Objective:To construct and screen the high efficiency interference plasmid of TFAIP8-shRNA-pSIREN-RetroQ.Methods:Selected and synthesized three Target Sequence of TNFAIP8 shRNA1,TNFAIP8 shRNA2,TNFAIP8 shRNA3,and construct the TNFAIP8 interference plasmid.Transfection TNFAIP8-shRNA-pSIREN-RetroQ interference plasmid to A549 cells.Filter out the highest interference efficiency plasmid by detecting the mRNA and protein levels using RT-PCR and Western blot methods.Results:We successfully design and built three TNFAIP8-shRNA-pSIREN-RetroQ interference plasmids,and screen out the highest efficiency interference plasmid.Conclusion: Three interference plasmids targeting the TNFAIP8 gene have been constructed successfully and provide a useful tool for studying the function of TNFAIP8.

Objective To investigate the effect of miR-30a on human osteosarcoma cell 143B in migration,invasion andcellviability.Methods 143BcellswereinfectedortransfectedwithrecombinantadenovirusmiR-30a(Ad-miR30a) and miR-30a inhibitor respectively .Wound healing assay was performed to detect the cell healing ability ( P<0.05 ) .Cell migration and invasion ability were determined by Transwell assay ( P<0.05 ) .The cell viability was analyzed by MTT assay ( P<0.01 ) .Real-time quantitative PCR was performed to analyze the expression of RUNX2 mRNA level and confirmed the adenovirus miR-30a expressed in 143B cells.The expression of RUNX2 was analyzed by Western blot .miR-30a target to RUNX2 was verified by luciferase reported gene assay .Results The ability of migration and invasion was suppressed in osteosarcoma cell 143B by overexpression miR-30a,and the cell viability also decreased .After the endogenous miR-30 a being inhibited , the cell motility and invasion enhanced and the cell viability was promoted .The RUNX2 protein decreased after overexpression miR-30 a as compared with controlgroup.TheluciferaseactivityofRUNX2decreasedbyaddingmiR-30a.Conclusions 143Bcellmigration, invasion and viability were suppressed by miR-30a,and this process is potentially achieved via suppressing RUNX 2 protein expression .

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.

Abnormal changes during fat formation are closely related to the prevalence of many diseases. In order to understand the formation mechanism of fat, we used atomic force microscopy (AFM) to characterize the morphology and mechanical properties of porcine preadipocytes during the differentiation. Preadipocytes and adipocytes were different morphologically. The surface roughness of adipocytes was less than preadipocytes by detection of the ultrastructure. The mechanical properties of preadipocytes were changed during differentiation with AFM-based force spectroscopy. Preadipocytes were 20% higher than adipocytes in the adhesion force, stiffness and Young's modulus. Therefore, AFM analysis of membrane changes related to adipocytes formation provided quantitative data in the nanometer level for further studying the formation mechanism of the adipocytes.
Adipocytes ; cytology ; ultrastructure ; Adipogenesis ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Microscopy, Atomic Force ; Swine

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

Objective: To explore the expression of FAT1 in esophageal squamous cell carcinoma (ESCC) tissues, and its effect on cell proliferation. Methods: The expression levels of FAT1 protein in human ESCC tissues and matched adjacent normal tissues were determined by immunohistochemistry (IHC). Lentivirus based knockdown of FAT1 was carried out in YSE2 and Colo680N cell lines and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assays was performed to examine the effect of FAT1 on the proliferation of these ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle and apoptosis. The expression levels of cell cycle markers in FAT1 knock out ESCC cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot. Results: The relative expression of FAT1 in ESCC tissues was 66.97±21.53, significantly lower than 78.13±16.76 of adjacent normal tissues(P<0.05). Knockdown of FAT1 promoted cell proliferation and colony formation. In YSE2 cell, the division time in negative control (NC) group was (1 570±51) min, significantly longer than (1 356±31) min in shFAT1 group. In Colo680N cell, division time in NC group was (1 532±53) min, significantly longer than (1 290±30) min in shFAT1 group (P<0.05). Knockdown of FAT1 promoted G1-to S-phase transition and resulted in the upregulation of CDK4/CDK6/CCND1. Conclusion FAT1 inhibits the proliferation and G1-to S-phase transition of ESCC cells through regulating the protein expression of CDK4/CDK6/CCND1 complex.

Objective: To explore the effect of c-fos on multidrug resistance of laryngeal cancer TU177 cells. Method: Increasing drug concentration gradient is adopted to establish the stability of the laryngeal cancer drug resistance in cell line; RT-PCR and Western blot were used to detect difference of the c-fos between TU177 and TU177/VCR cells; plasmids with human c-fos knockdown or over expression were transfected into TU177/VCR and TU177 cells respectively, and the effects of different treatment on cell proliferation were investigated with MTT. Results: The drug resistance of TU177/VCR cells was 26.25-fold in vincristine (VCR), 7.33-fold in Paclitaxel (TAX), 2.41 in cisplatin (DDP), and 5.50 in 5-fluorouracil (5-FU), comparing with TU177( P<0.05). The TU177/VCR cells had significantly higher c-fos expression compared to TU177 cells( P<0.05). The results showed that the IC₅₀ values of 5-FU for the NC group and c-fos shRNA group were (306.2±6.3)μmol/L and (81.3±3.9)μmol/L, respectively, which was decreased by 73% in the c-fos shRNA group compared to that in the NC group (P<0.05). Similarly, the results showed that the IC₅₀ values for 5-FU were (55.3±9.4) μmol/L in NC group and (288.1±7.3)μmol/L in c-fos WT group, which was increased 5.21-fold in c-fos WT cells. Conclusion C-fos plays important role in multidrug resistance of larynx cancer cell TU177/VCR, and might become a new molecular target for laryngeal cancer treatment.