MicroRNA-10b Promotes Migration And Invasion Through KLF4 In Human Esophageal Cancer Cell Lines

MicroRNAs are endogenous noncoding RNA molecules, on average only 22 nucleotides long. In metazoans, miRNAs bind to imperfect complementary sequences on target messenger RNA transcripts, resulting in translational repression or mRNA cleavage. Lately, emerging evidence suggests important roles for miRNAs in cell growth, proliferation and apoptosis. Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis.In this study, we have analyzed the level of miR-10b through real-time quantitative RT-PCR in eight human esophageal squamous cell carcinoma (ESCC) cell lines., and found that miR-10b levels varied considerably among the cell lines. Next, we performed Transwell assay to investigate the cell motility and invasiveness in these cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility.KLF4 is a transcription factor that is involved in cell cycle regulation, apoptosis, and differentiation; its expression can be increased by DNA damage, serum deprivation, and contact inhibition. Recently, KLF4 has been shown to inhibit cell migration and invasion in TE2 of human ESCC cell line. In addition, KLF4 can inhibit pancreatic cancer metastasis in vivo. Consistent with these results, KLF4 overexpression reduces cell migration and invasion in RKO colon cancer cells. Bioinformatics software predicted KLF4 as a target of miR-10b. Then, we constructed pIS0-KLF4-3'UTR which has a full length KLF4 mRNA 3'UTR, and pIS0-KLF4-3 -UTR-mut in which there are four-nucleotide substitution in the core binding sites. We identified KLF4 mRNA as a direct target of miR-10b through dual-luciferase reporter system.Furthermore, overexpression of miR-10b in KYSE140 and KYSE450 cells led to a reduction of endogenous KLF4 protein, whereas silencing of miR-10b in EC9706 cells caused up-regulation of KLF4 protein. Also, we measured the half-life of KLF4 by treatment of cyclohexamide and found that miR-10b does not change the rate of KLF4 protein degradation. The decreased KLF4 expression in miR-10b overexpressing cells was due to the post-transcriptional regulation of miR-10b.Next, we investigated whether KLF4 suppressed cell migration and invasion in KYSE140 and EC9706 cells. Silencing of KLF4 by siRNA in KYSE140 cells led to increased cell migration and invasiveness, whereas overexpression of KLF4 in EC9706 cells resulted in a decrease in cell migration and invasion, demonstrating a negative role for KLF4 protein in the migration and invasion in human ESCC cells. To further establish a functional connection between miR-10b and KLF4. we tested whether KLF4 deregulation was required for regulation of miR-10b on cell migration and invasion. Coexpression of miR-10b and KLF4 in KYSE140 cells and coexpression of small interfering RNA for KLF4 mRNA and miR-10b-AS in EC9706 cells partially abrogated the effect of miR-10b on cell migration and invasion.Finally, analyses of the miR-10b level in 40 human esophageal cancer samples and their paired normal adjacent tissues revealed an elevated expression of miR-10b in 95% (38 of 40) of cancer tissues, although no significant correlation of the miR-10b level with clinical metastasis status was observed in these samples.