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Long non-coding RNA OIP5-AS1 inhibition upregulates microRNA-129-5p to repress resistance to temozolomide in glioblastoma cells via downregulating IGF2BP2

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Abstract

Objective

Long non-coding RNAs (lncRNAs) and miRNAs (miRNAs) participate in tumors, while the effects of lncRNA OIP5 antisense RNA 1 (OIP5-AS1) and miR-129-5p on glioblastoma (GBM) remain to be further studied. We aim to explore the role of OIP5-AS1/miR-129-5p/insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) axis in GBM progression.

Methods

OIP5-AS1, miR-129-5p and IGF2BP2 expression in tissues was determined. Temozolomide (TMZ)-resistant GBM cells were established and transfected with relative plasmid to alter OIP5-AS1, IGF2BP2 or miR-129-5p expression. Then, the viability, proliferation, apoptosis and in vivo tumor growth were assessed. The subcellular localization of OIP5-AS1 was determined, and the binding relationships between OIP5-AS1 and miR-129-5p, and between miR-129-5p and IGF2BP2 were confirmed.

Results

OIP5-AS1 and IGF2BP2 were upregulated whereas miR-129-5p was downregulated in GBM. OIP5-AS1 silencing or miR-129-5p overexpression inhibited GBM cell chemoresistance to TMZ and proliferation, and promoted cell apoptosis. MiR-129-5p downregulation or IGF2BP2 upregulation reversed the role of OIP5-AS1 silencing on GBM cells. OIP5-AS1 sponged miR-129-5p and miR-129-5p targeted IGF2BP2.

Conclusion

OIP5-AS1 inhibition upregulated miR-129-5p to repress resistance to TMZ in GBM cells via downregulating IGF2BP2.

Graphical Abstract

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Abbreviations

lncRNAs:

Long non-coding RNAs

miRNAs:

miRNAs

OIP5-AS1:

OIP5 antisense RNA 1

GBM:

Glioblastoma

TMZ:

Temozolomide

DMEM:

Dulbecco’s modified Eagle medium

FBS:

Fetal bovine serum

P/S:

Penicillin-streptomycin

NC:

Negative control

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

CCK-8:

Cell counting kit-8

FISH:

Fluorescent in situ hybridization

WT:

Wild type

MUT:

Mutant type

RIP:

RNA Immunoprecipitation

ANOVA:

Analysis of variance

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Acknowledgement

We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.

Availability of data and material

Not applicable.

Code availability

Not applicable.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 81974390) and Natural Science Foundation of Hubei Province (Grant No. 2019CFB717).

Author information

Authors and Affiliations

Authors

Contributions

Xiaobing Jiang study design; Xuan Wang, Xudong Li, Yan Zhou finished experimental studies; Xuan Wang, Xing Huang, Xiaobing Jiang finished data analysis; Xuan Wang finished manuscript editing. All authors read and approved the final manuscript

Corresponding author

Correspondence to Xiaobing Jiang.

Ethics declarations

Ethical statement

This study was approved and supervised by the animal ethics committee of Union Hospital of Tongji Medical College Huazhong University of Science and Technology. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

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The authors declare that they have no conflicts of interest.

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Supplementary Information

Supplementary Figure 1.

OIP5-AS1 inhibition suppresses TMZ-resistance and growth of U251 cells. A OIP5-AS1 expression in transfected U251 cells. B IC50 values of TMZ-treated U251 cells (0, 50, 100, 150, 200 and 250 μmol/mL). C Cell viability was determined using CCK-8 assay. D Proliferation of cells was detected by colony formation assay. E Apoptosis of cells was assessed by flow cytometry. F Expression of Caspase-3, Bax and Bcl-2 in cells was determined using RT-qPCR; N = 3, * P < 0.05 vs the pcDNA3.1-NC group, # P < 0.05 vs the si-NC group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 799 kb)

Supplementary Figure 2.

Overexpressed OIP5-AS1 reduces TMZ-resistance and growth of U251 cells. A OIP5-AS1 expression in U251TR was determined using RT-qPCR. B OIP5-AS1 expression in TMZ-resistant cells was determined using RT-qPCR; C, IC50 values of TMZ-treated U251 cells (0, 50, 100, 150, 200 and 250 μmol/mL). D Cell viability was determined using CCK-8 assay after TMZ treatment. E Proliferation of cells was detected by colony formation assay after TMZ treatment. F Apoptosis of cells was assessed by flow cytometry after TMZ treatment; G, expression of Caspase-3, Bax and Bcl-2 in cells was determined using RT-qPCR after TMZ treatment; N = 3, * P < 0.05 vs the pcDNA3.1-NC group, # P < 0.05 vs the si-NC group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 2009 kb)

Supplementary Figure 3.

OIP5-AS1 sponges miR-129-5p. A Distribution of OIP5-AS1 was confirmed using FISH assay. B Binding relationship between OIP5-AS1 and miR-129-5p was confirmed using dual luciferase reporter gene assay. C miR-129-5p expression in U251TR cells was determined by RT-qPCR. D The spongy relation between OIP5-AS and miR-129-5p was confirmed by RNA pull-down assay; * P < 0.05 vs the NC-mimic group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, unpaired t-test was performed for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 636 kb)

Supplementary Figure 4.

MiR-129-5p overexpression promotes U251 cell chemosensitivity to TMZ. A miR-129-5p expression in U251 and U251TR cells was determined by RT-qPCR. B miR-129-5p expression in transfected U251TR cells was determined by RT-qPCR. C IC50 value of transfected U251TR cells. D Cell viability after TMZ treatment. E Proliferation of TMZ-treated cells was assessed using colony formation assay. F Apoptosis of TMZ-treated cells was assessed using flow cytometry. G Expression of Caspase-3, Bax and Bcl-2 was determined by RT-qPCR; * P < 0.05 vs the NC-mimic group, # P < 0.05 vs the NC-inhibitor group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, unpaired t-test was performed for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 2103 kb)

Supplementary Figure 5.

OIP5-AS1 binds to miR-129-5p to regulate IGF2BP2 expression. A Targeting relationship between miR-129-5p and IGF2BP2 was confirmed using dual luciferase reporter gene assay. B Binding among OIP5-AS1, miR-129-5p and IGF2BP2 was verified by RIP assay. C IGF2BP2 expression in U251 and U251TR cells was determined by RT-qPCR and Western blot analysis. D IGF2BP2 expression in U251TR cells was determined by RT-qPCR and Western blot analysis after OIP5-AS1 was knocked down. E IGF2BP2 expression in U251TR cells was determined by RT-qPCR and Western blot analysis after miR-129-5p was upregulated; * P < 0.05 vs the NC-mimic group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation and the unpaired t-test was performed for comparisons between two groups. (JPG 831 kb)

Supplementary Figure 6.

OIP5-AS1/miR-129-5p axis regulates viability and TMZ-resistance of U251 cells. A IGF2BP2 expression in U251TR cells detected by RT-qPCR and Western blot analysis. B Survival rate of transfected U251TR cells after TMZ treatment. C cell viability after TMZ treatment was detected by CCK-8 assay. D Proliferation after TMZ treatment was detected by colony formation assay. E Apoptosis of U251TR after TMZ treatment was detected by flow cytometry. F Expression of Caspase-3, Bax and Bcl-2 was determined by RT-qPCR. G Protein expression of Caspase-3, Bax and Bcl-2 in cells was determined using Western blot analysis; * P < 0.05 vs the si-OIP5-AS1 group; # P < 0.05 vs the NC group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 1120 kb)

Supplementary Figure 7.

OIP5-AS1 silencing suppresses TMZ-resistance of U251 cells in vivo. A Representative images of xenografts from nude mice. B Tumor growth curve. C Tumor weight in each group; * P < 0.05 vs the si-OIP5-AS1 group; the measurement data conforming to the normal distribution were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. (JPG 254 kb)

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Wang, X., Li, X., Zhou, Y. et al. Long non-coding RNA OIP5-AS1 inhibition upregulates microRNA-129-5p to repress resistance to temozolomide in glioblastoma cells via downregulating IGF2BP2. Cell Biol Toxicol 38, 963–977 (2022). https://doi.org/10.1007/s10565-021-09614-z

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  • DOI: https://doi.org/10.1007/s10565-021-09614-z

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