| Objective:With the economic and social development and environmental changes,skin squamous cell carcinoma(SCC)not only in our country but also in the world's incidence increased year by year,which poses a huge threat to human health.At present,the treatment of SCC mainly focused on surgery and radiation therapy,and poor efficacy of drug treatment.Metformin is widely used in the clinical treatment of type II diabetes.Epidemiological analysis of patients with diabetes showed a significant association between metformin and various types of solid tumors.Because metformin can enhance the body's sensitivity to insulin and reduce the absorption of glucose,it can inhibit the metabolism of tumor cells.With the deepening of the research,the effect of metformin on anti-tumor activity in vitro and in vivo has been paid more and more attention.Studies have shown that metformin can inhibit the growth of SCC tumor in mice,but the mechanism of inhibiting the growth of tumor cells and inducing apoptosis of cancer cells has not been fully elucidated.Nuclear transcription factor(NF-?B)controls the expression of many key physiological response genes,including immune and acute inflammatory responses,cell adhesion,differentiation,oxidative stress,and apoptosis.NF-?B /p65,as an important member of the NF-?B family,plays an important role in the development and progression of many tumors.It is also widely expressed in A431 tumor cells.C-myc as NF-?B downstream gene overexpression on tumor cell proliferation and metastasis plays a significant role.In this study,we investigated the effect of metformin on the expression of p65,p-p65 and c-myc in A431 tumor cells,and explored the new mechanism of metformin on SCC.Methods: 1 Cell: The cells used in this experiment were derived from human skin squamous cell carcinoma cell line A431.2 Experimental grouping and intervention: The experiment was grouped with different concentrations of metformin in IMDM medium,and 1m M,2.5m M,5m M and 10 m M were used in 4 experimental groups.With no drug group as the control group.3 CCK-8 method was used to detect the cell inhibition rate at 24 h,48 and 72 h by OD.4 Immunofluorescence was used to detect the expression of p65 in control and 10 m M group.5 The protein expression of total p65,p65 of cytoplasm and c-myc and p-p65 in nucleus were detected by Western blot.6 The expression of p65 and c-myc gene levels in each group was detected by real-time quantitative PCR(q RT-PCR).7 Statistical analysis was performed with SPSS19.0.Measurement data were expressed as mean ± standard deviation,One-way ANOVA test was used to analyze statistical differences between groups.SNK-q test between the two groups and P<0.05 was statistically significant difference.Results:1 At the same time,the effect of the increase in the concentration of metformin(1m M,2.5 m M,5m M,10 m M)on the inhibition of A431 cells: Metformin significantly inhibited the proliferation of A431 cells with 0m M and 10 m M metformin for 24 h and 72 h by morphological analysis.After 24 hours of drug stimulation,the inhibition rates were:(5.18±0.76)%,(8.05±2.08)%,(14.39±1.77)%,(17.85±0.60)%.Differences between groups were statistically significant(F=63.913,P=0.000<0.05).After 48 hours of drug stimulation,the inhibition rates were:(7.79±0.27)%,(11.97±0.70)%,(19.14±0.80)%,(32.06±2.07)%.Differences between groups were statistically significant(F=330.990,P=0.000<0.01).After 72 hours of drug stimulation,the inhibition rates were:(9.27±0.70)%,(14.93±1.32)%,(21.31±0.95)%,(37.09±2.80)%.Differences between groups were statistically significant(F=209.890,P=0.000<0.01).It can be seen at the same time,the effect of metformin on cell inhibition rate has a certain concentration-dependent.2 Under the same drug concentration,the effect of the drug on the cells over time: 1m M group: 24 h,48h,72 h cell inhibition rate was statistically significant(F=45.532,P=0.000<0.01);2.5m M group: 24 h,48h,72 h cell inhibition rate was statistically significant(F=21.806,P=0.000<0.01);5m M group: 24 h,48h,72 h cell inhibition rate was statistically significant(F=32.237,P=0.000<0.01);10m M group: 24 h,48h,72 h cell inhibition rate was statistically significant(F=95.631,P=0.000<0.01).It can be seen that the inhibition rate of metformin on cells is time dependent on the same drug concentration.3 Immunofluorescence assay was used to observe the distribution of p65 in the nucleus and cytoplasm after metformin was applied to the cells: After treated with metformin for 24 hours,the expression of p65 in the nucleus of 10 m M drug concentration was significantly less than that in the control group.4 Effects of drug-stimulated cells on the expression of total p65 after 24 hours of drug concentration(0m M,1m M,2.5m M,5m M,10 m M): The relative expression of each group of proteins: 1.11±0.07,1.09±0.06,0.93±0.04,0.92±0.04,0.43±0.03.the difference was statistically significant(F=89.018,P=0.000<0.01).It can be seen that the relative expression of total p65 decreased with the increase of drug concentration,which was concentration-dependent.5 Effects of drug-stimulated cells on the expression of p65 in cytoplasm after 24 hours of drug concentration(0m M,1m M,2.5m M,5m M,10 m M): The relative expression of each group of proteins: 0.50±0.04,0.53±0.02,0.78±0.04,0.79±0.03,0.82±0.02.the difference was statistically significant(F=76.789,P=0.000<0.01).It can be seen that the relative expression of p65 in cytoplasm increased with the increase of drug concentration,and it was concentration-dependent.6 Effects of drug-stimulated cells on the expression of p-p65 in nucleus after 24 hours of drug concentration(0m M,1m M,2.5m M,5m M,10 m M): The relative expression of each group of proteins: 0.22±0.01,0.18±0.02,0.17±0.01,0.14±0.02,0.12±0.01.the difference was statistically significant(F=23.844,P=0.000<0.01).It can be seen that the relative expression of p-p65 in nucleus decreased with the increase of drug concentration,which was concentration-dependent.7 Effects of drug-stimulated cells on the expression of c-myc in nucleus after 24 hours of drug concentration(0m M,1m M,2.5m M,5m M,10 m M): The relative expression of each group of proteins: 0.85±0.07,0.79±0.06,0.50±0.02,0.27±0.04,0.18±0.03.the difference was statistically significant(F=130.910,P=0.000<0.01).It can be seen that the relative expression of c-myc in nucleus decreased with the increase of drug concentration,which was concentration-dependent.8 The expression of p65 m RNA were test in A431 cells at different concentrations of metformin(0m M,1m M,2.5m M,5m M,10 m M)by q RT-PCR.The relative expression of each group is as follows: 1.00±0.00,0.85±0.12,0.60±0.13,0.51±0.14,0.24±0.09.the difference was statistically significant(F=22.808,P=0.000<0.01).With the gradual increase in drug concentration,p65 m RNA expression decreased.The relative expression of c-myc m RNA of each group is as follows: 1.00±0.00,0.83±0.05,0.70±0.07,0.70±0.16,0.44±0.13.the difference was statistically significant(F=13.208,P=0.001<0.01).Conclusion:1 Metformin inhibits A431 cell proliferation.2 Metformin reduced the expression of p65 and c-myc in A431 cells and inhibited phosphorylation of p65. |
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