Yonsei Med J.  2020 Jan;61(1):20-29. 10.3349/ymj.2020.61.1.20.

Curcumin Inhibits the Proliferation, Migration, Invasion, and Apoptosis of Diffuse Large B-Cell Lymphoma Cell Line by Regulating MiR-21/VHL Axis

Affiliations
  • 1Department of Pathology, Xianning Central Hospital, The First Affiliated Hospital of Hubei University of Science and Technology, Hubei, China.
  • 2Department of Gastrointestinal Surgery, Xianning Central Hospital, The First Affiliated Hospital of Hubei University of Science and Technology, Hubei, China.
  • 3Cancer Center of Guangzhou Medical University, Guangzhou, China.
  • 4Administrative Department, Xianning Central Hospital, The First Affiliated Hospital of Hubei University of Science and Technology, Hubei, China. hongyuzjhz@163.com

Abstract

PURPOSE
Curcumin exerts its anti-cancer effects, partly by targeting special microRNAs, in human cancers. MiR-21 is a key oncomir in carcinogenesis of multiple human cancers. Here, we aimed to further explore the mechanistic insight into the link between curcumin and miR-21 on diffuse large B-cell lymphoma (DLBCL).
MATERIALS AND METHODS
Quantitative real-time PCR assays were performed to assess the levels of miR-21 and Von Hippel-Lindau (VHL) mRNA. In situ hybridization assay was used for miR-21 expression visualization in lymphoma tissues. Western blot was used for determination of VHL protein, Ki-67, caspase-3, and cleaved caspase-3 levels. Dual-luciferase reporter assay and RNA immunoprecipitation assay were employed to confirm the direct target of miR-21. MTT assay, flow cytometric analysis, and transwell assay were used to evaluate cell proliferation, apoptosis, and migration and invasion capacities, respectively.
RESULTS
Curcumin repressed the proliferation, migration, and invasion abilities and promoted apoptosis in SU-DHL-8 cells. Curcumin inhibited miR-21 expression and curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis effects by miR-21 in SU-DHL-8 cells. VHL was a direct target of miR-21. Moreover, curcumin exerted its regulatory effects on SU-DHL-8 cells by VHL.
CONCLUSION
Curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis functions, at least partly, by repressing miR-21 and regulating VHL expression in DLBCL cell line. Our findings provided a possible molecular mechanism of curcumin-mediated anti-cancer effect.

Keyword

Diffuse large B-cell lymphoma (DLBCL); curcumin; miR-21; Von Hippel-Lindau (VHL)

MeSH Terms

Apoptosis*
B-Lymphocytes*
Blotting, Western
Carcinogenesis
Caspase 3
Cell Line*
Cell Proliferation
Curcumin*
Humans
Immunoprecipitation
In Situ Hybridization
Lymphoma
Lymphoma, B-Cell*
MicroRNAs
Real-Time Polymerase Chain Reaction
RNA
RNA, Messenger
Caspase 3
Curcumin
MicroRNAs
RNA
RNA, Messenger

Figure

  • Fig. 1 Curcumin inhibited proliferation and promoted apoptosis of SU-DHL-8 cells. SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin. (A) At 0, 24, 48, 72, and 96 h treatment, cell proliferation was detected by MTT assay. (B) After 24 h treatment, cell apoptosis rate was measured by flow cytometric analysis. (C) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3 in curcumin-treated cells. *p<0.05 vs. control.

  • Fig. 2 Curcumin repressed the migration and invasion of SU-DHL-8 cells. SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the measurement of cell migration (A) and invasion (B) abilities by transwell assay (×100 magnification). *p<0.05 vs. control.

  • Fig. 3 Curcumin inhibited miR-21 expression of SU-DHL-8 cells. (A) MiR-21 expression was detected in 45 cases of DLBCL tissues and 23 cases of reactive lymphoid hyperplasia tissues. (B) Representative ISH of miR-21 in DLBCL tissues and reactive lymphoid hyperplasia tissues (×100 magnification). (C) MiR-21 expression was assessed in DLBCL cell lines (SU-DHL-8, OCI-LY1 and SU-DHL-10) and normal control CD19+ cells by qRT-PCR. (D) SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the determination of miR-21 expression. *p<0.05 vs. corresponding control. DLBCL, diffuse large B-cell lymphoma; ISH, in situ hybridization.

  • Fig. 4 The effects of curcumin on proliferation and apoptosis were mediated by miR-21 in SU-DHL-8 cells. SU-DHL-8 cells were transfected with NC mimics or miR-21 mimics, and then were treated with or without curcumin (20 µmol/L). (A) At 0, 24, 48, 72, and 96 h treatment, MTT assay was performed to detect cell proliferation ability. (B) At 24 h treatment, flow cytometric analysis was used to assess cell apoptosis. (C) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3 in treated cells. *p<0.05 vs. NC mimics or NC mimics+curcumin.

  • Fig. 5 Curcumin exerted its anti-migration and anti-invasion effects by miR-21 in SU-DHL-8 cells. SU-DHL-8 cells were transfected with NC mimics or miR-21 mimics, and then were treated with or without curcumin (20 µmol/L) for 24 h, followed by the determination of cell migration ability (A) and invasion capacity (B) by transwell assay. *p<0.05 vs. NC mimics or NC mimics+curcumin.

  • Fig. 6 VHL was a direct target of miR-21. (A) Schematic illustration of the putative binding sites between miR-21 and VHL, and mutation in binding sequence in 3′-UTR of VHL. (B) VHL wild-type or mutant-type reporter plasmids (VHL-WT or VHL-MUT) were constructed and transfected into SU-DHL-8 cells together with NC mimics, miR-21 mimics, anti-NC, or anti-miR-21. Relative luciferase activities were detected. (C) SU-DHL-8 cells were transfected with miR-21 mimics, followed by the detection of VHL mRNA enrichment with anti-Ago2 or anti-IgG. SU-DHL-8 cells were transfected with NC mimics, miR-21 mimics, anti-NC, or anti-miR-21, followed by the measurement of VHL mRNA by qRT-PCR assay (D) and VHL protein by western blot (E). The expression of VHL protein was detected by western blot in DLBCL tissues and reactive lymphoid hyperplasia tissues (F), and in DLBCL cell lines (SU-DHL-8, OCI-LY1, and SU-DHL-10) and normal control CD19+ cells (G). *p<0.05 vs. NC mimics or anti-NC. VHL, Von Hippel-Lindau; DLBCL, diffuse large Bcell lymphoma.

  • Fig. 7 Curcumin exerted its regulatory effects by VHL in SU-DHL-8 cells. (A) SU-DHL-8 cells were treated with different concentrations (0, 5, 10, 20, 40, and 60 µmol/L) of curcumin for 24 h, followed by the measurement of VHL mRNA expression. SU-DHL-8 cells were transfected with si-VHL or si-NC and then were treated with 20 µmol/L of curcumin. (B) After 24 h treatment, VHL protein expression was determined by western blot analysis. (C) At 0, 24, 48, 72, and 96 h treatment, cell proliferation ability was assessed by MTT assay. (D) At 24 h treatment, cell apoptosis was detected by flow cytometric analysis. (E) Western blot was performed to determine the levels of Ki-67, caspase-3, and cleaved caspase-3. At 24 h treatment, cell migration ability was determined by transwell assay (F) and cell invasion capacity was evaluated by transwell assay (G). *p<0.05 vs. control or si-NC+curcumin. VHL, Von Hippel-Lindau.


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