miR-137 and miR-491 Negatively Regulate Dopamine Transporter Expression and Function in Neural Cells

Background/Aims: Human hedgehog-interacting protein (HHIP) is a negative regulator of the hedgehog (HH) signaling pathway. It is deregulated in gastric cancer. The underlying molecular mechanism of HHIP-induced inhibition of HH signaling remains to be determined. Methods: A lentiviral HHIP expression vector (“LV-HHIP”) was established to exogenously over-express HHIP in gastric cancer cells. HHIP protein and mRNA were tested by Western blotting assay and quantitative real-time PCR assay, respectively. Cell survival was tested by the Cell Counting Kit-8 (CCK-8) assay. Cell proliferation was examined by the BrdU ELISA assay and [H³] Thymidine DNA incorporation assay. Cell invasion and migration were tested by the phagokinetic track assay and the “Transwell” assay. The bisulfite-sequencing PCR was applied to test HHIP promoter methylation. Results: In the established (AGS cell line) and primary human gastric cancer cells, LV-HHIP transfection increased HHIP expression and inhibited cancer cell survival and proliferation as well as cell migration and invasion. Furthermore, LV-HHIP significantly attenuated promoter methylation of the endogenous HHIP gene in AGS cells, causing it upregulation. Inhibition of methylation by 5-aza-dc similarly induced HHIP expression in gastric cancer cells, which inhibited cancer cell proliferation and migration. Conclusions: Our results suggest that inhibition of HHIP promoter methylation can efficiently inhibit human gastric cancer cell proliferation and migration.

Section: Methodsmentioning

confidence: 99%

Inhibition of HHIP Promoter Methylation Suppresses Human Gastric Cancer Cell Proliferation and Migration

Zuo

Qian

et al. 2018

“…Western blotting analysis was carried out using the described method [27-30]. Same set of lysate samples were run in sister gels to examine different proteins.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of Lymphocyte Antigen 6 Complex, Locus E in Gastric Cancer Promotes Cancer Cell Growth and Metastasis

Chen

et al. 2018

Background/Aims: Lymphocyte antigen 6 complex, locus E (LY6E) is a member of the lymphostromal cell membrane Ly6 superfamily protein. The present study investigated the clinical significance and potential biological function of LY6E in gastric cancer (GC). Methods: LY6E mRNA and protein expressions in human GC tissues and GC cells were tested. Relationship between LY6E expression and the GC patients’ clinicopathologic characteristics was analyzed. LY6E was silenced by siRNA in the cultured GC cells. Results: The RNA expression microarray profiling assay results demonstrated that LY6E mRNA was significantly increased in multiple human GC tumor tissues. Immunohistochemistry (IHC) staining analysis revealed that 59 of 75 (78.7%) GC specimens were LY6E positive. LY6E over-expression in human GC was correlated with the histology grade, AJCC stage, N classification, lymphatic invasion, and tumor location. Notably, functional LY6E expression was also detected in AGS and other established GC cell lines. LY6E knockdown by targeted-siRNA inhibited AGS cell survival and proliferation. Meanwhile, the LY6E siRNA induced G1-S cell cycle arrest and apoptosis in AGC cells. Additionally, AGC cell migration was also inhibited by LY6E knockdown. Expressions of tumor-suppressing proteins, including PTEN (phosphatase and tensin homolog) and E-Cadherin, were increased in LY6E-silenced AGS cells. Conclusion: LY6E over-expression in GC is potentially required for cancer cell survival, proliferation and migration.

“…The amount of mRNA in each sample was calculated and normalized to the levels of GAPDH mRNA. [17-19]…”

Section: Methodsmentioning

confidence: 99%

Berbamine Exerts Anti-Inflammatory Effects via Inhibition of NF-κB and MAPK Signaling Pathways

Jia

Wang

et al. 2017

Self Cite

Background/Aims: This study aimed to investigate the anti-inflammatory activity of Berbamine (BER), a bisbenzylisoquinoline alkaloid extracted from Berberis amurensis (Xiao Bo An), and the underlying mechanisms. Methods: Macrophages and neutrophils were treated with BER in vitro and stimulated with LPS and fMLP. The effects of BER on the expression of pro-inflammatory mediators in macrophages were evaluated with quantitative RT-PCR and ELISA. The effects of BER on the activation and superoxide release of neutrophils were determined with flow cytometry and WST-1 reduction test. The inhibitory effects of BER on the activation of signaling pathways related to inflammatory response in macrophages were evaluated by western blot analysis. In addition, a mouse peritonitis model was made by peritoneal injection of thioglycollate medium and anti-inflammatory effects of BER were investigated in vivo by quantitative analysis of pro-inflammatory factor production and leukocyte exudation. Results: BER significantly inhibited inflammatory factor expression by LPS-stimulated macrophages and suppressed activation and superoxide release of fMLP-stimulated neutrophils. In the mouse peritonitis model, BER significantly inhibited the activation of macrophages and exudation of neutrophils. According to analysis, BER significantly suppressed phosphorylation of NF-κB and MAPK (JNK and ERK1/2) signaling pathways in LPS-stimulated macrophages. Conclusions: Collectively, data from this study suggest that BER has anti-inflammatory potential, which is effected via inhibition of NF-κB and MAPK signaling pathways, and thus holds promise for treatment of inflammatory disease.