CCAT-1 promotes proliferation and inhibits apoptosis of cervical cancer cells via the Wnt signaling pathway

Abstract

Though the long noncoding RNA colon cancer associated transcript-1 (CCAT-1) has been shown to be involved in tumors of other tissues, its involvement in cervical cancer is still unknown. Therefore, the aim of this study was to investigate the molecular mechanism of CCAT-1 in cervical cancer. We quantified the expression of CCAT-1 long noncoding RNA in samples of cervical cancer tissue by real-time PCR. Effects of CCAT-1 expression on the proliferation and apoptosis of HeLa and CaSki cells were assessed by cell-count, colony-formation, and flow cytometry assays. Binding of the c-Myc protein to the CCAT-1 promoter was confirmed by chromatin immunoprecipitation. Finally, TOP-Flash and western blotting were used to examine the regulation of the Wnt/β-catenin pathway by CCAT-1. The results showed that compared with adjacent normal tissue, the expression of CCAT-1 in cervical cancer tissue was significantly upregulated. CCAT-1 expression was related to the stage and size of the tumor and recurrence prognosis. Then, we showed through functional assays that CCAT-1 could promote proliferation and inhibit apoptosis of cervical cancer cells. Furthermore, chromatin immunoprecipitation showed that c-Myc protein could promote CCAT-1 expression by binding to its promoter. Finally, fluorescent-reporter assays and western blotting showed that CCAT-1 could activate the Wnt/β-catenin pathway. In conclusion, we showed that CCAT-1 can be activated by the c-Myc protein and it can promote proliferation and inhibit apoptosis in cervical cancer cells by regulating the Wnt/β-catenin pathway. CCAT-1 might serve as a good prognostic indicator and target for treatment of cervical cancer.

Keywords: CCAT-1; Wnt pathways; c-Myc; cervical cancer; long non-coding RNA.

Figure 1. The relative expression of CCAT-1 in cervical cancer tissues

(A) The expression of CCAT-1 in cervical cancer tissues (n=94) was significantly higher than that in adjacent normal tissues. (B) The expression of CCAT-1 in patients with tumor size <4 cm was significantly lower than that in patients with tumor size ≥ 4 cm. (C) The expression of CCAT-1 in patients of FIGO stage I was significantly lower than that in FIGO stage II. (D) The recurrence-free survival of low-CCAT-1 group was significantly higher than that of high-CCAT-1 group. Each assay was performed in triplicate. *P < 0.05, ^*P < 0.01.

Figure 2. The relative expression of CCAT-1 and c-Myc after transfection of cervical cancer cells

(A) pcDNA-CCAT-1 could significantly increase the expression of CCAT-1 in HeLa and CaSki cells. (B, C) si-CCAT-1 and sh-CCAT-1 could down-regulate the expression of CCAT-1 in HeLa and CaSki cells. (D) pcDNA-c-Myc can significantly increase the expression of c-Myc protein in HeLa and CaSki cells, and si-c-Myc can significantly decrease the c-Myc protein expression in HeLa and CaSki cells. Each assay was performed in triplicate. *P < 0.05.

Figure 3. The effect of CCAT-1 on the proliferation of cervical cancer cells in vivo

(A) sh-CCAT-1 significantly inhibited the tumorigenic ability of HeLa and CaSki cells in vivo. (B) Expression levels of CCAT-1 were detected in xenograft tumor tissues by qRT-PCR. Each assay was performed in triplicate. *P < 0.05.

Figure 4. The effect of CCAT-1 on the proliferation of cervical cancer cells in vitro

(A) The growth curve and cell proliferative potential (CCK-8) were determined in HeLa and CaSki cells after pcDNA-CCAT-1 and si-CCAT-1 transfection compared to negative control. (B) Colony-formation assay was used to determine the cell proliferation ability at 48 h post-transfection. Each assay was performed in triplicate. *P < 0.05.

Figure 5. Flow cytometry was used to assess the early apoptotic rate of cervical cancer cells

The proportion of early apoptotic cells in pcDNA-CCAT-1 group was significantly lower than that in pcDNA-NC group. And the percentage of early apoptotic cells in HeLa and CaSki cells transfected with si-CCAT-1 was significantly higher than that in control cells. Each assay was performed in triplicate. *P < 0.05.

Figure 6. Expression of CCAT-1 was up-regulated by c-Myc protein

(A) There was a positive correlation between CCAT-1 and c-Myc mRNA expression in cervical cancer tissues (n=94). (B, C) Up-regulation of the expression of c-Myc protein in HeLa and CaSki cells could promote the transcription of CCAT-1. And down-regulated the expression of c-Myc protein in HeLa and CaSki cells could inhibit the transcription of CCAT-1. (D) ChIP assay was used to detect the binding of c-Myc protein to E-box of CCAT-1 promoter. Each assay was performed in triplicate. *P < 0.05.

Figure 7. The regulation of Wnt pathway by CCAT-1

(A) TOP-Flash assays showed that CCAT-1 might up-regulate the Wnt pathway activity and down-regulation of CCAT-1 might significantly decrease the Wnt pathway activity of HeLa and CaSki cells. (B) Western blot showed that up-regulation of CCAT-1 expression in HeLa and CaSki cells resulted in a significant increase of β-catenin protein level, while down-regulation of CCAT-1 in HeLa and CaSki cells resulted in a significant decrease of β-catenin protein level. Each assay was performed in triplicate. *P < 0.05.

Figure 8. CCAT-1 achieved its biological functions via regulating Wnt pathway

(A) Wnt pathway antagonist Dkk-1 and activator LiCl just reversed the regulatory effect of pcDNA-CCAT-1 and si-CCAT-1 on Wnt pathway. (B) The CCAT-1 biological function was impaired after reversing the regulatory effect of CCAT-1 on Wnt pathway. Each assay was performed in triplicate. *P < 0.05.