| ? Objective ? To verify the effect of Wnt/Lin28 negatively regulating the expression of micro RNA-let-7a on metastasis of esophageal squamous cell carcinoma and further explore its related mechanisms.?Methods?(1)Quantitative PCR detection for microRNA-let-7a,microRNA-let-7b,and micro RNA-let-7c expression in cancer tissues and adjacent normal tissues of 70 patients with esophageal squamous cell carcinoma;agarose gel electrop Horesis to detect the expression of micro RNA-let-7a in cancer tissues and paracancerous tissues of recurrent or non-recurrent esophageal squamous cell carcinoma patients.Quantitative PCR was used to detect the expression of micro RNA-let-7a in cancer tissues and paracancerous tissues of patients with recurrent and non-recurrent squamous cell carcinoma.(2)Quantitative PCR detection for micro RNA-let-7a expression in human normal esophageal squamous cell Het-1a and TE-1,ECA109,KYSE-150 cells;CCK8 assay detect for the proliferation of esophageal squamous cell carcinoma cells at different time after overexpression or inhibition of micro RNA-let-7a treatment and the expression of Ki-67 was detected by Western blotting.Scratch test and Transwell TM assay for the migration and infiltration ability of ECA109 cells after up-rehulation of micro RNA-let-7a.(3)Morphological changes of ECA109 and TE-1 cells after overexpression or inhibition of micro RNA-let-7a were observed by light microscope;Changes of E-cadherin and Vimentin expression in esophageal squamous cell carcinoma cells were detected by immunofluorescence and Western blotting after overexpression of micro RNA-let-7a;Upregulation of mi RNA-let-7a or interfering with Lin28 in quantitative PCR analysis of Slug,Snail m RNA in ECA109 cells and detection of Lin28,MMP-2,MMP-9,VEGF-C,VEGF-A protein expression changes by Western blotting;ELISA was used to detect the expression of VEGF-C,MMP-2and MMP-9 in the supernatant of ECA109 cells before and after up-rehulation of micro RNA-let-7a.(4)Western blotting was used to detect the activation of Wnt signaling pathway between Het-1a cells and esophageal squamous cell carcinoma TE-1 and ECA109 cells,and the effect on Wnt signaling in ECA109 cells after upregulating or inhibiting micro RNA-let-7a;combined with Wnt signaling activator Li Cl and inhibitor ICG-001 to verify the regulation of micro RNA-let-7a by Lin28 protein expression in esophageal squamous cell carcinoma cells;further quantitative PCR assay and Western blotting assay were to detected changes in ability of infiltration and invasion after Wnt/Lin28 negatively regulating the expression of MicroRNA-let-7a.?Results?(1)The expressions of microRNA-let-7a,microRNA-let-7b and micro RNA-let-7c in esophageal cancer patients were lower than those in paracancerous tissues,and the expression of micro RNA-let-7a was significantly different.The low expression of micro RNA-let-7a in esophageal cancer tissues was closely related to TMN staging and tumor recurrence but was no related to the patient's gender,age,tumor differentiation,tumor size,and tumor location.(2)The expression of microRNA-let-7a in esophageal squamous cell carcinoma cell lines was significantly lower than that of normal esophageal epithelial cells;the up-regulation or inhibition of micro RNA-let-7a in esophageal squamous cell carcinoma ECA109 cells had no effect on proliferation;inhibition of micro RNA-let-7a expression can promote the migration and invasion of esophageal squamous carcinoma cells.(3)There was no significant change in the morp Hology of micro RNA-let-7a after overexpression or inhibition in ECA109 and TE-1 cells;immunofluorescence and immunoblot results showed that the up-regulation of micro RNA-let-7a in ECA109 cells increased the expression intensity of E-cadherin and decreased Vimentin Expression;interfering with Lin28 expression or up-regulating micro RNA-let-7a could down-regulate theexpression levels of Slug and Snail of EMT-related transcription factors,while micro RNA-let-7a expression had no significant effect on Lin28 protein;interfering with Lin28 expression or up-regulating micro RNA-let-7a could significantly decreased expression of MMP9 and VEGF-C in ECA109 cells;upregulation of micro RNA-let-7a in ECA109 cells can downregulate the secretion of VEGF-C and MMP9;(4)In esophageal squamous cell carcinoma,changes in micro RNA-let-7a expression had no significant effect on Wnt pathway activation;activation or inhibition of Wnt pathway could down-or up-regulate micro RNA-let-7a expression by up-or down-regulation of Lin28 protein expression.Concomitant with the occurrence,invasion and EMT.?Conclusions?The abnormal activation of Wnt pathway in esophageal squamous cell carcinoma can promote the metastasis through inhibiting the expression of micro RNA-let-7a by Lin28. |
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