ABSTRACT
Objective To screen the critical genes related to the development of esophageal squamous cell carcinoma ( ESCC ) by weighted gene co-expression network analysis ( WGCNA ) and to verify by experiments.Methods Gene expression data of ESCC were downloaded from gene expression omnibus (GEO) database based on gene chip platform ( GPL) 570, GPL571, GPL96/97 or GPL14613 platform, respectively. Meanwhile, the obtained differentially expressed genes together with gene expression data of 81 ESCC patients from the cancer genome atlas ( TCGA ) and clinical data were analyzed by WGCNA to set up co-expression networks including mRNA and microRNA ( miRNA ) . The expression of miRNA in ESCC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction ( RT-PCR ) .And the expression of target protein Kruppel like factor 4 ( KLF4 ) and desmocollin 2 ( DSC2 ) were detected by immunohistochemistry .After ESCC cell line ECA-109 cells were transfected with miRNA-92b-3p mimic, cell cycle was tested by flow cytometry ,the cell invasion and migration ability was measured by Transwell chamber assay and scratch-wound assay.The expression of KLF4 and DSC2 was observed by confocal laser scanning microscopy and Western blotting .The target genes were verified by luciferase assay .T-test, rank sum test, chi-square test and Pearson correlation analysis were performed for statistical analysis .Results A total of 4023 differential expression gene ( DEG) and 328 differential expression miRNA ( DEM) were screened and 11 gene modules were set up by WGCNA .Among them, the brown modules were negatively associated with tumor grade and T stage (r=-0.340 and -0.268, P=0.002 and 0.016).Meanwhile, has-miR-92b and the potential target genes KLF4 and DSC2 were all in the brown module .Furthermore, the results of RT-PCR showed the expression of miRNA-92b-3p in ESCC tissues was higher than that in paracancerous tissues (3.052(1.652, 5.371) vs.0.985(0.558, 2.032)), and the difference was statistically significant (Z=-4.021,P<0.01). The results of immunohistochemistry demonstrated that the positive rates of KLF 4 and DSC2 in ESCC tissues were 43.3％(13/30) and 20.0％(6/30), respectively, which were lower than those of paracancerous tissues (70.0％(21/30) and 63.3％(19/30)), and the differences were statistically significant (χ2 =4.344 and 1.589, both P<0.05).After ECA-109 cells were transfected with miRNA-92b-3p mimics, the percentage of cells at G0/G1 phase decreased ((63.71 ±2.83)％vs.(54.62 ±4.00)％) and the percentage of cells at the S phase and G2/M phase increased ((31.81 ±2.88)％vs.(41.20％±2.87)％, and (3.87 ±1.75)％vs. (8.10 ±1.71)％, respectively), and the differences were statistically significant (t =3.215, 4.000 and 2.998;P=0.032, 0.016 and 0.040).The invasion and migration ability of the cells were significantly improved (79.67 ±27.54 vs.280.33 ±46.18, (69.72 ±3.91)％ vs.(84.90 ±5.25)％), and the differences were statistically significant (t=6.465 and 4.019, P=0.003 and 0.016).The results of Western blotting indicated that, compared with control mimic group , the expression of KLF4 and DSC2 was both dramatically downregulated after transfected with miRNA-92b-3p mimics transfected (1.00 ±0.23 vs.0.42 ±0.03, 1.00 ±0.20 vs.0.55 ± 0.21), and differences were statistically significant (t=4.470 and 5.493, P=0.042 and 0.032).The results of luciferase assay demonstrated that miRNA-92b-3p could directly bind KLF4 and DSC2. Conclusion WGCNA is an efficient systemic biological approach by which miRNA-92b-3p is identified as a new cancer-promoting gene .