| ObjectiveThe aim of this study is to test the protein expression of NLRP3 in renal cell carcinoma tissues and clarify the role of NLRP3 inflammasome and Sirt1 in cell proliferation, migration, invasion and apoptosis after RSV treatment.Method1. Human renal carcinoma 786-O cells were randomly divided into control group, RSV treatment groups. RSV treatment groups were treatment with RSV(25u M, 50 u M, 100 u M) for12 h, 24 h, 48 h. The cell proliferation rates were checked by CCK-8 method. The morphological changes of 786-O cells were observed under inverted microscope. The cell migration rates were evaluated in vitro by wound-healing assay. The cell invasion rates were determined by Transwell assay. Cell apoptosis rates were determined by flow cytometry. The protein expression of the apoptosis associated protein Bax was analyzed by Western blot after treatment with RSV.2. The protein expression of NLRP3 in renal cell carcinoma tissues and normal tissues were determined by Western blot. Human renal carcinoma 786-O cells were randomly divided into control group, LPS+ATP treatment group, LPS+ATP+RSV treatment group(RSV treatment group). Cells were pretreated with 50 u M RSV for 30 min in RSV treatment group, subsequently 1 mg/ml LPS was added into cell cultures for 5.5h, followed by 5m M ATP for 30 min. The effects of RSV on the m RNA expression of NLRP3, Caspase-1 and IL-1β were determined by Real-time PCR. In addition, small interfering RNA(si RNA) was used to knock down Sirt1 gene expression, the expression of Sirt1 and NLRP3 in protein and m RNA levels were determined by Real-time PCR and Western blot.Results1. After treatment with RSV in 25 u M, 50 u M, 100 u M for 12 h, 24 h, 48 h, the proliferation of 786-O cells were significantly inhibited. The inhibition rates were 10.22±4.02%、23.81±5.19%、31.45±6.15%. After treatment with 50 u M RSV for 12h, 24 h, 48 h, the inhibition rates were 19.05±4.89%、44.85±3.38%、52.54±6.55%at the time point of 12 h, 24 h, 48 h after treatment with 25 u M RSV. After treatment with 100 u M RSV for 12 h, 24 h, 48 h, the inhibition rates were 27.47±4.84%、53.55±13.03%、67.02±10.78%. 50 u M was selected as the final concentration in the following experiment according IC50. The volume of 786-O cells was reduced compared with control after treatment with RSV for 24 h. The wound healing assay showed that the width of cell migration in RSV treated group is narrower than the control group at the same time. Transwell assay showed the number of cells that invaded through the microporous member was 11.21±4.19, and the control group was 39.23±3.64 after treated with 50 u M RSV for 24 h, indicating that RSV has the potential to inhibit migration and invasion ability of 786-O cells in vitro. There was great statistical significance between two groups(P<0.05). The apoptotic rates of 786-O cells treated with 50 u M RSV after 24 h were 3.56%, and the control group was 15.27%. The differences were statistically significance(P<0.05). The protein level of Bax is gradually increased in a time dependent manner compared with control group.2. The protein expression of NLRP3 in renal cell carcinoma tissues were elevated significantly compared to the normal tissues(P<0.05). The m RNA level of NLRP3, Caspase-1 and IL-1β were significantly increased in 786-O cells after stimulation with LPS and ATP(P<0.05). Adding RSV into medium inhibits the enhancement effect of LPS and ATP. And the protein level of Sirt1 was higher than control group(P<0.01). But the inhibition effect of RSV was attunated after silencing Sirt1 gene. The differences was statisticance significantly(P<0.05). The Bax expression is decreased after RSV treatment. But this effect was attunated after Sirt1 gene silencing.ConclusionIn a word, NLRP3 plays important role in the proliferation, migration and invasion, promoting the apoptosis progression of 786-O cells. The NLRP3 inflammasome and Sirt1 play important roles in the apoptotic promotion effects of RSV. |
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