The effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell_Chinese Journal of Ocular Fundus Diseases

Authors：

Bai Ru , Zhou Yue , Lu Xiaohong , Cai Jinjin ,  Yao Qing

Department of Pathobiology and Immunology, School of Basic Medicine, Ningxia Medical Uuniversity, Yinchuan 750004, China;

Corresponding author：

Yao Qing, Email: yaoqing726@163.com

Keywords：

Silent information regulator proteins, saccharomyces cerevisiae; Lentivirus infections; Retinal ganglion cells; Cells, cultured

DOI：

10.3760/cma.j.issn.1005-1015.2017.05.015

Video：

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Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×10⁸ TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.

Citation： Bai Ru, Zhou Yue, Lu Xiaohong, Cai Jinjin, Yao Qing. The effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell. Chinese Journal of Ocular Fundus Diseases, 2017, 33(5): 503-507. doi: 10.3760/cma.j.issn.1005-1015.2017.05.015 Copy

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2. Maloney SC, Antecka E, Odashiro AN, et al. Expression of SIRT1 and DBC1 in developing and adult retinas[J/OL]. Stem Cells Int, 2012, 2012: 908183[2012-08-30]. https://dx.doi.org/10.1155/2012/908183. DOI: 10.1155/2012/908183.
3. Kim SJ, Sung MS, Heo H, et al. Mangiferin protects retinal ganglion cells in ischemic mouse retina viaSIRT1[J]. Curr Eye Res, 2016, 41(6): 844-855. DOI: 10.3109/02713683.2015.1050736.
4. Zhang Y, Li H, Cao Y, et al. Sirtuin 1 regulates lipid metabolism associated with optic nerve regeneration[J]. Mol Med Rep, 2015, 12(5): 6962-6968. DOI: 10.3892/mmr.2015.4286.
5. Sun G, Yeh SY, Yuan CW, et al. Molecular properties, functional mechanisms, and applications of sliced siRNA[J/OL]. Mol Ther Nucleic Acids, 2015, 4: 221[2015-01-20]. https://linkinghub.elsevier.com/retrieve/pii/mtna201473. DOI: 10.1038/mtna.2014.73.
6. Wang F, Chen W, Liu P, et al. Lentivirus-mediated RNAi knockdown of LMP2A inhibits the growth of the Epstein-Barr-associated gastric carcinoma cell line GT38 in vitro[J]. Exp Ther Med, 2017, 13(1): 187-193. DOI: 10.3892/etm.2016.3954.
7. Zhou L, Wang Q, Chen X, et al. AML1-ETO promotes SIRT1 expression to enhance leukemogenesis of t (8;21) acute myeloid leukemia[J]. Exp Hematol, 2017, 46: 62-69. DOI: 10.1016/j.exphem.2016.09.013.
8. Zhang W, Wu H, Yang M, et al. SIRT1 inhibition impairs non-homologous end joining DNA damage repair by increasing Ku70 acetylation in chronic myeloid leukemia cells[J]. Oncotarget, 2016, 7(12): 13538-13550. DOI: 10.18632/oncotarget.6455.
9. Lovaas JD, Zhu L, Chiao CY, et al. SIRT1 enhances matrix metalloproteinase-2 expression and tumor cell invasion in prostate cancer cells[J]. Prostate, 2013, 73(5): 522-530. DOI: 10.1002/pros.22592.
10. Zhao G, Cui J, Zhang JG, et al. SIRT1 RNAi knockdown induces apoptosis and senescence, inhibits invasion and enhances chemosensitivity in pancreatic cancer cells[J]. Gene Ther, 2011, 18(9): 920-928. DOI: 10.1038/gt.2011.81.
11. Choi HN, Bae JS, Jamiyandorj U, et al. Expression and role of SIRT1 in hepatocellular carcinoma[J]. Oncol Rep, 2011, 26(2): 503-510. DOI: 10.3892/or.2011.1301.
12. 莫小亮, 罗殿中, 莫凌昭, 等.人端粒酶逆转录酶基因 shRNA慢病毒表达载体的构建及其病毒包装鉴定[J]. 中国妇幼保健, 2014, 29(4): 580-585. DOI: 10.7620/zgfybj.j.issn.1001-4411.2014.04.37.Mo XL, Luo DZ, Mo LZ, et al. Construction and identification of human telomerase reverse transcriptase targeted small hairpin RNA expressing plasmid and its stable transfected cervical canc-er line Caski[J]. Maternal & Child Health Care of China, 2014, 29(4): 580-585. DOI: 10.7620/zgfybj.j.issn.1001-4411.2014.04.37.
13. 武剑, 庞春艳, 尹芳蕊, 等.CD40-siRNA慢病毒载体的构建及其对小鼠巨噬细胞的感染[J]. 贵阳医学院学报, 2015, 40(8): 801-805.Wu J, Pang CY, Yin FR, et al. Construction of siRNA lentiviral vector against CD40 and measurement of its titer and infection efficiency on mouse macrophages cells[J]. Journal of Guizhou Medical University, 2015, 40(8): 801-805.

Chinese Journal of Ocular Fundus Diseases

The effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell

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