Curcumin attenuates lncRNA H19‑induced epithelial‑mesenchymal transition in tamoxifen‑resistant breast cancer cells

Abstract

The H19 long non‑coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen‑resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF‑7/TAMR cell epithelial‑mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19‑mediated effects. Reverse transcription‑quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit‑8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF‑7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E‑cadherin and markedly increased the expression levels of mesenchymal marker N‑cadherin in MCF‑7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose‑dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19‑induced alterations in N‑cadherin and E‑cadherin expression levels. Curcumin also prevented H19‑induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF‑7/TAMR cells, suggesting that curcumin may prevent H19‑associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.

Keywords: tamoxifen‑resistance; epithelial‑mesenchymal transition; breast cancer; H19 imprinted maternally expressed transcript; curcumin.

Figure 1.

H19 induces MCF-7/tamoxifen-resistant cell epithelial-mesenchymal transition. Transfection efficiency of (A) siRNAH19 and (B) H19-epv. The effects of (C) siRNAH19 and (D) H19-epv on E-cadherin and vimentin mRNA expression levels were determined via reverse transcription-quantitative PCR. (E) The effects of siRNAH19 and H19-epv on E-cadherin and vimentin protein expression levels were determined via western blotting. **P<0.05 vs. siRNAH19-NC or H19-epv-NC. E-cad, E-cadherin; siRNA, small interfering RNA; epv, expression vector; NC, negative control.

Figure 2.

H19 induces MCF-7/tamoxifen-resistant cell migration and invasion. Cells were transfected with H19-epv or siRNAH19 for 24 h. Cell (A) migration (magnification, ×40) and (B) invasion (Magnification, ×200) were assessed by performing wound healing and Transwell invasion assays, respectively. **P<0.05 vs. siRNAH19-NC, ^##P<0.01 vs. H19-epv-NC. NC, negative control; epv, expression vector; siRNA, small interfering RNA.

Figure 3.

H19 promotes MCF-7/tamoxifen-resistant cell epithelial-mesenchymal transition via regulating Snail. Cells were transfected with H19-epv or siRNAH19 for 24 h. Snail (A) protein and (B) mRNA expression levels were measured via western blotting and reverse transcription-quantitative PCR, respectively. **P<0.05 vs. siRNAH19-NC, ^##P<0.05 vs. H19-epv-NC. NC, negative control; epv, expression vector; siRNA, small interfering RNA.

Figure 4.

Curcumin influences MCF-7/tamoxifen-resistant cell viability and epithelial-mesenchymal transition. (A) The effect of curcumin on cell viability was determined by performing a Cell Counting Kit-8 assay. *P<0.05 and **P<0.01 vs. 0 µM curcumin groups. E-cadherin and N-cadherin (B) protein and (C) mRNA expression levels were determined via western blotting and reverse transcription-quantitative PCR, respectively. **P<0.01 vs. Con. Con, control; E-cad, E-cadherin; N-cad, N-cadherin.

Figure 5.

Curcumin inhibits H19 mRNA expression in MCF-7/tamoxifen-resistant cells. Cells were treated with various concentrations of curcumin for 48 h. Subsequently, H19 mRNA expression levels were determined via reverse transcription-quantitative PCR. **P<0.05, ***P<0.01 vs. 0 µM curcumin.

Figure 6.

Curcumin attenuates H19-induced epithelial-mesenchymal transition in MCF-7/tamoxifen-resistant cells. Following transfection with H19-epv, cells were treated with or without curcumin for 48 h. (A) E-cadherin and N-cadherin protein expression levels were determined via western blotting. **P<0.01 vs. H19-epv-NC; ^##P<0.01 vs. H19-epv. (B) Cytolocalization of E-cadherin and N-cadherin in MCF-7/TAMR cells was detected by performing immunofluorescence staining. Magnification, ×200. E-cad, E-cadherin; N-cad, N-cadherin; epv, expression vector; NC, negative control.

Figure 7.

Curcumin inhibits H19-induced MCF-7/tamoxifen-resistant cell migration and invasion. Following transfection with H19-epv for 24 h, cells were treated with or without curcumin for 48 h. Cell (A) migration (magnification, ×40) and (B) invasion (magnification, ×200) were determined by performing wound healing and Transwell invasion assays, respectively. **P<0.05 vs. H19-epv-NC; ^##P<0.01 vs. 19-epv. NC, negative control; epv, expression vector.