J Korean Med Sci.  2016 Jun;31(6):836-842. 10.3346/jkms.2016.31.6.836.

Silencing of ABCG2 by MicroRNA-3163 Inhibits Multidrug Resistance in Retinoblastoma Cancer Stem Cells

Affiliations
  • 1Department of Ophthalmology, Linzi District People's Hospital, Zibo City, Shandong Province, P. R. China. jiaming_20150401@163.com

Abstract

To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma.

Keyword

Retinoblastoma; RCSCs; ABCG2; miR-3163

MeSH Terms

3' Untranslated Regions
ATP Binding Cassette Transporter, Sub-Family G, Member 2/antagonists & inhibitors/genetics/*metabolism
Antagomirs/metabolism
Antineoplastic Agents/toxicity
Apoptosis/drug effects
Base Sequence
Cell Line, Tumor
Cell Proliferation/drug effects
Drug Resistance, Neoplasm/drug effects
Gene Silencing
Humans
MicroRNAs/antagonists & inhibitors/genetics/*metabolism
Neoplasm Proteins/antagonists & inhibitors/genetics/*metabolism
Neoplastic Stem Cells/*metabolism
Retinoblastoma/metabolism/pathology
Sequence Alignment
Transfection
3' Untranslated Regions
ATP Binding Cassette Transporter, Sub-Family G, Member 2
Antagomirs
Antineoplastic Agents
MicroRNAs
Neoplasm Proteins

Figure

  • Fig. 1 Identification of RCSCs in the WERI-Rb1 retinoblastoma cell line. (A) ABCG2 positive (ABCG2+) and negative (ABCG2-) cells were sorted by flow cytometry based on expression of the RCSC surface marker ABCG2. For FACS sorting, the blank group was incubated with IgG2a antibody, whereas the ABCG2+ group was incubated with ABCG2 antibody. The area of R3 was the ABCG2+ cells sorted for further research, the rest cells was ABCG2- cells. (B) Expression levels of ABCG2, OCT4 and NANOG were detected in ABCG2+ and ABCG2- cells by Western blot.

  • Fig. 2 miRNA prediction and expression of miR-3163 in RCSCs. (A) Putative miR-3163-binding sites on the ABCG2 3'UTR with potential complementary residues shown in black. (B) Expression levels of miR-3163 were detected in ABCG2+ and ABCG2- cells by qRT-PCR. * P < 0.05 compared with the control. The data represent the results of three independent experiments.

  • Fig. 3 Verification of miRNA target sites. (A) miR-3163 could be overexpressed by Mimics (Mim) as measured by qRT-PCR. Scramble (Scr) was used as the negative control. (B) Western blot analysis showing the protein levels of ABCG2 in RCSCs treated with Scramble or miR-3163 Mimics. GAPDH was used as a loading control. (C) Luciferase activity was measured in HeLa cells co-transfected with miR-3163 and either wide-type Reporter ABCG2 3'UTR (grey) or mutated Reporter ABCG2 3’UTR (black). Renilla activity was used as an internal control. * P < 0.05 compared with the control. The data represent the results of three independent experiments.

  • Fig. 4 Effects of miR-3163 on RCSC proliferation and apoptosis. (A) The effect of miR-3163 on RCSC growth was detected by CCK-8 assay at the indicated time points. (B) Representative images of flow cytometry analysis showing Annexin V/PI-stained RCSC cells with Scramble or miR-205 Mimics in the presence of cisplatin. Results from three independent experiments quantifying the percentage of Annexin V-positive cells were counted. * P < 0.05 compared with the control. The data represent the results of three independent experiments.

  • Fig. 5 Effects of miR-3163 on RCSC multidrug resistance. (A) The effect of miR-3163 on RCSC multidrug resistance was detected by CCK-8 assay of cells treated with different drugs. CIS, cisplatin; CAR, carboplatin; VIN, vincristine; DOX, doxorubicin; ETO, etoposide. (B) The effect of miR-3163 on RCSC ATPase activity was detected by ABCG2 ATPase assay. * P < 0.05 compared with the control. † P < 0.05 compared with the control. The data represent the results of three independent experiments.


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