| Objective: To study the expression of Linc ROR in esophageal squamous cell carcinoma and normal esophageal epithelium with different differentiation degree,to preliminarily research and discuss the function thereof in the process of esophageal squamous carcinoma invasion and metastasis,and to discuss the molecular mechanism thereof through detecting the gap-associated proteins of mi RNA145/ZEB2 pathway.Methods:(1)q RT-PCR was used to detect the expression of Linc ROR in esophageal squamous carcinoma cell lines TE-1,、TE-13、 Eca109 and normal esophageal epithelial cell line Het-1a.(2)The cells with the highest expression of the Linc ROR were selected and the silencing related genes were transfected by si RNA.(3)CCK-8,scratch test,flow cytometry,Transwell assay,and colony formation assay were used to compare the proliferation,invasion,migration and apoptosis of Linc ROR before and after silencing.(4)Detect the variation of the related molecular levels of mi RNA145/ZEB2 pathway before and after Linc ROR silencing through western blot and q RT-PCR,and meanwhile detect the variation of the expression of EMT gap-associated proteins(E-cadherin,β-catenin,N-cadherin and Vimentin)before and after Linc ROR silencing through western blot.Resaults: 1.The q RT-PCR showed that the expression of Linc ROR in each cell lines of TE-1,TE-13,Eca109 and Het-1a were 1.02 ± 0.24、0.74 ± 0.04、0.90 ± 0.07、0.58 ± 0.04,Compared with the normal esophageal epithelial cell,the esophageal squamous carcinoma cell has high Linc ROR expression therein(P<0.05),and esophageal squamous cell carcinoma lines TE-1 are the highest expression..2.Effects of silencing Linc ROR gene on cell survival rate of TE-1 cells measured by CCK-8 at different time points.Results showed si-ROR group:(82.72±1.90)%(24h)、(63.75±3.67)%(48h)、(62.14±1.56)%(72h);si-NC group:(98.74±1.35)%(24h)、(97.23±6.53)%(48h)、(92.45±1.99)%(72h).The clone formation rates of each group after 15 days cell culture in the clone formation experiment were as follows: si-ROR group(18.00±1.73)%、si-NC group and control group(75.67±7.02)%.In Transwell cell invasion assay,the number of cells in si-ROR group and control group passing through matrigel were as follows: 68.00±2.65、137.33±6.43、135.33±4.93.The results of scratch test showed that the cell migration distances of si-ROR group and control group were(362±3.6)μm、(512±6.7)μm、(514±3.6)μm respectively.The apoptotic rates of si-ROR group and control group were determined by flow cytometry.It was(17.15±0.49)%、(6.99±0.49)%、(5.13±0.13)% respectively.These results suggested that targeted silencing of Linc ROR could inhibit the proliferation and migration and invasion of TE-1 cells,and increased the rate of cell apoptosis.3.The q RT-PCR showed that the expression of mi RNA 145 in groups of si-ROR,si-NC,control were4.99±0.26,1.10±0.09,1.00±0.07,and the expression of ZEB2 were 0.27±0.01,0.95±0.04,1.00±0.02.Western blot showed that the group si-ROR can increase epithelial molecular marker(E-cadherin,β-catenin)expression and reduce Targeted Linc ROR silencing can increase mi RNA145 expression and epithelial molecular marker(E-cadherin,β-catenin)expression,but reduce ZEB2 expression and mesenchyme marker(N-cadherin,Vimentin)expression.Conclusions: 1.Compared with the normal esophageal epithelial cell,the esophageal squamous carcinoma cell has the increased expression level.2.In the esophageal squamous carcinoma cell strains,Linc ROR can activate EMT process through mi RNA145/ZEB2 signal pathway and promote the esophageal squamous carcinoma cell invasion and metastasis. |
