A comprehensive evaluation of circ_0065214/ miR-188-3p/GPNMB axis in breast cancer.

CircRNAs are involved in multiple aspects during carcinogenesis, including tumorigenesis, vascularization, apoptosis and others. Exploring the role of circRNAs in breast cancer (BC) enables us to understand the development mechanism of BC more comprehensively. Here, we screened out and verified an up-regulated circRNA in BC from GEO data. Quantitative Real-time PCR (qRT-PCR) showed that circ_0065214 had a high expression level in BC patients. Besides, circ_0065214 had good diagnostic value in BC serum, and the area under the diagnostic curve, sensitivity and specificity were 0.78, 0.63 and 0.85, respectively. The combined application of circ_0065214 with CEA and CA-153 can further improve the diagnostic efficiency. The knockdown of circ_0065214 in vivo and in vitro inhibited the proliferation, migration and invasion of BC, but promoted autophagy. At last, dual-luciferase reporter assay and rescue assays revealed that circ_0065214 acted as a decoy to adsorb miR-188-3p, and then relieved the repressive effect of miR-188-3p on its target GPNMB. Our results demonstrated that circ_0065214 regulated the expression of GPNMB by competitively binding to miR-188-3p, thus promoting the proliferation, migration and invasion of breast cancer cells and inhibiting autophagy. These findings provided an original therapeutic strategy for BC.

Exosomal miR-1910-3p promotes proliferation, metastasis, and autophagy of breast cancer cells by targeting MTMR3 and activating the NF-κB signaling pathway.

Exosomes are key mediators of intercellular communication and play a role in the pathogenesis and progression of cancer. Exosomes in circulating body fluids serve as molecular markers for cancer diagnosis. This study aimed to investigate the role of exosomal microRNA (miR)-1910-3p in breast cancer and determine its clinical diagnostic value. MiR-1910-3p promoted proliferation and migration of breast cancer cells in vitro and in vivo. In vitro, exosomes enriched in miR-1910-3p transferred miR-1910-3p to mammary epithelial cells and breast cancer cells, promoting proliferation and migration, inhibiting apoptosis, and inducing autophagy. In vivo, exosomes enriched in miR-1910-3p promoted the proliferation and migration of breast cancer cells. MiR-1910-3p downregulated myotubularin-related protein 3, activated the NF-κB and wnt/ß-catenin signaling pathway, and promoted breast cancer progression. Serum miR-1910-3p in exosomes was an effective diagnostic marker that improved the sensitivity of breast cancer diagnosis when used in combination with the traditional tumor marker CA153. In conclusion, breast cancer cell-derived exosomes promoted the growth, metastasis, and autophagy of breast cancer cells by transferring miR-1910-3p. MiR-1910-3p in serum exosomes may serve as a novel molecular marker for breast cancer diagnosis.

Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth.

Bromodomain-containing protein 4 (BRD4) and PI3K-AKT are both important for renal cell carcinoma (RCC) development and progression. SF2523 is a BRD4 and PI3K-AKT dual inhibitor. The present study demonstrated that SF2523 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. SF2523 induced activation of caspase and apoptosis in RCC cells. Further, SF2523 disrupted RCC cell cycle progression and inhibited cell migration in vitro. At the signaling level, SF2523 in-activated PI3K-AKT-mTOR, and downregulated BRD4-dependent proteins, Bcl-2 and Myc, in RCC cells. Remarkably, SF2523 was more efficient than Wortmannin (the PI3K inhibitor) and JQ1 (the BRD4 specific inhibitor) in killing RCC cells. In vivo, SF2523 administration at well-tolerated doses suppressed 786-O xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that concurrent blockage of BRD4 and PI3K-AKT signalings by SF2523 efficiently inhibits RCC cell growth in vitro and in vivo.

microRNA-302c-3p inhibits renal cell carcinoma cell proliferation by targeting Grb2-associated binding 2 (Gab2).

The expression and biological function of Grb2-associated binding 2 (Gab2) in renal cell carcinoma (RCC) cells was tested here. We showed that Gab2 expression was significantly elevated in human RCC tissues and RCC cells. It was correlated with over-activation of Akt and downregulation of microRNA-302c-3p ("miR-302c-3p"), a putative Gab2-targeting microRNA. Knockdown of Gab2 inhibited Akt activation and 786-O RCC cell proliferation. Reversely, forced over-expression of Gab2 led to Akt hyper-activation to facilitate 786-O cell proliferation. Exogenous expression of miR-302c caused Gab2 downregulation, Akt inhibition and 786-O cell proliferation inhibition. On the other hand, miR-302c-3p depletion by expressing its anti-sense ("antagomiR-302c") led to Gab2 upregulation, Akt activation and increased 786-O cell proliferation. Significantly, miR-302c-3p failed to affect the proliferation of 786-O cells with shRNA-depleted Gab2. Together, we suggest that miR-302c-3p depletion in human RCC cells leads to Gab2 over-expression, Akt hyper-activation and cell proliferation.

Concurrent inhibition of mTORC1 and mTORC2 by WYE-687 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Mammalian target of rapamycin (mTOR)in renal cell carcinoma (RCC) represents a valuable oncotarget for treatment. We here tested the potential anti-RCC activity by a novel mTOR kinase inhibitor WYE-687in vitro and in vivo.WYE-687 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. Yet, it was non-cytotoxic toHK-2 tubular epithelial cells.WYE-687 provoked caspase-dependent apoptosis in the RCC cells. At the molecular level, WYE-687 almost completely blocked mTORC1 (p-S6K1 and p-S6) and mTORC2 (p-Akt Ser 473) activation in both 786-Ocells and primary human RCC cells, where it downregulated both hypoxia-inducible factor (HIF)-1α and HIF-2α expression. Significantly, oral administration of WYE-687 potently suppressed786-O tumor xenograft growth in nude mice. mTORC1/2 activation and HIF-1α/2α expression were also remarkably downregulated in WYE-687-treated tumor tissues. Thus, our preclinical results imply that WYE-687 may have important translational value for the treatment of RCC.

Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation.

Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation.

Pre-clinical evaluation of AZD-2014, a novel mTORC1/2 dual inhibitor, against renal cell carcinoma.

Here we found that dual mTORC1/2 inhibitor AZD-2014 significantly inhibited RCC cell survival and growth, with higher efficiency than conventional mTORC1 inhibitors rapamycin and RAD001. RCC cell apoptosis was also induced by AZD-2014. AZD-2014 disrupted mTORC1/2 assembly and activation, while downregulating HIF-1α/2α and cyclin D1 expressions in RCC cells. Meanwhile, AZD-2014 activated autophagy, detected by p62 degradation, Beclin-1/ATG-5 upregulation and light LC3B-I/-II conversion. Autophagy inhibition by pharmacologic or siRNA-based means increased AZD-2014 activity in vitro, causing substantial RCC cell apoptosis. In vivo, AZD-2014 was more efficient than RAD001 in inhibiting 786-0 xenografts and downregulating HIF-1α/2α or p-AKT (Ser-473). Finally, AZD-2014's activity in vivo was further enhanced by co-administration of the autophagy inhibitor 3-methyaldenine. We provide evidence for clinical trials of using AZD-2014 in RCC treatment.

The role of Toll-like receptor 4 on inflammation and Aß formation in cortex astrocytes.

To investigate the role and possible molecular mechanism of astrocytes in inflammation and amyloid ß-protein (Aß) formation, in this research, by using LPS to stimulate cultured rat astrocytes in vitro with or without anti-Toll-like receptor 4 (TLR4) antibody pretreatment, we first detected the TLR4, TNF-α, IL-1ß, ß-amyloid precursor protein (ß-APP) and ß-site APP clearing enzyme 1 (BACE1) mRNA with real-time PCR, and TLR4, NF-κB/P65 protein in cultured astrocytes by Western blot, and then further probed the translocation of NF-κB/P65 using immunofluorescence and the contents of TNF-α, IL-1ß and Aß in culture supernatant through ELISA. We found that all of these indexes increased at different degrees after LPS-stimulation. However, if pretreatment with anti- TLR4 antibody, such stimulating effects of LPS on the nuclear translocation of NF-κB/P65 and TNF-α, IL-1ß, Aß contents in astrocytic culture supernatant were reduced significantly or disappeared in comparison with the group with only LPS-administration. Our results suggest that TLR4 in astrocytes might play an important role in the inflammation and Aß formation through the TLR4/NF-κB signaling pathway, thus providing new knowledge and understanding of the inflammatory hypothesis of AD pathogenesis.

[The role of TLR4-mediated MyD88-dependent pathway in neuroinflammation in hippocampal neurons of rats].

OBJECTIVE: To investigate weather there is a toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent pathway in hippocampal neurons of rats and the probable role of the pathway in neuroinflammation. METHODS: To establish the proper model, primarily cultured hippocampal neurons were treated with lipopolysaccharides (LPS), or pretreated with TLR4 antibody then co-treated with LPS. The expression of mRNA of MyD88 and TNF-alpha receptor associated factor 6 (TRAF6) were tested by RT-qPCR. The content of MyD88 and TRAF6 were tested by Western blot. The nuclear translocation of nuclear factor-kappaB/P65 (NF-kappaB/p65) was tested by immunofluorescence. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and nitric oxide (NO) were tested by ELISA. RESULTS: LPS could increase MyD88 and TRAF6 mRNA, upregulate protein level of MyD88 and TRAF6 and increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. LPS also could promote NF-kappa B/p65 translation to the nucleus. The pretreatment with TLR4 antibody reduced the translocation to nucleus for NF-kappaB/P65 and the contents of TNF-alpha, IL-1beta and NO in the culture supernatant. CONCLUSION: There is a TLR4-mediated MyD88-dependent pathway in hippocampal neurons. The activation of this pathway can increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. TLR4-mediated MyD88-dependent pathway in hippocampal neurons participate in neuroinflammation, that means neurons are not passive in inflammation.

[The effect of meloxicam on the inflammatory reaction induced by beta amyloid protein in Alzheimer's disease rats].

OBJECTIVE: To investigate the effect and mechanism of meloxicam on the inflammatory reaction induced by beta amyloid protein (AB) in Alzheimer's disease (AD) rats. METHODS: The rat model was established by microinjection of Abeta(1-40) into hippocampus. The expression of NF-kappaB p65 and glial fibrillary acidic protein (GFAP) in hippocampus were detected by immunohistochemistry. The content of GFAP in cortex was tested by Western-blot. The content of TNF-alpha in cortex was tested by ELISA. The expression of IL-1beta mRNA was tested by RT-PCR. RESULTS: The expression of NF-kappaB p65, GFAP and TNF-alpha as well as IL-1beta mRNA were decreased by meloxicam. CONCLUSION: Meloxicam can reduce the proliferation of astrocyte by decreasing the expression of GFAP in AD model rat's hippocampus and cortex. And the depression of NF-kappaB p65 may significantly decrease the expression of TNF-alpha1 and IL-1beta to lessen the inflammatory reaction in cerebral tissue.

[Effects of Chinese herb compound Naoyikang on expression of choline acetyltransferase in brain of rats with Alzheimer's disease].

OBJECTIVE: To observe the effects of Naoyikang (NYK) on expression of choline acetyltransferase (ChAT) in brain of rats with Alzheimer' s disease (AD). METHOD: Bilateral infusions of Ibotenic acid (IBO) into nucleus basalis of Meynert (NBM) using hamilton syringe and stereotaxic apparatus were adopted to establish the rat model of AD. After intragastrically administrated with different solution for 28 days, immunohistochemistry and Western-blot were adopted to study the expression of ChAT in frontal cortex of AD rats. RESULT: NYK could improve the morphology and increase the number of ChAT immunoreactive neurons, and significantly promote ChAT protein expression. CONCLUSION: NYK may be able to increase the synthesis of acetylcholine (ACh) through elevating the expression of ChAT protein, thus improving the level of brain ACh so as to protect central cholinergic neurons.

[Protective effect of Naoyikang on the Alzheimer's disease model mice induced by D-galactose and NaNO2].

AIM: To investigate the mechanisms of Naoyikang (Traditional Chinese Medicine) on the Alzheimer's Disease (AD) model mice induced by D-galactose (D-gal) and NaNO2. METHODS: The mouse model was established by intraperitoneal injection of D-gal and NaNO2. The capacity of learning and memory was tested on mice with electrical maze; the content of nitric oxide (NO) and the activity of monoamine oxidase-B (MAO-B), glutathione peroxidase (GSH-PX), Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme in cerebral cortex and hippocampus were assayed by biochemical methods; expression of Bax and Bcl-2 mRNA was detested by RT-PCR. RESULTS: Naoyikang could ameliorate the capacity of learning and memory of AD model mice and reduce MAO-B activity in the brain tissue and activate the activity of Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme in the brain tissue and decrease the expression of Bax mRNA, but increase the expression of Bcl-2 mRNA in the model brain tissue. CONCLUSION: Naoyikang could protect AD model mice induced by D-gal and NaNO2. It could modify the metabolism of monoamine neurotransmitter in brain through reducing MAO-B activity and protect neurons by activating the activity of Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme and decrease Bax expression and increase Bcl-2 expression in the model brain tissue.

[Effect of acupoint-injection of oxymatrine on experimental hepatic carcinoma and study on the mechanism].

OBJECTIVE: To investigate the effect of acupoint injection of oxymatrine (OM) on experimental hepatocellular carcinoma and the mechanism. METHODS: The rats of hepatocellular carcinoma induced by 2-acetoaminoflurence (2-AAF) were randomly divided into a normal control group (group N), a model group (group M), a control group of oxymatrine intraperitoneal injection (OM ip group) and a treatment group of small dose oxymatrine injection into Zusanli (OM ZSL group). At the end of 12h week, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (gamma-GT) were determined. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expressions of cyclin D1 and cyclin-dependent kinase 4 (CDK4) mRNA in hepatocellular carcinoma tissues. RESULTS: The number of cancer nodes on the surface of liver in th Om ip group and the Om ZSL group was lower than in the group M, with the serum ALT, AST, and gamma-GT levels significantly decreased (P<0. 01), and significantly inhibited expressions of cyclin D1, CDK4 mRNA (P<0. 01). CONCLUSION: OM ip and small dose oxymatrine injection into ZSL can treat or delay hepatocarcinogenisis of hepatocellular carcinoma induced by 2-AAF. Partial mechanism of this anti-carcinoma is protecting hepatocytes possibly through improving hepatic functions, and inhibiting excessive proliferation of liver cancer cells via inhibiting the expressions of cyclin Dl, CDK4 mRNA.

[Protective effect of Naoyikang on the damage induced by glutamate in hippocampal neuron].

AIM: To investigate the effect of Naoyikang serum on the damage induced by glutamate in hippocampal neuron. METHODS: Morphological observation, MTT assay and nuclear DNA-associated fluorescence with DAPI dye were applied to evaluate the viability of hippocampal neuron, immunocytochemistry and RT-PCR were used to determine the expression of PTEN. RESULTS: A decreased viability and increased expression of PTEN were shown in hippocampal neuron in response to the treatment with glutamate. It was shown that the percentage of cell death and the expression of PTEN were reduced by the treatment with Naoyikang serum. CONCLUSION: These results suggest that Naoyikang may prevent the toxicity of glutamate by suppressing the expression of PTEN.