ABSTRACT
@#[Abstract] Objective: To investigate the influences of human lung adenocarcinoma PC-9 cells on tight junction proteins of blood brain barrier (BBB) under CXCR4/SDF-1 axis by establishing a model of BBB in vitro. Methods: The immortalized mouse brain microvascular endothelial Bends cells were used to establish a model of BBB in vitro by monolayer culture; Subsequently, transendothelial electric resistance (TEER) and fluorescein sodium permeability experiment were used to detect the function of in vitro BBB model and observe the effect of PC-9 cells on the function of BBB model, respectively. Western blotting was used to detect the effect of PC-9 cells on function of BBB model and expressions of endothelial tight junction proteins under the treatment of single or combined AMD3100 and SDF-1 (1 μg/ml AMD3100,100 ng/ml SDF-1, AMD3100+SDF-1). Transwell assay was used to detect the influence of CXCR4/SDF-1 axis on the ability of PC-9 cells transmigrating the cell layer of BBB model. Results: Monolayer culture of Bends cells can form tightly connected BBB withhighTEER,which reached (182.13±5.19) Ω.cm2 at the 96 h; in the meanwhile, fluorescein sodium permeability experiment showed that BBB had significantly lower permeability than that of control group ([40.31±2.43]% vs [150.10±3.17]%, P<0.05). The TEER of BBB decreased to (46.7±4.35) Ω·cm2 after coculture with PC-9 cells for 24 h, and at the same time the sodium fluorescein permeability of BBB significantly increased than that of pre-treatment ([136.32±4.93]% vs [50.24±6.21]%, P<0.05). PC-9 cells up-regulated the expressions of tight junction proteins of Bends cells under the treatment of AMD3100 (P<0.05). The number of PC-9 cells transmigrating the BBB inAMD3100 treatment group was significantly lower than that of CON group (43±2 vs 81±2, P<0.05). Conclusion: AMD3100 can reduce the ability of PC-9 cells destroying the tight junction of the BBB model established in vitro by Bends cells.
ABSTRACT
@# Objective: To investigate the effects of long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) on the proliferation of lung adenocarcinoma PC-9 cells and to explore its mechanism. Methods: qPCR was used to detect the expression level of lncRNA NEAT1 in human lung adenocarcinoma PC-9 cells and human embryonic lung diploid 2BS cells. The sequence of small interfering RNA(siRNA) targeting lncRNANEAT1 gene was designed and synthesized, and then transfected into PC-9 cells by liposome method. The expression level of NEAT1 in PC-9 cells before and after transfection was detected by qPCR. MTT and flow cytometry were used to detect the effect of lncRNANEAT1 knockdown on proliferation and cell cycle distribution of PC-9 cells, respectively. WB assay was used to detect the expressions of DNA damage-related proteins, namely, double-stranded DNA breaks (DSBs) biomarker γ-H2AX and ataxia-telangiectasia mutated (ATM), before and after transfection. Results: Compared with 2BS cells, lncRNA NEAT1 was highly expressed in PC-9 cells (P<0.05). The PC-9 cells with lncRNA NEAT1 knock-down were successfully established. After being transfected with siRNA for 12 h, the proliferation of PC-9 cells in siNEAT1 group and siNEAT2 group significantly decreased as compared with the blank control group and the empty transfection group (P<0.05). In the interference groups, cell cycle was arrested in G1 phase ([88.97±2.64]%, [88.15±1.48]% vs [84.5±1.72]%, P<0.05) and G2/M phase ([8.35±2.02]%, [8.11± 1.36]% vs [4.28±1.28]%, P<0.05). The expression levels of DNA damage-related proteinsATM and γ-H2AX in the interference groups were significantly increased (all P<0.05). Conclusion: lncRNA NEAT1 is highly expressed in lung adenocarcinoma PC-9 cells. lncRNA NEAT1 inhibits DNA damage and causes cell cycle at G1/M phase switch, and thus promotes the proliferation of lung adenocarcinoma cells.
ABSTRACT
OBJECTIVE： To study the effects of bezafibrate （BEZ） and fenofibrate （FEN） on the proliferation of lung adenocarcinoma PC-9 cells and the expression of c-myc. METHODS： The effects of BEZ and FEN （12.5， 25， 50， 100， 200 μmol/L） on the survival rate of PC-9 cells were detected by CCK8 method. PC-9 cells were divided into administration group and control group. Administration group was given low， medium and high concentration （25， 50， 100 μmol/L） of BEZ and FEN； control group was treated with dimethyl sulfoxide for 48 h. Cell cycle distribution and apoptosis were detected by flow cytometry. qRT-PCR was used to detect mRNA relative expression of c-myc in cells. The protein relative expression of c-myc in cells were detected by Western blot assay. RESULTS： The survival rates of PC-9 cells were （80.76±3.2）％， （74.35±5.06）％， （62.8±1.23）％， （59.03±1.55）％， （39.8±1.01）％ under the action of above concentration of BEZ； and the survival rates of PC-9 cells were （74.46±1.30）％， （61.91±4.77）％， （48.95±2.8）％， （37.05±1.55）％， （32.49±1.36）％ under the action of FEN. Compared with control group， G1 phase cell ratio increased significantly in medium and high concentration groups of BEZ and FEN； the apoptotic rate of PC-9 cells was increased significantly in low， medium and high concentration groups of BEZ and FEN； mRNA and protein relative expression of c-myc were decreased significantly， with statistical significance （P＜0.05）. CONCLUSIONS： BEZ and FEN can inhibit the proliferation of PC-9 cells， and down-regulate c-myc expression.