Downregulation of LINC01021 by curcumin analog Da0324 inhibits gastric cancer progression through activation of P53

Abstract

Curcumin is a safe, cost-effective natural agent with multiple targets that displays therapeutic potential in cancer. Recently, we reported a novel curcumin analog, Da0324, which exhibited significantly improved stability and anti-cancer activity. However, the molecular mechanism underlying the anti-cancer activity of Da0324 remains largely unknown. Long non-coding RNAs have been shown to play important roles in cancer development and progression and may be potential targets for cancer therapy. Here, we showed that Da0324 treatment down-regulated the expression of LINC01021 in gastric cancer cells. Da0324 treatment or knockdown of LINC01021 by antisense oligos significantly inhibited gastric cancer cell growth, and also up-regulated P53 expression and down-regulated Bcl-2 expression in vitro and in vivo. Furthermore, Da0324 treatment or knockdown of LINC01021 in gastric cancer cells suppressed cell migration, invasion and epithelial-mesenchymal transition (EMT), as well as induced apoptosis and autophagy. In addition, overexpression of LINC01021 promoted growth and EMT, inhibited P53 expression and increased Bcl-2 expression in gastric cancer cells. Finally, overexpression of LINC01021 reversed the anti-cancer effect of Da0324. Our findings indicate a novel anti-cancer mechanism for Da0324, and that LINC01021 might be a potential therapeutic target for the treatment of gastric cancer.

Keywords: Curcumin analog; Da0324; LINC01021; gastric cancer.

Figure 1

Da0324 inhibits proliferation and induces apoptosis and autophagy in gastric cancer cells. A. Gastric cancer cell lines (BGC823, SGC7901 and KATO III) were treated with the indicated concentration of Da0324 for 24 h, 48 h, or 72 h. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and Growth inhibition rates of Da0324 on GC cells were in comparison with untreated cells. B. BGC823, SGC7901 and KATO III cells were treated with 0, 0.5, 1, 2, 4, 6, 8, 10, or 12 mM Da0324 for 48 h, and cell viability was measured by CCK-8 assays. The half maximal inhibitory concentration (IC50) values were calculated with GraphPad statistical software. C. Clonogenic assay showed the effects of Da03224 treatment on clonogenic formation in gastric cancer cells. BGC823, SGC7901 and KATO III cells were treated with Da0324 (1 or 2 µM) for 48 h, then cultured for two weeks in complete medium and the colony density calculated. Representative images of clonogenic assay (left panel) and quantitative analysis (right panel). All data are representative of three independent experiments and are presented as the means ± SD. *, P < 0.05; **, P < 0.01. D. BGC823, SGC7901 and KATO III cells were treated with 4 μM Da0324 for 48 h. Cells were fixed and stained with Annexin V/PI and apoptosis was analyzed by flow cytometry. Representative dot plots of Annexin V/PI staining are shown in the left panel, and quantitative data are presented in the right panel. All data are representative of three independent experiments and are presented as the means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E. BGC823 and SGC7901 cells were treated with 4 μM Da0324 or control for 48 h and then harvested for analysis by western blot using P53, Bcl-2, p-mTOR, mTOR, P62, and LC3B antibodies. GAPDH or β-actin was used as an internal control.

Figure 2

Da0324 inhibits migration, invasion, and EMT in GC cells. A. BGC823, SGC7901 and KATO III cells were treated with Da0324 (1 or 2 µM) for 24 h and transwell migration and matrigel invasion assays were performed. Representative image (left panel) and quantitative data (right panel) of cell migration and invasion are shown. Bar graphs are representative of three independent experiments and the data are presented as the means ± SD. **, P < 0.01; ***, P < 0.001. B. Western blot analysis for the expression of E-cadherin, vimentin, and N-cadherin in BGC823 and SGC7901 cells treated with or without 2 µM Da0324. GAPDH was used as an internal control.

Figure 3

Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log₂ of fold changes. The Y-axis represents -log₁₀ of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P < 0.001; n.s. means no significant difference. D. LINC01021 expression by qRT-PCR in BGC823, SGC7901 and KATO III cells treated with Da0324 or DMSO (control) for 48 h. All bars represent relative expression levels and data represent mean ± SD. ***, P < 0.001. E. TCGA data for the expression of LINC01021 in gastric cancer tissues (n = 375) and normal tissues (n = 32). F. LINC01021 expression by qRT-PCR in the normal gastric epithelial cell line GES-1 and in gastric cancer cell lines (BGC823, SGC7901, and KATO III). All bars represent relative expression levels and data represent mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G. The subcellular location of LINC01021 in SGC7901 and KATO III cells was determined by qRT-PCR. GAPDH was used as a positive control for cytoplasmic RNA localization, U6 for nuclear RNAs. The data are shown as means ± SD. lncRNA, long non-coding RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 4

ASO-mediated silencing of LINC01021 inhibits cell proliferation and induces apoptosis and autophagy in gastric cancer cells. A. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021-specific ASO-1, or LINC01021-specific ASO-2 for 48 h. LINC01021 expression were determined by qRT-PCR. B. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021-specific ASO-1, or LINC01021-specific ASO-2. At 0, 24, 48 or 72 h post-transfection, cell viability was determined by the CCK-8 assays. The data were expressed as mean ± SD. All data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001. C. The proliferation of gastric cancer cells (BGC823 and SGC7901) transfected with LINC01021-ASOs or ASO-control was determined by colony formation assays. The data were expressed as mean ± SD. All data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001. D. Flow cytometry analysis of cell apoptosis in gastric cancer cells (BGC823 and SGC7901). Cells were transfected with 50 nM LINC01021-ASOs or ASO-control for 48 h and stained with Annexin V/PI. Representative dot plots of Annexin V/PI staining are shown in the left panel, and quantitative data are presented in the right panel. All data are representative of three independent experiments and are presented as the means ± SD. ***, P < 0.001. E, F. Western blot analysis of P53, Bcl-2, mTOR, p-mTOR, P62, and LC3B expression in BGC823 and SGC7901 cells transfected with LINC01021-ASOs or ASO-control. qRT-PCR, quantitative reverse transcription-polymerase chain reaction; ASO, antisense oligo; LC3B, light chain3B; p-mTOR, phosphorylated mTOR.

Figure 5

ASO-mediated silencing of LINC01021 inhibits gastric cancer cell migration, invasion, and EMT. A. BGC823 and SGC7901 cells were transfected with 50 nM ASO-control, LINC01021-specific ASO-1 or LINC01021-specific ASO-2 for 48 h and transwell migration and matrigel invasion assays were performed. Representative image (left panel) and quantitative data (right panel) of cell migration and invasion are shown. All data are presented as the means ± SD. ***, P < 0.001. B. Western blot analysis of the expression of E-cadherin, N-cadherin and vimentin proteins in BGC823 and SGC7901 cells transfected with ASO-control, LINC01021-specific ASO-1 or LINC01021-specific ASO-2. ASO-ctrl, ASO-control; EMT, epithelial-mesenchymal transition.

Figure 6

Da0324 or LINC01021-specific ASO treatment suppresses tumor growth of GC cells in vivo. A. Effects of Da0324 treatment on tumor volume in subcutaneous xenograft mouse models using KATO III cells. Tumor volume as measured by caliper of Da0324 (20 mg/kg, intraperitoneal injection every day) or vehicle administered for 18 days in a subcutaneous xenograft model. The data were expressed as mean ± SD. *, P < 0.05. B. Tumors were excised after 18 days of treatment. Images and weight of the dissected xenograft tumors are shown. C. Body weight of nude mice. D. Determination of tumor LINC01021 expression by qRT-PCR for both the control and treatment groups. The data were expressed as mean ± SD. **, P < 0.01. E. Western blot analysis of the protein expression of P53 and Bcl-2 in control or Da0324-treated mouse xenograft tumors. GAPGH was used as an internal control. F. Tumor volume of subcutaneous xenograft mouse models using KATO III cells treated with LINC01021-specific ASO or ASO-control. Tumor volume as measured by caliper of LINC01021-specific ASO or ASO-control (250 nmol/kg, intratumoral injection at three-day intervals) administered for 24 days in a subcutaneous xenograft model. The data were expressed as mean ± SD. *, P < 0.05. G. Tumors were excised after 24 days of treatment. Images and weight of the dissected xenograft tumors are shown. The data were expressed as mean ± SD. **, P < 0.01. H. Body weight of nude mice from ASO-control and ASO-LINC01021 treatment groups. I. Determination of LINC01021 expression by qRT-PCR in ASO-control and ASO-LINC01021 treatment xenografts. *, P < 0.05. J. Western blot analysis of the protein expression of P53 and Bcl-2 in tumor tissues from ASO-control and ASO-LINC01021 treatment groups. GAPGH was used as an internal control. ASO-ctrl, ASO-control; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 7

Overexpression of LINC01021 reverses Da0324-mediated cytotoxic effects in gastric cancer cells. A. BGC823 and SGC7901 cells were transfected with negative control vector (pLVX-NC) or overexpressing LINC01021 vector (pLVX-LINC01021) for 48 h. LINC01021 expression was determined by qRT-PCR analysis. The data represent the mean ± SD from three independent experiments. ***, P < 0.001. B. BGC823 and SGC7901 cells were transfected with pLVX-NC or pLVX-LINC01021 for 48 h. Cell viability was evaluated by CCK-8 assays. The data represent the mean ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001. C, D. Overexpression of LINC01021 reversed Da0324-mediated growth inhibition of gastric cancer cells. BGC823 and SGC7901 cells were transfected with pLVX-NC or pLVX-LINC01021 for 24 h and then treated with Da0324 (4 µM) for 24 h. Cell viability was determined by CCK-8 assays. The data represent the mean ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. E. BGC823 and SGC7901 cells transfected with pLVX-NC or pLVX-LINC01021 were exposed to Da0324 for 48 h and colony formation was assessed. F. BGC823 and SGC7901 cells were transfected with pLVX-NC or pLVX-LINC01021 for 48 h and the expression levels of P53, Bcl-2, E-cadherin, N-cadherin and vimentin were determined by western blot analysis. GAPGH was used as an internal control. qRT-PCR, quantitative reverse transcription-polymerase chain reaction.