Effect and mechanism of low ENPP1 expression on the epithelial-mesenchymal transition of oral squamous cell carcinoma cell line
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摘要:
目的 探讨外核苷酸焦磷酸酶/磷酸二酯酶1(ENPP1)低表达对口腔鳞状细胞癌(简称口腔鳞癌)细胞上皮间质转化的作用。 方法 取正常人口腔黏膜上皮细胞系、人口腔鳞癌细胞系CAL-27进行培养、传代，检测ENPP1 mRNA表达。取对数生长期CAL-27细胞进行转染，分为空白组(未处理)、对照组(转染NC-siRNA质粒)及转染组(转染ENPP1-siRNA)，检测ENPP1 mRNA、蛋白表达。取对数生长期CAL-27细胞进行转染，分为A组(未转染)、B组(转染NC-siRNA质粒)、C组(转染ENPP1-siRNA质粒+MAPK激动剂)、D组(转染ENPP1-siRNA质粒)、E组(MAPK激动剂)，检测CAL-27细胞增殖活性、侵袭细胞数、迁移能力及磷酸化丝裂原活化蛋白激酶(p-MAPK)、磷酸化胞外信号调节蛋白激酶1/2(p-ERK1/2)蛋白表达。 结果 各时段吸光度(A)值，D组＜C组＜A组、B组＜E组(均P＜0.05)；A组、B组、C组、D组、E组侵袭细胞数分别为(85.23±2.22)个/视野、(85.41±1.59)个/视野、(53.67±2.10)个/视野、(37.39±2.06)个/视野、(111.08±5.20)个/视野，D组＜C组＜A组、B组＜E组(均P＜0.05)；划痕宽度百分比，E组＜A组、B组＜C组＜D组(均P＜0.05)；p-MAPK、p-ERK1、p-ERK2蛋白相对表达量，D组＜C组＜A组、B组＜E组(均P＜0.05)。 结论 ENPP1低表达可抑制口腔鳞癌细胞上皮间质转化，机制可能与抑制MAPK信号通路有关。 -
关键词:
- 口腔鳞状细胞癌 /
- 上皮间质转化 /
- 核苷酸焦磷酸酶/磷酸二酯酶1
Abstract:Objective To investigate the effect of low expression ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) on the epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma. Methods The normal human oral mucosal epithelial cell line and human oral squamous cell carcinoma CAL-27 cells were cultured and subcultured, the expression of ENPP1 mRNA was detected. CAL-27 cells in logarithmic growth stage were transfected and divided into blank group (without treatment), control group (transfected with NC-siRNA plasmid) and transfection group (transfected with ENPP1-siRNA). The expression of ENPP1 mRNA and protein were detected. CAL-27 cells in logarithmic growth stage were transfected and divided into group A (not transfected), group B (transfected with NC-siRNA plasmid) and group C (transfected with ENPP1-siRNA plasmid + MAPK agonist), group D (transfected with ENPP1-siRNA plasmid), group E (MAPK agonist). The proliferation activity, number of invasive cells, migration ability and protein expression of phosphorylated-mitogen activated protein kinase (p-MAPK) and phosphorylated-extracellular regulated kinase 1/2 (p-ERK1/2) were detected. Results Absorbance (A) of each period, group D < group C < group A, group B < group E (all P < 0.05). The number of invasive cells in five groups were (85.23±2.22) cells/field, (85.41±1.59) cells/field, (53.67±2.10) cells/field, (37.39±2.06) cells/field and (111.08±5.20) cells/field cells/field, respectively, group D < group C < group A, group B < group E (all P < 0.05). The percentage of scratch width: group E < group A, group B < group C < group D (all P < 0.05). The relative expression levels of p-MAPK, p-ERK1 and p-ERK2 proteins: group D < group C < group A, group B < group E (all P < 0.05). Conclusion Low expression of ENPP1 can inhibit EMT, the mechanism may be related to the inhibition of MAPK signaling pathway. -
图 2 各组细胞侵袭能力(HE染色，×200)
Figure 2. Invasion ability of cells in each group (HE staining, ×200)
图 4 各组细胞p-MAPK、p-ERK1、p-ERK2的蛋白表达情况
Figure 4. Protein expression of p-MAPK, p-ERK1 and p-ERK2 in cells of each group
表 1 各组细胞ENPP1 mRNA、蛋白相对表达量比较(x±s)
Table 1. Comparison of the relative expression levels of ENPP1 mRNA and protein in each group of cells (x±s)
组别 n ENPP1 mRNA ENPP1蛋白 空白组 5 0.64±0.02 0.73±0.03 对照组 5 0.63±0.02 0.72±0.03 转染组 5 0.31±0.02ab 0.24±0.02ab F值 328.086 524.770 P值 < 0.001 ＜0.001 注：与空白组比较，aP＜0.05；与对照组比较，bP＜0.05。 
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表 2 各组细胞MTT实验A值比较(x±s，%)
Table 2. Comparison of A value in MTT experiment of cells in each group(x±s)
组别 n 24 h 48 h 72 h A组 5 30.68±1.65 39.76±1.69e 52.22±3.61ef B组 5 30.35±1.35 39.86±1.74e 51.49±2.51ef C组 5 19.09±1.02ab 29.62±1.59abe 40.42±2.25abef D组 5 8.33±0.70abc 14.70±1.44abce 21.04±1.86abcef E组 5 38.94±1.93abcd 54.91±1.60abcde 65.55±1.79abcdef F值 289.917 337.243 178.047 P值 ＜0.001 ＜0.001 ＜0.001 注：与A组比较，aP＜0.05；与B组比较，bP＜0.05；与C组比较，cP＜0.05；与D组比较，dP＜0.05；与本组培养24 h时比较，eP＜0.05；与本组培养48 h时比较，fP＜0.05。
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表 3 各组CAL-27细胞侵袭细胞数比较(x±s，个/视野)
Table 3. Comparison of the number of invasive cells of CAL-27 cells in each group (x±s, pcs/field of view)
组别 n 侵袭细胞数 A组 5 85.23±2.22 B组 5 84.51±1.59 C组 5 53.67±2.10ab D组 5 37.39±2.06abc E组 5 111.08±5.20abcd F值 390.643 P值 ＜0.001 注：与A组比较，aP＜0.05；与B组比较，bP＜0.05；与C组比较，cP＜0.05；与D组比较，dP＜0.05。
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表 4 各组CAL-27细胞划痕宽度百分比比较(x±s，%)
Table 4. Comparison of the percentage of scratch width of CAL-27 cells in each group (x±s, %)
组别 n 划痕宽度 A组 5 44.73±1.78 B组 5 44.05±2.09 C组 5 57.99±1.17ab D组 5 95.59±2.15abc E组 5 31.08±2.21abcd F值 666.858 P值 ＜0.001 注：与A组比较，aP＜0.05；与B组比较，bP＜0.05；与C组比较，cP＜0.05；与D组比较，dP＜0.05。
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表 5 各组细胞p-MAPK、p-ERK1、p-ERK2蛋白相对表达量比较(x±s)
Table 5. Comparison of the relative expression levels of p-MAPK, p-ERK1 and p-ERK2 proteins in each group of cells (x±s)
组别 n p-MAPK p-ERK1 p-ERK2 A组 5 0.37±0.02 0.66±0.03 0.52±0.02 B组 5 0.36±0.02 0.65±0.02 0.52±0.02 C组 5 0.27±0.01ab 0.32±0.02ab 0.33±0.01ab D组 5 0.12±0.01abc 0.14±0.01abc 0.15±0.01abc E组 5 0.75±0.02abcd 1.01±0.02abcd 1.13±0.05abcd F值 845.950 1 042.375 844.352 P值 ＜0.001 ＜0.001 ＜0.001 注：与A组比较，aP＜0.05；与B组比较，bP＜0.05；与C组比较，cP＜0.05；与D组比较，dP＜0.05。
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