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. 2018 Nov;16(5):5543-5550.
doi: 10.3892/ol.2018.9327. Epub 2018 Aug 20.

Effects of let-7c on the proliferation of ovarian carcinoma cells by _targeted regulation of CDC25a gene expression

Wei Zhang  1 , Qingru Zeng  2 , Zhenying Ban  1 , Jing Cao  1 , Tianjiao Chu  1 , Dongmei Lei  1 , Chi Liu  3 , Wentao Guo  4 , Xianxu Zeng  1
Affiliations

Effects of let-7c on the proliferation of ovarian carcinoma cells by _targeted regulation of CDC25a gene expression

Wei Zhang et al. Oncol Lett. 2018 Nov.

Abstract

MicroRNAs serve a role in the development of ovarian cancer (OC). The present study investigated whether let-7c is able to regulate the proliferation of OC cells by _targeting cell division cycle 25A (CDC25a). The reverse transcription-quantitative polymerase chain reaction was performed to detect the expression of let-7c in OC specimens. Let-7c agomir was transfected into OC cells, and the proliferation and apoptosis of OC cells were detected. A dual-luciferase assay and western blotting were performed to analyze whether CDC25a was the _target gene of let-7c as well as its interaction site. The results revealed that, in OC tissue, let-7c was downregulated when compared with normal ovarian tissue. A Cell Counting Kit-8 (CCK8) assay, colony formation assay and flow cytometry demonstrated that increased expression of let-7c was able to inhibit the proliferation and increase the apoptosis of OC cells. Western blotting revealed that upregulated let-7c is able to decrease the expression of CDC25a, and a dual-luciferase assay and a recovery assay demonstrated that let-7c was able to regulate the expression of the 3' untranslated region of CDC25a. Therefore, the roles of let-7c in inhibiting the proliferation and promoting the apoptosis of OC cells may be realized through the regulation of the expression of CDC25a. The results of the present study revealed that let-7c may be a novel _target in the diagnosis and treatment of OC.

Keywords: cell division cycle 25a; let-7c; ovarian cancer; proliferation.

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Figures

Figure 1.
Figure 1.
Detection of let-7c expression level in OC tissue using the reverse transcription-quantitative polymerase chain reaction. (A) Let-7c was significantly increased in OC tissue compared with normal tissue (*P<0.05). (B) Let-7c expression levels are significantly decreased in metastatic lymph nodes compared with non-metastatic lymph node tissue (*P<0.05). (C) In OC tissues, the higher the clinical stage, the lower the let-7c expression level (*P<0.05). OC, ovarian cancer.
Figure 2.
Figure 2.
Let-7c agomir significantly inhibits the proliferation of OC cells. A Cell Counting Kit-8 assay revealed that compared with the blank group and the control group, the proliferation abilities of (A) SKOV3 and (B) ES2 were significantly decreased at 2, 3 and 4 days after let-7c agomir transfection compared with the control (*P<0.05). OD, optical density. OC, ovarian cancer.
Figure 3.
Figure 3.
Overexpression of let-7c inhibits the colony formation ability of OC cells. The let-7c agomir-transfected OC cell lines (A) SKOV3 and (B) ES2 exhibited significantly decreased cell colony numbers compared with control cells (*P<0.05). OC, ovarian cancer.
Figure 4.
Figure 4.
Overexpression of let-7c may lead to the apoptosis of SKOV3 and ES2 cells. The apoptotic rates of (A) SKOV3 and (B) ES2 cells were significantly increased when compared with the control group following transfection with let-7c agomir (*P<0.05). PI, propidium iodide.
Figure 5.
Figure 5.
Let-7c acts on CDC25a 3′UTR and regulates its expression. (A) Let-7c acts on CDC25a 3′UTR seed region sequence. (B) A dual-luciferase reporter gene assay demonstrated that the co-transfection of let-7c agomir and pmirGLO-CDC25a-wt significantly decreases the luciferase activity of SKOV3 cells when compared with the control group (miR-NC and pmirGLO-CDC25a-wt co-transfection group; *P<0.05); the co-transfection of let-7c agomir and pmirGLO-CDC25a-mut does not significantly alter the luciferase activity in comparison with the control group (negative control and pmirGLO-CDC25a-mut co-transfection group) (*P>0.05). (C) Western blot analysis revealed that the overexpression of let-7c suppresses the CDC25a protein level in SKOV3 cells. CDC25a, cell division cycle 25a; Hsa, Homo sapiens; WT, wild-type; Mut, mutant.
Figure 6.
Figure 6.
Overexpression of CDC25a may recover the pro-apoptotic effects of let-7c towards OC cells. (A) The co-transfection of CDC25a 3′UTR-free pcDNA3.1-CDC25a and let-7c agomir into SKOV3 cells significantly decreased the apoptotic rate of the cells (*P<0.05). (B) Western blot analysis revealed that CDC25a 3′UTR-free pcDNA3.1-CDC25a is able to recover the let-7c agomir-inhibited expression of CDC25a protein in SKOV3 cells, thus resulting in the upregulation of CDC25a protein. CDC25a, cell division cycle 25a.
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