| Objective:To examine the effect of the autophagy of human HT-29 cells induced by VPA in vitro,and explore the mechanism of VPA’s impaction human colon cancer HT-29 cells.Methods:Culture human HT-29 cells in vitro,use media of different VPA concentrations(0mmol/L,2mmol/L,4mmol/L,8mmol/L,16mmol/L) to intervene HT-29 cells. Durations of intervention are 0h, 12 h, 24 h, and 48 h.Observe the growth of cells in each group underan inverted microscope. CCK-8 method is used to the examinecorrelation between human HT-29 cell proliferation and VPA concentrations, durations of intervention. Real-time PCR Analysisis used to detect the changes of m RNA mounts of LC3 and Beclin-1.Western Blot Analysis is used to detect the expressions of autophagy-related genes LC3, Beclin-1,m TOR and p-m TOR.Results:1.Morphological signatures under inverted microscope:the control group is in full shape, has abundant cytoplasm, adheres firmly, grows actively. The experimental group is not in good condition. Cells are shrunken, not firmly adherent. Some floating dead ones can be observed.2. Inhibition ratios of cell proliferation detected by CCK8:VPA fluid of four different concentrations(0mmol/L,2mmol/L,4mmol/L,8mmol/L,16mmol/L) are used to intervene HT-29 human colon adenocarcinoma cells’ growth.After 12 hours of intervention, theratios of inhibition are(0.03±0.01)%,(6.58±0.36)%,(8.74±0.66)%,(16.45±0.8)% and(18.49±0.43)% respectively. After 24 hours of intervention, the ratios of inhibition are(0.02±0.02)%,(10.43±0.45)%,(15.29±0.5)%,(28.94 ±0.3)%,(40.23±0.25)%. After 48 hours of intervention, the ratios are respectively(0.02±0.01)%,(19.22±0.32)%,(26.53±0.34) %,(41.82±0.25) % and(44.54±0.62)%. It is statistically significant that VPA inhibits HT-29 cell proliferation.3. The changes of gene expressions: LC3 and Beclin-1 are detected by fluorescence quantitative PCR. While concentrations of VPA in the fluids are 0mmol/L,2mmol/L,4mmol/L, 8mmol/L, 16mmol/L, gene expression levels of LC3 are(0.95±0.04),(1.42±0.34),(1.53±0.35),(1.98±0.26), and(2.13±0.31)levels of Beclin-1 are(0.98±0.01),(1.73±0.28),(1.88±0.19),(2.07±0.04), and(2.11±0.29). The difference is statistically significant(P<0.05).4. The expression of autophagy-related genes, LC3, Beclin-1, m TOR, and p-m TORdetected by Western Blot Analysis: the expression levels of LC3 in the control group and ‘16mmol/LVPA’ group are respectively(0.99±0.12)and(2.39±0.37). the expression levels of Beclin-1 are(1.01±0.15) to(1.52±0.21), those of m TOR are(1.04±0.17)to(1.16±0.21), those of P-m TOR are(1.12±0.27) to( 0.74±0.04). Compared m TOR expression levels of VPA group with control group, the result is statistically nonsignificant(p>0.05).Compared with the control group,the expression levels of p-m TOR is statistically significant(p<0.05).Conclusion:.1.VPA is effective in inhibiting HT-29 cell proliferation,inhibition is positively correlated with VPA concentration and time of interaction.2.VPA induces HT-29 cell autophagic cell death, its mechanism may have a connection with negatively regulated m TOR signal path. |
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