 mRNA and in turn induced p21 expression and caused cell cycle arrest at the G2/M phase, further inhibiting BC tumor growth. Collectively, our results improve our understanding of the role of miR-411 in BC tumor growth and suggest miR-411 and MLLT11 as potential new targets for the treatment of BC patients.

XIAP overexpression promotes bladder cancer invasion in vitro and lung metastasis in vivo via enhancing nucleolin-mediated Rho-GDIß mRNA stability.

Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.

NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein.

BACKGROUND: Since invasive bladder cancer (BC) is one of the most lethal urological malignant tumors worldwide, understanding the molecular mechanisms that trigger the migration, invasion, and metastasis of BC has great significance in reducing the mortality of this disease. Although RelA/p65, a member of the NF-kappa B transcription factor family, has been reported to be upregulated in human BCs, its regulation of BC motility and mechanisms have not been explored yet. METHODS: NF-κBp65 expression was evaluated in N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced high invasive BCs by immunohistochemistry staining and in human BC cell lines demonstrated by Western Blot. The effects of NF-κBp65 knockdown on BC cell migration and invasion, as well as its regulated RhoGDIα and FBW7, were also evaluated in T24T cells by using loss- and gain-function approaches. Moreover, the interaction of FBW7 with RhoGDIα was determined with immunoprecipitation assay, while critical role of ubiquitination of RhoGDIα by FBW7 was also demonstrated in the studies. RESULTS: p65 protein was remarkably upregulated in the BBN-induced high invasive BCs and in human BC cell lines. We also observed that p65 overexpression promoted BC cell migration by inhibiting RhoGDIα expression. The regulatory effect of p65 on RhoGDIα expression is mediated by its upregulation of FBW7, which specifically interacted with RhoGDIα and promoted RhoGDIα ubiquitination and degradation. Mechanistic studies revealed that p65 stabilizing the E3 ligase FBW7 protein was mediated by its attenuating pten mRNA transcription. CONCLUSIONS: We demonstrate that p65 overexpression inhibits pten mRNA transcription, which stabilizes the protein expression of ubiquitin E3 ligase FBW7, in turn increasing the ubiquitination and degradation of RhoGDIα protein and finally promoting human BC migration. The novel identification of p65/PTEN/FBW7/RhoGDIα axis provides a significant insight into understanding the nature of BC migration, further offering a new theoretical support for cancer therapy.

[Effects of electroacupuncture on the function of respiration chain in hippocampal mitochondria of SAMP8 mice ].

OBJECTIVE: from the perspective of mitochondria. METHODS: Tweleve SAMP8 mice aged 8 months were randomly divided into a model group (n = 6) and an electroacupuncture group (n = 6), and six SAMR1 mice aged 8 months were selected as a control group. The electroacupuncture group was treated with electroacupuncture at "Baihui" (GV 20), "Dazhui" (GV 14), "Shenshu" (BL 23) and "Taixi" (KI 3) for 20 min, once each day, 10 days for a course, and lasted for 3 courses. The other two groups were grasped and fixed in the same way at the same time without the other treatment. After the end of treatment, the learning and memory abilities of the mice were measured by Morris water maze, the activity of hippocampal mitochondrid respiratory chain enzyme complex was performed by spectrophotometry, and the levels of adenosine triphosphate (ATP) were detected by a reverse-phase high performance liquid chromatography (HPLC) method. RESULTS: Compared with the control group, the average escape latency significantly lengthened, the residence time on the plateau phase shortened, the activity of respiratory chain enzyme complexe I, II, III, and IV was decreased, and ATP concentration was decreased in the model group. Compared with the model group, the average escape latency significantly shortened, the residence time on the plateau phase lengthened, the activity of hippocampal mitochondrid respiratory chain complexes I, II, III, and IV was significantly increased, and ATP concentration was increased in the electroacupuncture group. CONCLUSION: Electroacupuncture can increase the activity of hippocampal mitochondrid respiratory chain enzyme complexe and ATP concentration and improve mitochondrial function, which may be one of underlying mechanisms of electroacupuncture in treatment of AD.