The Expression And Role Of KLF4 In Esophageal Squamous Cancer Cells

Objective: In the early stage of the work study, we found that the sample Kruppel factor 4(KLF4) is lower expression in esophageal squamous carcinoma, and is highly expressed in normal esophageal mucosa through clinical research, the expression and the staging of esophageal squamous carcinoma is related to the prognosis of patients.In addition,the result of detection by methylation specific PCR, Western blot of esophageal squamous cancer cells showed: CAES17, EC109, KYSE140, and KYSE KYSE70,KYSE30 are relatively high expression of KLF4, and it's relatively low expression in TE1 and KYSE 510. The results found that KYSE140 cell line was low methylation but high expression KLF4, and TE1 was high methylation but lower expression KLF4. This topic mainly experiments in vitroby cytological, and further study of KLF4 expression in esophageal squamous cancer cells and its role, and to explore its influence on PI3K/AKT signal pathway. Purpose is to explore KLF4 initial role in the occurrence and development in esophageal squamous carcinoma and its mechanism, we hope found target genes for the gene therapy of esophageal squamous cell carcinomas.Methods: 1.Constructed lentivirus vector which carries targeting KLF4 interfering RNA(LV-sh KLF4-A ~ C) and overexpressing lentivirus vector which contains plasmid with full length KLF4 expression(LV-KLF4), screened lentivirus vector with high infection rate, transfected and selected stable esophageal cancer cell lines.Whether the lentivirus vector was successfully constructed was certified by sequencing, fluorescence, RT-PCR, Western blot. 2.The expression level of KLF4 protein before and after interference by KYSE140 and TE1 was measured by Western blot.Scratching test was used for the detection of migration ability of esophageal cancer cell before and after interference by KYSE140 and TE1.Invasion ability of esophageal cancer cell before and after interference by KYSE140 and TE1 was judged by Transwell assay. Proliferation ability of esophageal cancer cell before and after interference by KYSE140 and TE1 was detected by MTT assay. 3.Western blot was used to detect the expression level of P-AKT and AKT protein before interfered or overexpressed by KYSE140 and TE1 in esophageal cancer cell.Results: 1. Lentivirus vector which carries targeting KLF4 interfering RNA and overexpressing lentivirus vector which contains plasmid with full length KLF4 expression was successfully constructed, among which the LV-sh KLF4-C group got highest interference efficiency. The expression efficiency of KLF4 in the LV-KLF4 group were significantly higher than those in TE1 group and Ctrl- LV-KLF4 group.2.Western blot showed that KLF4 protein expression level in sh KLF4-C group was lower than KYSE140 group and Ctrl-LV-sh RNA group. Scratching test revealed that LV-sh KLF4-C group got a higher migration ability than KYSE140 group and Ctrl-LV-sh RNA group. The results of Transwell showed that the invasion ability of LV-sh KLF4-C group was higher than that of KYSE140 group and Ctrl-LV-sh RNA group. MTT results showed that the proliferation ability of LV-sh KLF4-C group was higher compared with KYSE140 group and Ctrl-LV-sh RNA group. There was no significant difference between the KYSE140 group and the Ctrl-sh RNA group. The results showed that the expression of KLF4 was negatively correlated with the migration, invasion and proliferation of esophageal cancer cells.3. Western blot showed that expression level of p-Akt protein in LV-sh KLF4-C group were higher than KYSE140 group and Ctrl-LV-sh RNA group, no significant difference were found in the expression of AKT among the three groups. Expression level of p-Akt protein in LV-KLF4 group were lower than TE1 group and Ctrl-LV-KLF4 group, no significant difference were found in the expression of AKT.Conclusion 1.Lentivirus vector which carries targeting KLF4 interfering RNA and overexpressing lentivirus vector which contains plasmid with full length KLF4 expression was successfully constructed.2.KLF4 plays an important role in the migration, invasion and proliferation of esophageal cancer cell line.3.P-AKT expression was significantly increased in KYSE140's esophageal cancer cell line after KLF4 was silenced. P-AKT expression was decreased after KLF4 overexpression in TE-1 esophageal cancer cell line. The expression of KLF4 in esophageal cancer cell line can affect the PI3K/Akt signaling pathway.