Role and mechanism of microRNA-92b-3p in esophageal squamous cell carcinoma analyzed by weighted gene co-expression network analysis

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 4 023 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 KLF4 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. Key words: Computational biology; Weighted gene co-expression network analysis; Esophageal squamous cell carcinoma; microRNA-92b; Kruppel like factor 4; Desmocollin 2