Our present data, together with published results, suggested that ciRS-7 might be a potential target for ESCC treatment. Funding Statement This research was supported by the Basic research project (Natural science funds) of Jiangsu Province (NO. our present study revealed that ciRS-7 can trigger the migration and invasion of ESCC cells via miR-7/KLF4 and NF-B signals. Targeted inhibition of ciRS-7 might be a potential approach for ESCC treatment. migration of both KYSE150 (Physique 2C) and Eca9706 (Physique 2D) cells. This was confirmed by transwell analysis that over expression of ciRS-7 also significantly increased the invasion of both KYSE150 and Eca9706 cells (Physique 2E). In addition, increased expression of MMP-2 and MMP-9 was observed in ciRS-7 over expressed KYSE150 and Eca9706 cells (Physique 2F). Collectively, our data showed that ciRS-7 can promote the migration and invasion of ESCC cells. Open in a separate window Physique 2. ciRS-7 increased the migration and invasion of ESCC cells. (A) KYSE150 and Eca9706 cells were transfected with pLCDH-ciR/ciRS-7 or vector control for 24?h, the expression of ciRS-7 was detected by use of qRT-PCR; After transfected with pLCDH-ciR/ciRS-7 or vector control for 48?h, the cell proliferation was detected by CCK-8 kit (B), the migration (C, KYSE150; D, Eca9706) was measured by wound healing assay, the invasion was measured by transwell assay (E), and the expression of MMP-2, MMP-9, and vimentin was measured by western blot analysis (F). ** p?0.01. Mir-7 was involved in cirs-7 induced migration and invasion of ESCC cells Previous studies indicated that ciRS-7 can act as a ceRNA of miR-7.14,15,17 We evaluated the effects of ciRS-7 around the expression of miR-7 in ESCC cells. Our data confirmed that over expression of ciRS-7 can significantly inhibit the expression of miR-7 in both KYSE150 and Eca9706 cells Mouse monoclonal to IgG2b/IgG2a Isotype control(FITC/PE) (Physique 3A). To verify whether miR-7 is usually involved in ciRS-7 induced migration and invasion of ESCC cells, we over expressed miR-7 in KYSE150 and Eca9706 cells (Physique 3B). Our data showed that over expression of miR-7 can partially attenuate ciRS-7 induced invasion of both KYSE150 (Physique 3C) and Eca9706 (Physique 3D) cells. This was confirmed by Paroxetine HCl western blot analysis that over expression of miR-7 reversed ciRS-7 induced over expression of MMP-2 and MMP-9 in KYSE150 cells (Physique 3E). These data indicated that miR-7 is usually involved in ciRS-7 induced migration and invasion of ESCC cells. Open in a separate window Physique 3. miR-7 was involved in ciRS-7 induced migration and invasion of ESCC cells. (A) KYSE150 and Eca9706 cells were transfected with pLCDH-ciR/ciRS-7 or vector control for 24?h, the expression of Paroxetine HCl miR-7 was detected by use of qRT-PCR; (B) KYSE150 and Eca9706 cells were transfected with miR-7 construct or vector control for 24?h; KYSE150 (C) and Eca9706 (D) cells were transfected with pLCDH-ciR/ciRS-7, miR-7 construct or vector control alone or together for 48?h, the cell invasion was Paroxetine HCl evaluated by use of transwell assay; (E) KYSE150 cells were treated as (C), the protein expression was measured by western blot analysis. ** p?0.01. KLF-4 mediated the effects of ciRS-7/miR-7 axis on invasion of ESCC cells We analyzed the potential targets of miR-7 by use of two widely-used bioinformatics tools, PicTar (http://pictar.mdc-berlin.de) and miRNA.org (http://www.microrna.org/microrna/home.do). Among the recognized targets, three interesting genes EGFR, X-linked inhibitor of apoptosis protein (XIAP), KLF4, and NOTCH1 were prioritized because of their functions in cancer progression.18 We found that over expression of ciRS-7 can significantly increase the mRNA expression of EGFR and KLF4 in KYSE150 cells (Physique 4A). While ciRS-7 only increased the mRNA expression of KLF4 in Eca9706 cells (Physique 4B). Western blot analysis confirmed that ciRS-7 can increase the expression of KLF4 in both KYSE150 and Eca9706 cells (Physique 4C). We therefore knocked down the expression of KLF4 in KYSE150 cells via its specific siRNA (Physique 4D). Our data showed that si-KLF4 can also reverse.
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