薯莨提取物通过KTN1反义RNA 1对食管癌细胞生物行为的影响

Effect of Dioscorea cirrhosa extract on biological behavior of esophageal cancer cells through KTN1-AS1

目的研究薯莨提取物是否影响食管癌细胞的生物学行为。方法自2019年1月至2020年1月,采用(10、20和30 mg/L)浓度的薯莨提取物处理食管癌Eca109细胞,分别记为薯莨-L组、薯莨-M组、薯莨-H组,另以未加薯莨提取物的Eca109细胞作为对照组。细胞计数试剂盒8(CCK-8)测定细胞增殖,克隆形成实验分析细胞克隆能力,蛋白质印迹法检测周期素依赖激酶抑制剂p21(P21)、上皮钙黏素(E-cadherin)、基质金属蛋白酶-2(MMP-2)蛋白表达,Transwell分析细胞迁移、侵袭,实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测长链非编码RNA(lncRNA)KTN1反义RNA 1(KTN1-AS1)表达。在Eca109细胞中转染KTN1-AS1小干扰RNA(si-KTN1-AS1),或转染KTN1-AS1过表达质粒(pcDNA3.1-KTN1-AS1)并使用薯莨提取物处理,观察其对细胞增殖、迁移和侵袭的影响。结果与对照组相比,薯莨-L组、薯莨-M组、薯莨-H组Eca109细胞的抑制率[(1.06±0.21)%比(19.25±1.68)%比(38.57±3.69)%比(62.14±6.06)%]、P21、E-cadherin蛋白水平明显提高,细胞的克隆形成数、迁移细胞数[(142.00±11.59)比(121.00±11.07)比(94.00±9.26)比(73.00±7.15)]、侵袭细胞数[(81.00±8.03)比(67.00±6.21)比(50.00±5.04)比(37.00±3.56)]、MMP-2蛋白表达量、KTN1-AS1表达量显著减少(P<0.05),均呈浓度依赖性。抑制KTN1-AS1明显增加Eca109细胞的抑制率[(1.04±0.17)%比(47.81±4.55)%]、E-cadherin、P21蛋白表达量,显著降低迁移细胞数[(144.00±13.52)比(85.00±8.51)]、侵袭细胞数[(80.00±8.11)比(47.00±4.36)]、克隆形成数和MMP-2蛋白水平(P<0.05)。过表达KTN1-AS1能减弱薯莨提取物对食管癌细胞增殖、迁移、侵袭的抑制作用。结论薯莨提取物通过下调食管癌细胞中KTN1-AS1的表达,发挥抑制细胞增殖、迁移和侵袭的作用。

Objective To investigate whether the extract of Dioscorea cirrhosa extract affects the biological behavior of esophageal cancer cells through long non-coding RNA(lncRNA)KTN1 antisense RNA 1(KTN1-AS1).Methods From January 2019 to January 2020,esophageal cancer Eca109 cells were treated with(10,20,and 30 mg/L)Dioscorea cirrhosa extract,which were designated as Di⁃oscorea cirrhosa-L group,Dioscorea cirrhosa-M group and Dioscorea cirrhosa-H group,respectively,and Eca109 cells without Dioscorea cirrhosa extract were used as control group.Cell proliferation was measured by Cell Counting Kit-8(CCK-8),the cloning formation experiment analyzed the cloning ability of the cells.Western blotting was used to detect the protein expression of P21,E-cadherin and matrix metalloproteinase-2(MMP-2),Transwell analyzed cell migration and invasion,and real-time fluorescence quantitative PCR(qRTPCR)detected KTN1-AS1 expression.Eca109 cells were transfected with KTN1-AS1 small interfering RNA(si-KTN1-AS1),or transfected with KTN1-AS1 overexpressed plasmid(pcDNA3.1-KTN1-AS1)and treated with Dioscorea cirrhosa extract to observe the effects on cell proliferation,migration,and invasion.Results Compared with the control group,the inhibition rate[(1.06±0.21)%vs.(19.25±1.68)%vs.(38.57±3.69)%vs.(62.14±6.06)%]of Eca109 cells in Dioscorea cirrhosa-L group,Dioscorea cirrhosa-M group and Di⁃oscorea cirrhosa-H group,P21,and E-cadherin protein levels were significantly increased,and the number of colony formation,number of migrating cells[(142.00±11.59)vs.(121.00±11.07)vs.(94.00±9.26)vs.(73.00±7.15)],number of invading cells[(81.00±8.03)vs.(67.00±6.21)vs.(50.00±5.04)vs.(37.00±3.56)],MMP-2 protein expression levels,and KTN1-AS1 expression were evidently reduced(P<0.05),with concentration dependence.Inhibition of KTN1-AS1 greatly increased the inhibition rate[(1.04±0.17)%vs.(47.81±4.55)%]of Eca109 cells,as well as the expression of E-cadherin,and P21 protein,and obviously reduced the number of migrating cells[(144.00±13.52)vs.(85.00±8.51)],invading cells[(80.00±8.11)vs.(47.00±4.36)],colony formation,and MMP-2 protein levels(P<0.05).Overexpression of KTN1-AS1 can reduce the inhibitory effect of Dioscorea cirrhosa extract on the proliferation,migration and invasion of esophageal cancer cells.Conclusion Dioscorea cirrhosa extract can inhibit cell proliferation,migration and invasion by down-regulating the expression of KTN1-AS1 in esophageal cancer cells.