Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs

Baharak Abdolhossein Zadeh; Kamal Yavari; Mehdi Banan; Ali Fallah; Leila Nasehi; Moloud Absalan; Gholamreza Tavoosidana

doi:10.14715/cmb/2018.64.11.5

Issue

Vol. 64 No. 11: Issue 11

Issue Published : August 31, 2018

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Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs

https://doi.org/10.14715/cmb/2018.64.11.5

Baharak Abdolhossein Zadeh

Department of Molecular Medicine, School of Advance Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

Kamal Yavari

Department of Radiochemistry, Nuclear Sciences and Technology Research Institute (NSTRI), Tehran, Iran

Mehdi Banan

Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

Ali Fallah

RNAx Ltd., London, United Kingdom

Leila Nasehi

Department of Medical Laboratory Sciences, School of Sciences, Zanjan University of Medical Sciences, Zanjan, Iran

Moloud Absalan

Department of Molecular Medicine, School of Advance Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

Gholamreza Tavoosidana

Department of Molecular Medicine, School of Advance Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

Corresponding Author(s) : Gholamreza Tavoosidana

g-tavoosi@tums.ac.ir

Cellular and Molecular Biology, Vol. 64 No. 11: Issue 11
Article Published : August 30, 2018

Abstract

B-cell lymphoma 2 (BCL2) family proteins play a critical role in tuning cell death processes. Almost in half of all human cancers, a dysregulation in BCL2 family gene expression has been shown which made it an impressive target for human gene therapy as a novel approach in cancers. In this study we will optimize lentiviral-mediated RNA interference (RNAi), recombinant lentiviruses accommodating anti-BCL2 micro adaptive short hairpin RNAs (shRNAs), to downregulate BCL2 in human embryonic kidney 293T (HEK293T) cells to produce stable cell lines. We tested 4 different Dharmaconâ„¢ GIPZâ„¢ shRNAmir lentiviral vectors targeting BCL2 in different positions and a pGIPZ non-silencing shRNAmir lentiviral vector (as a negative control). Lentivirus packaging was performed by the calcium phosphate precipitation method. HEK293T cells were transduced by each type of recombinant lentiviruses individually and selected by puromycin within 10 days. The relative mRNA level and protein expression were assayed by using real-time polymerase chain reaction (PCR) technic and western blotting, respectively. Lentivirus (LV) packaging was performed in high efficiency (transfection rate was > 90%). Recombinant viruses of 4 expression vector addition to a control vector were produced then transduced to HEK293T cells successfully. All the 4 cell groups showed a significant down regulation of BCL2 gene (~90-95%) at mRNA level compared to the control group (p<0.01) but differences between silenced groups were not significant (P > 0.05). We showed that the lentivirus-mediated RNAi technique is an efficient method to establish HEK293 cell lines with stable down-regulation of BCL2 gene.

Keywords

BCL2 Lentivirus RNA interference HEK293 Cells Genetic Transductions.

Abdolhossein Zadeh, B., Yavari, K., Banan, M., Fallah, A., Nasehi, L., Absalan, M., & Tavoosidana, G. (2018). Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs. Cellular and Molecular Biology, 64(11), 25–30. https://doi.org/10.14715/cmb/2018.64.11.5

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References

Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, et al. Cloning the chromosomal breakpoint of t (14; 18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell. 1985;41(3):899-906.
Vaux D, Cors S. Bcl-2 Gene Promotes Haemopoietic Cell Survival And Cooperates With C-Myc To Immortalize. Pre-B Cells Nature. 1988;335(S 440).
Certo M, Moore VDG, Nishino M, Wei G, Korsmeyer S, Armstrong SA, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer cell. 2006;9(5):351-65.
Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene. 2003;22(53):8590.
Reed JC. Mechanisms of apoptosis. The American journal of pathology. 2000;157(5):1415-30.
Del Bufalo D, Trisciuoglio D, Scarsella M, Zangemeister-Wittke U, Zupi G. Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity. Oncogene. 2003;22(52):8441-7.
Schwandner O, Schiedeck T, Bruch H-P, Duchrow M, Windhoevel U, Broll R. p53 and Bcl-2 as significant predictors of recurrence and survival in rectal cancer. European Journal of Cancer. 2000;36(3):348-56.
Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance?Oncotarget. 2015;6(6):3519.
Buchholz TA, Davis DW, McConkey DJ, Symmans WF, Valero V, Jhingran A, et al. Chemotherapy"Induced Apoptosis and Bcl"2 Levels Correlate with Breast Cancer Response to Chemotherapy. The Cancer Journal. 2003;9(1):33-41.
Lin Y, Fukuchi J, Hiipakka RA, Kokontis JM, Xiang J. Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. Cell research. 2007;17(6):531-6.
Han B, Park D, Li R, Xie M, Owonikoko TK, Zhang G, et al. Small-molecule Bcl2 BH4 antagonist for lung cancer therapy. Cancer cell. 2015;27(6):852-63.
Reed JC. Bcl-2–family proteins and hematologic malignancies: history and future prospects. blood. 2008;111(7):3322-30.
Devi G. siRNA-based approaches in cancer therapy. Cancer gene therapy. 2006;13(9):819-29.
Taxman DJ, Moore CB, Guthrie EH, Huang MT-H. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. RNA Therapeutics: Function, Design, and Delivery. 2010:139-56.
Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer cell. 2002;2(1):17-23.
Manjunath N, Wu H, Subramanya S, Shankar P. Lentiviral delivery of short hairpin RNAs. Advanced drug delivery reviews. 2009;61(9):732-45.
Hosseini A, Estiri H, Niaki HA, Alizadeh A, Ghaderian SM, Farjadfar A, et al. Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model. Cell J (Yakhteh). 2017;19(3).
Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of pharmacological and toxicological methods. 2005;51(3):187-200.
Salmon P, Trono D. Production and titration of lentiviral vectors. Current protocols in human genetics. 2007:12.0. 1-.0. 24.
Hanahan D, Weinberg RA. The hallmarks of cancer. cell. 2000;100(1):57-70.
Reed JC, Cuddy M, Slabiak T, Croce CM, Nowell PC. Oncogenic potential of bcl-2 demonstrated by gene transfer. Nature. 1988;336(6196):259-61.
Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002;108(2):153-64.
Takahashi H, Chen MC, Pham H, Matsuo Y, Ishiguro H, Reber HA, et al. Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2013;1833(12):2980-7.
Lin Y-L, Jiang G, Zhang Z, Nör JE, ElSayed ME. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using "Smart” Particles. Journal of functional biomaterials. 2014;5(3):167-82.
Connolly J. Lentiviruses in gene therapy clinical research. Gene therapy. 2002;9(24):1730.
Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nature reviews Drug discovery. 2010;9(1):57.
Boden D, Pusch O, Silbermann R, Lee F, Tucker L, Ramratnam B. Enhanced gene silencing of HIV"1 specific siRNA using microRNA designed hairpins. Nucleic acids research. 2004;32(3):1154-8.
Nasehi L, Ghahremani M, Yavari K, Hadjati J, Fallah A, Zadeh BA, et al. Stable silencing of IGF1R using Lentiviral-mediated shRNA in HEK293T cells. Cell Mol Biol (Noisy le Grand). 2017;63(2).
Zhao Y, Zhang C-l, Zeng B-f, Gao T-T, Oda Y. Enhanced chemosensitivity of drug-resistant osteosarcoma cells by lentivirus-mediated Bcl-2 silencing. Biochemical and biophysical research communications. 2009;390(3):642-7.
Low SY, Tan BS, Choo HL, Tiong KH, Khoo AS-B, Leong C-O. Suppression of BCL-2 synergizes cisplatin sensitivity in nasopharyngeal carcinoma cells. Cancer letters. 2012;314(2):166-75.
Lamers F, Schild L, den Hartog IJ, Ebus ME, Westerhout EM, Ora I, et al. Targeted BCL2 inhibition effectively inhibits neuroblastoma tumour growth. European Journal of Cancer. 2012;48(16):3093-103.
Waters JS, Webb A, Cunningham D, Clarke PA, Raynaud F, di Stefano F, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. Journal of Clinical Oncology. 2000;18(9):1812-23.
amp A, Profile D. Augmerosen, BCL-2 Antisense Oligonucleotide”Genta, G 3139, GC 3139, Oblimersen Sodium. Drugs. 2007;8(5):321-34.

References

Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, et al. Cloning the chromosomal breakpoint of t (14; 18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell. 1985;41(3):899-906.

Vaux D, Cors S. Bcl-2 Gene Promotes Haemopoietic Cell Survival And Cooperates With C-Myc To Immortalize. Pre-B Cells Nature. 1988;335(S 440).

Certo M, Moore VDG, Nishino M, Wei G, Korsmeyer S, Armstrong SA, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer cell. 2006;9(5):351-65.

Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene. 2003;22(53):8590.

Reed JC. Mechanisms of apoptosis. The American journal of pathology. 2000;157(5):1415-30.

Del Bufalo D, Trisciuoglio D, Scarsella M, Zangemeister-Wittke U, Zupi G. Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity. Oncogene. 2003;22(52):8441-7.

Schwandner O, Schiedeck T, Bruch H-P, Duchrow M, Windhoevel U, Broll R. p53 and Bcl-2 as significant predictors of recurrence and survival in rectal cancer. European Journal of Cancer. 2000;36(3):348-56.

Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance?Oncotarget. 2015;6(6):3519.

Buchholz TA, Davis DW, McConkey DJ, Symmans WF, Valero V, Jhingran A, et al. Chemotherapy"Induced Apoptosis and Bcl"2 Levels Correlate with Breast Cancer Response to Chemotherapy. The Cancer Journal. 2003;9(1):33-41.

Lin Y, Fukuchi J, Hiipakka RA, Kokontis JM, Xiang J. Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. Cell research. 2007;17(6):531-6.

Han B, Park D, Li R, Xie M, Owonikoko TK, Zhang G, et al. Small-molecule Bcl2 BH4 antagonist for lung cancer therapy. Cancer cell. 2015;27(6):852-63.

Reed JC. Bcl-2–family proteins and hematologic malignancies: history and future prospects. blood. 2008;111(7):3322-30.

Devi G. siRNA-based approaches in cancer therapy. Cancer gene therapy. 2006;13(9):819-29.

Taxman DJ, Moore CB, Guthrie EH, Huang MT-H. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. RNA Therapeutics: Function, Design, and Delivery. 2010:139-56.

Paddison PJ, Hannon GJ. RNA interference: the new somatic cell genetics? Cancer cell. 2002;2(1):17-23.

Manjunath N, Wu H, Subramanya S, Shankar P. Lentiviral delivery of short hairpin RNAs. Advanced drug delivery reviews. 2009;61(9):732-45.

Hosseini A, Estiri H, Niaki HA, Alizadeh A, Ghaderian SM, Farjadfar A, et al. Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model. Cell J (Yakhteh). 2017;19(3).

Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of pharmacological and toxicological methods. 2005;51(3):187-200.

Salmon P, Trono D. Production and titration of lentiviral vectors. Current protocols in human genetics. 2007:12.0. 1-.0. 24.

Hanahan D, Weinberg RA. The hallmarks of cancer. cell. 2000;100(1):57-70.

Reed JC, Cuddy M, Slabiak T, Croce CM, Nowell PC. Oncogenic potential of bcl-2 demonstrated by gene transfer. Nature. 1988;336(6196):259-61.

Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002;108(2):153-64.

Takahashi H, Chen MC, Pham H, Matsuo Y, Ishiguro H, Reber HA, et al. Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2013;1833(12):2980-7.

Lin Y-L, Jiang G, Zhang Z, Nör JE, ElSayed ME. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using "Smart” Particles. Journal of functional biomaterials. 2014;5(3):167-82.

Connolly J. Lentiviruses in gene therapy clinical research. Gene therapy. 2002;9(24):1730.

Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nature reviews Drug discovery. 2010;9(1):57.

Boden D, Pusch O, Silbermann R, Lee F, Tucker L, Ramratnam B. Enhanced gene silencing of HIV"1 specific siRNA using microRNA designed hairpins. Nucleic acids research. 2004;32(3):1154-8.

Nasehi L, Ghahremani M, Yavari K, Hadjati J, Fallah A, Zadeh BA, et al. Stable silencing of IGF1R using Lentiviral-mediated shRNA in HEK293T cells. Cell Mol Biol (Noisy le Grand). 2017;63(2).

Zhao Y, Zhang C-l, Zeng B-f, Gao T-T, Oda Y. Enhanced chemosensitivity of drug-resistant osteosarcoma cells by lentivirus-mediated Bcl-2 silencing. Biochemical and biophysical research communications. 2009;390(3):642-7.

Low SY, Tan BS, Choo HL, Tiong KH, Khoo AS-B, Leong C-O. Suppression of BCL-2 synergizes cisplatin sensitivity in nasopharyngeal carcinoma cells. Cancer letters. 2012;314(2):166-75.

Lamers F, Schild L, den Hartog IJ, Ebus ME, Westerhout EM, Ora I, et al. Targeted BCL2 inhibition effectively inhibits neuroblastoma tumour growth. European Journal of Cancer. 2012;48(16):3093-103.

Waters JS, Webb A, Cunningham D, Clarke PA, Raynaud F, di Stefano F, et al. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma. Journal of Clinical Oncology. 2000;18(9):1812-23.

amp A, Profile D. Augmerosen, BCL-2 Antisense Oligonucleotide”Genta, G 3139, GC 3139, Oblimersen Sodium. Drugs. 2007;8(5):321-34.

Author biographies is not available.