Objective To explore the molecular mechanism of BRAFV600E inducing chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.Methods The endogenous Mps1 in stable Sbcl2-and SK-MEL31-B-RafV600E expression cells were depleted by siRNA approach.To test the effect of B-RafV600E on the centrosome amplification and the formation of multipolar spindles,cells at S-phase with HU-treatment were arrested and then the centrosomes and mitotic spindles structure were detected through immunofluoresence.Results The percentage of B-RafV600E expressing Sbcl2 and SK-MEL31 cells (Sbcl2-B-RafV600E and SKMEL31-B-RafV600E) with centrosome amplification and multipolar spindle was reduced from 36 ％ to 6 ％ when Mps1 was absent.Conclusion B-RafV600E leads to centrosome amplification and multipolar spindle through Mps1,thus results in chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.

Objective To investigate the mechanism of Lnc001209 and phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and amyloid β-protein (Aβ) in aluminum-induced cognitive impairment. Methods Specific pathogen free adult male SD rats were randomly divided into control group, empty vector group, adeno-associated virus (AAV) group, aluminum maltolate (Al-maltolate) group, and AAV+Al-maltolate group, with 10 rats in each group. AAV and AAV+Al-maltolate group rats were injected with 5 μL of AAV overexpressing Lnc001209 with a titer of 1×10¹² vg/mL into the lateral ventricle using stereotaxic coordinates. Empty vector group rats were injected with 5 μL of empty vector AAV with a titer of 1×10¹² vg/mL into the lateral ventricle using stereotaxic coordinates. Control and Al-maltolate group rats underwent the same surgical procedure without any injections. The rats were intraperitoneally injected at the volume of 1 mL/kg body weight after 14 days of single cage feeding. Rats in the Al-maltolate group and AAV+Al-maltolate group were treated with 20 μmol/kg body weight Al-maltolate, while rats in the control group, empty vector group and AAV group were treated with an equivalent volume of 0.9% sodium chloride solution every other day for three months. The Morris water maze test was used to detect the learning and memory abilities of rats after treatment. The hippocampal tissues of the rats were collected to detect the expression of Aβ using the enzyme-linked immunosorbent assay and to detect the expression of microtubule-associated protein 1 light chain 3B (LC3B) using immunofluorescence. Western blot was used to detect the expression of phosphat idylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rap amycin (mTOR), and their corresponding phosphorylated proteins, and reverse transcription-polymerase chain reaction was used to detect the expression of Lnc001209. Resultsi) Compared with the rats in control group, the escape latencies of rats in the Al-maltolate group were longer from the second day of Morris water maze experiment (all P<0.05), the relative expression of Aβ40, Aβ42, phosphorylated PI3K (p-PI3K), phosphorylated AKT(p-AKT) and phosphorylated mTOR (p-mTOR) proteins increased in the hippocampal tissues (all P<0.05), the average fluorescence intensity of LC3B and the relative expression of PI3K, AKT and mTOR proteins decreased (all P<0.05), the relative expression of Lnc001209 decreased (P<0.05). ii) Compared with the rats in Al-maltolate group, the escape latencies of rats in AAV+Al-maltolate group were shortened from the second day of Morris water maze experiment (all P<0.05), and the relative expression of Aβ40, Aβ42, p-PI3K, p-AKT and p-mTOR proteins in hippocampus decreased (all P<0.05) , the average fluorescence intensity of LC3B and the relative expression of PI3K, AKT and mTOR proteins increased (all P<0.05), the relative expression of Lnc001209 increased (P<0.05). Conclusion Aluminum can affect the PI3K/AKT/mTOR protein signaling pathway through Lnc001209, leading to an increase in Aβ and a decrease in neuronal autophagy, ultimately resulting in cognitive impairment.

OBJECTIVETo study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.

METHODSMelanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression.

RESULTSIn melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector.

CONCLUSIONSBased on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Humans ; MAP Kinase Signaling System ; Melanoma ; genetics ; metabolism ; Mutation ; Phenotype ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins B-raf ; metabolism ; Signal Transduction ; Transfection