PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway
Abstract
:1. Introduction
2. Results
2.1. PRKAR1B-AS2 lncRNA Is Overexpressed in OC and Associated with Poor Survival
2.2. Silencing PRKAR1B-AS2 Inhibits Cell Viability and Improves the Response to Cisplatin In Vitro
2.3. PRKAR1B-AS2 Knockdown Suppresses Migration and Invasion of OC Cells In Vitro
2.4. PRKAR1B-AS2 Regulates the PI3K/AKT/mTOR Signaling Pathway
2.5. PRKAR1B-AS2 Silencing Improves OC Sensitivity to Cisplatin In Vivo
3. Discussion
4. Materials and Methods
4.1. OS Analysis of lncRNA Expression in TCGA OC Cohort
4.2. LncRNA–Protein Interactions
4.3. Cell Culture, Reagents, and siRNA Transfection
4.4. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR Analysis
4.5. Cell Viability Assay
4.6. Colony Formation Assay
4.7. Wound Healing Assay
4.8. Invasion Assay
4.9. Annexin V/PI Assay
4.10. Cell Extracts and Western Blot Analysis
4.11. RIP Assay
4.12. RPPA Analysis
4.13. Liposomal Nanoparticle Preparation
4.14. OC Xenograft Mouse Model Studies
4.15. Immunohistochemistry
4.16. TUNEL Assay
4.17. In Situ Hybridization
4.18. Collection of Mouse Tumor Specimens for Quantitative Reverse Transcription PCR
4.19. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AKT | Protein kinase B |
DOPC | 1,2-dioleoyl-sn-glycero-3-phosphocholine |
lncRNA | Long non-coding RNA |
mTOR | Mammalian target of rapamycin |
OC | Ovarian cancer |
OS | Overall survival |
PDK1 | Phosphoinositide dependent kinase 1 |
PI3K | Phosphoinositide 3-kinase |
PIP2 | Phosphatidylinositol-3,4-bisphosphate |
PIP3 | Phosphatidylinositol-3,4,5- triphosphate |
PRKAR1B | Protein Kinase CAMP-Dependent Type I Regulatory Subunit Beta |
PTEN | Phosphatase and tensin homolog |
RIP | RNA immunoprecipitation |
RPPA | Reverse phase protein array |
siRNA | Small interfering RNA |
TCGA | The cancer genome atlas |
TSC1/2 | Tuberous sclerosis complex 1 and 2 |
TUNEL | Terminal deoxynucleotidyl transferase dUTP nick-end labeling |
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siRNA Name | Sequence (Sense) 5′ to 3′ | Sequence (Antisense) 5′ to 3′ |
---|---|---|
PRKAR1B-AS2 siRNA 1 | GAGCACAAAUGCUGGUCUUdTdT | AAGACCAGCAUUUGUGCUCdTdT |
PRKAR1B-AS2 siRNA 2 | GGUCUUACUACUAAGCAAUdTdT | AUUGCUUAGUAGUAAGACCdTdT |
PRKAR1B-AS2 siRNA 3 | GCAAUAUGUCCCUCGGAUCdTdT | GAUCCGAGGGACAUAUUGCdTdT |
Gene Symbol | Forward Primer | Reverse Primer |
---|---|---|
PRKAR1B-AS2 | 5′-TTGGACACTGCCCATCTT-CC3′ | 5′-TGCAGCCACGGGATGTTT-AT3′ |
PRKAR1B-AS2 | 5′-CCTCGGATCCAAAGGAGA-GC-3′ | 5′-AAGATGGGCAGTGTCCAA-GG-3′ |
PIK3CA | 5′-GAGTAACAGACTAGCTAGAGAC-3′ | 5′-AGAAAATCTTTCTCCTGCTC-3′ |
GAPDH | 5′-GACAGTCAGCCGCATCTT-CT-3′ | 5′-GCGCCCAATACGACCAAA-TC-3′ |
U6 snRNA | 5′-CTCGCTTCGGCAGCACA-3′ | 5′-GGAACGCTTCACGAATTT-GC-3′ |
Primary Antibody | Purchasing Company (Catalog #) | Dilution Factor | Secondary Antibody | Purchasing Company | Dilution Factor |
---|---|---|---|---|---|
PI3 Kinase p110α (C73F8) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (4249) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
PI3 Kinase p110α Antibody | Cell Signaling Technology, Danvers, MA, USA (4255) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Phospho-mTOR (Ser2448) Antibody | Cell Signaling Technology, Danvers, MA, USA (2971) | 1:500 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
mTOR (7C10) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (2983) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Phospho-Akt (Ser473) (D9E) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (4060) | 1:400 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Akt (pan) (40D4) Mouse mAb | Cell Signaling Technology, Danvers, MA, USA (2920) | 1:1000 | Anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7076) | 1:2000 |
PDK1 Antibody | Cell Signaling Technology, Danvers, MA, USA (3062) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (3438) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
PTEN (D4.3) XP® Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (9188) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
p70 S6 Kinase (49D7) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (2708) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Phospho-p70 S6 Kinase (Thr389) (1A5) Mouse mAb | Cell Signaling Technology, Danvers, MA, USA (9206) | 1:500 | Anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7076) | 1:2000 |
Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (6943) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Src (36D10) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (2109) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Cleaved Caspase-9 (Asp315) Antibody (Human Specific) | Cell Signaling Technology, Danvers, MA, USA (9505) | 1:5000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Caspase-9 (C9) Mouse mAb | Cell Signaling Technology, Danvers, MA, USA (9508) | 1:1000 | Anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7076) | 1:2000 |
Cleaved Caspase-3 (Asp175) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (9661) | 1:300 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Caspase-3 (D3R6Y) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (14220) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Phospho-FAK (Tyr397) (D20B1) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (8556) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Cleaved Caspase-7 (Asp198) (D6H1) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (8438) | 1:400 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Caspase-7 (D2Q3L) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (12827) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (5625) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
PARP (46D11) Rabbit mAb | Cell Signaling Technology, Danvers, MA, USA (9532) | 1:1000 | Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology, Danvers, MA, USA (7074) | 1:2000 |
Anti-beta Actin Mouse Antibody | Abcam (ab6276) | 1:10000 | Anti-mouse IgG, HRP-linked | CST, Danvers, USA (7076) | 1:2000 |
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Elsayed, A.M.; Bayraktar, E.; Amero, P.; Salama, S.A.; Abdelaziz, A.H.; Ismail, R.S.; Zhang, X.; Ivan, C.; Sood, A.K.; Lopez-Berestein, G.; et al. PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway. Int. J. Mol. Sci. 2021, 22, 1882. https://doi.org/10.3390/ijms22041882
Elsayed AM, Bayraktar E, Amero P, Salama SA, Abdelaziz AH, Ismail RS, Zhang X, Ivan C, Sood AK, Lopez-Berestein G, et al. PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway. International Journal of Molecular Sciences. 2021; 22(4):1882. https://doi.org/10.3390/ijms22041882
Chicago/Turabian StyleElsayed, Abdelrahman M., Emine Bayraktar, Paola Amero, Salama A. Salama, Abdelaziz H. Abdelaziz, Raed S. Ismail, Xinna Zhang, Cristina Ivan, Anil K. Sood, Gabriel Lopez-Berestein, and et al. 2021. "PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway" International Journal of Molecular Sciences 22, no. 4: 1882. https://doi.org/10.3390/ijms22041882