Preliminary Exploration For The Mechanism Of LLGL2 Regulated By SOX2 In Esophageal Squamous Cell Carcinoma

Objective:To explore the roles of LLGL2 in the pathogenesis and progression of esophageal squamous cell carcinoma(ESCC)after knockdown of SOX2.To lay a foundation for the establishment of candidate schemes for the diagnosis and treatment of ESCC.Methods:1.The changes of LLGL2 in mRNA and protein levels after SOX2 knockdown were observed by real-time PCR and Western blot.2.The expression of LLGL2 were detected by immunofluorescence technology in each 15 clinical cases of ESCC and normal tissues.3.The changes of miR-142-3p expression after SOX2 knockdown were observed by real-time PCR to verify our previous miRNA microarray results.4.Luc iferase reporter plasmids containing the 3’UTR of target gene LLGL2 mRNA of miR-142-3p were constructed.Dual-luciferase reporter assays were used to verify the effect of miR-142-3p on LLGL2.5.The lentiviral vector was constructed,and thus KYSE450 and TE-1 cell lines stable expression of LLGL2 were established.6.The proliferation of ESCC cell lines with LLGL2 overexpression were analyzed by MTT and colony formation.The migration and invasion of ESCC cell lines with LLGL2 overexpression were detected by wound healing assay and transwell experiment.The tumor formation was observed in vivo after nude mice were xenografted with KYSE450 cells each subcutaneously.Results:1.The results of real-time PCR and Western blot showed that LLGL2 was up-regulated significantly at the protein level after SOX2 down-regulation,but there was no significant difference at the mRNA level,indicating that LLGL2 may be regulated at the post transcriptional level.2.Previous studies have shown that SOX2 is highly expressed in ESCC.The results of immunofluorescence showed that the expression of LLGL2 in cancer tissues was lower compared with the normal tissues,suggesting that LLGL2 may be a tumor suppressor gene.3.The results of real-time PCR showed that miR-142-3p was down-regulated after SOX2 down-regulation.4.The results of dual-enzyme digestion and sequencing showed recombinant plasmids containing the wide type or mutant LLGL2 gene 3’UTR were successfully constructed.Luciferase reporter assay indicated that LLGL2 is the target gene of miR-142-3p.5.The results of dual-enzyme digestion and sequencing showed the pCDH-CMV-LLGL2-IRES-GFP-EF 1-Puro lentiviral vector was successfully constructed.Western blot showed the expression level of LLGL2 protein was significantly increased in KYSE450 and TE-1 stable cell lines compared with the controls(P<0.05),indicating that two kinds of esophageal squamous cell carcinoma cell lines stably expressing LLGL2 are successfully established.6.The results of MTT and plate clone formation assay showed that the proliferation abilities of KYSE450 and TE-1 after LLGL2 overexpression were significantly lower compared with the control group(P<0.05).The results of stratch assay and invasion test showed that the overexpression of LLGL2 could inhibit the migration and invasion of tumor cells(P<0.05)compared with the control group.The result of xenograft transplantation assay showed that the tumor formation of KYSE450 with LLGL2 overexpressed in the nude mice was decreased compared with the control group.Conclusion:1.miR-142-3p is down-regulated and LLGL2 is up-regulated after SOX2 down-regulation in ESCC.2.LLGL2 is the target gene of miR-142-3p.3.LLGL2 can inhibit the proliferation,clone formation,migration and invasion of esophageal squamous cell carcinoma cell lines KYSE450 and TE-1 in vitro,and can inhibit the growth of KYSE450 cells in vivo.