Beclin-1基因shRNA慢病毒载体的构建及其对B16F10细胞自噬及活力的影响

doi:10.11843/j.issn.0366-6964.2021.09.024

Abstract

Abstract: In this study, we constructed a mouse melanoma cell line B16F10 with stable interference targeting Beclin-1 gene by RNA interference, and to explore autophagy and cell viability, which is the foundation for analysis of the relationship between Beclin-1 gene, autophagy, and tumor. Firstly, we designed the shRNA of mouse Beclin-l and constructed interference vectors. Then we transfected the lentiviral vector into 293T cells to obtain lentiviral particles, and B16F10 cells were infected with lentivirus packaging. To obtain the monoclonal cell line, the transfected cells were selected with the methods of pressurized screening and limited dilution. And then the monoclonal cells were cultured in fed-batch mode for quality evaluation. The interference effect of Beclin-l in the stable cell line was detected by RT-PCR and immunofluorescence technique, the expression of autophagy protein LC3 and the formation of autophagosomes were analyzed by Western blot and transmission electron microscopy, and cell viability were determined by CCK-8 assay. The results showed that the lentiviral vector targeting Beclin-l was successfully constructed, and the titer of lentivirus purified by packaging was 1×10⁸TU·mL^-1. Beclin-1 mRNA and protein were inhibited in the stable cell line, and the interference efficiency was around 75% (P<0.01). The results demonstrated that intervention of Beclin-1 expression could inhibit the expression of LC3-Ⅱ protein and the number of autophagosomes, and reduce the viability of B16F10 cells. These data indicated that the silence of Beclin-1 gene could effectively reduce the autophagy intensity and promote cell death in B16F10 cells, which lays the foundation for exploring the function of Beclin-1 gene and the role of autophagy in anti-tumor.

Key words: RNA interference, B16F10 cells, Beclin-1 gene, lentivirial vector, cell autophagy

CLC Number:

S852.2

JIA Yanyan, ZHAO Yingying, YU Zuhua, HE Lei, LIAO Chengshui, LI Jing, YU Chuan, ZHANG Chunjie, LI Yinju. Construction and Influence of Beclin-1 Gene shRNA Lentiviral Vector on Autophagy and Virability of B16F10 Cell[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2609-2616.

References 24

[1]	GERMIC N, FRANGEZ Z, YOUSEFI S, et al. Regulation of the innate immune system by autophagy:neutrophils, eosinophils, mast cells, NK cells[J]. Cell Death Differ, 2019, 26(4):703-714.
[2]	PECORARO A, PAGANO M, RUSSO G, et al. Role of autophagy in cancer cell response to nucleolar and endoplasmic reticulum stress[J]. Int J Mol Sci, 2020, 21(19):7334.
[3]	DAS M, KARNAM A, STEPHEN-VICTOR E, et al. Intravenous immunoglobulin mediates anti-inflammatory effects in peripheral blood mononuclear cells by inducing autophagy[J]. Cell Death Dis, 2020, 11(1):50.
[4]	KAUSHIK S, CUERVO A M. The coming of age of chaperone-mediated autophagy[J]. Nat Rev Mol Cell Biol, 2018, 19(6):365-381.
[5]	DENNY P, FEUERMANN M, HILL D P, et al. Exploring autophagy with gene ontology[J]. Autophagy, 2018, 14(3):419-436.
[6]	ONORATI A V, DYCZYNSKI M, OJHA R, et al. Targeting autophagy in cancer[J]. Cancer, 2018, 124(16):3307-3318.
[7]	KAUR S, CHANGOTRA H. The beclin 1 interactome:Modification and roles in the pathology of autophagy-related disorders[J]. Biochimie, 2020, 175:34-49.
[8]	SU B, WANG X T, SUN Y H, et al. Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway[J]. J Geriatr Cardiol, 2020, 17(5):284-293.
[9]	TOTON E, LISIAK N, SAWICKA P, et al. Beclin-1 and its role as a target for anticancer therapy[J]. J Physiol Pharmacol, 2014, 65(4):459-467.
[10]	ZHANG W, LI Q Y, SONG C, et al. Knockdown of autophagy-related protein 6, Beclin-1, decreases cell growth, invasion, and metastasis and has a positive effect on chemotherapy-induced cytotoxicity in osteosarcoma cells[J]. Tumor Biol, 2015, 36(4):2531-2539.
[11]	MATSUZAWA-ISHIMOTO Y, HWANG S, CADWELL K. Autophagy and inflammation[J]. Annu Rev Immunol, 2018, 36:73-101.
[12]	谢昆, 李密杰, 蒋成砚, 等. 自噬相关蛋白ATG5/BECLIN-1调控细胞自噬和凋亡的分子机理研究进展[J]. 中国人兽共患病学报, 2018, 34(3):272-275, 285.XIE K, LI M J, JIANG C Y, et al. Research progress on autophagy-related protein 5 and BECLIN-1 regulate autophagy and apoptosis[J]. Chinese Journal of Zoonoses, 2018, 34(3):272-275, 285. (in Chinese)
[13]	HU Y J, ZHONG J T, GONG L, et al. Autophagy-related Beclin 1 and head and neck cancers[J]. OncoTargets Ther, 2020, 13:6213-6227.
[14]	BRIOSCHI M, BANFI C. The application of gene silencing in proteomics:from laboratory to clinic[J]. Expert Rev Proteomics, 2018, 15(9):717-732.
[15]	WILSON R C, DOUDNA J A. Molecular mechanisms of RNA interference[J]. Annu Rev Biophys, 2013, 42:217-239.
[16]	SHENG P K, FLOOD K A, XIE M Y. Short hairpin RNAs for strand-specific small interfering RNA production[J]. Front Bioeng Biotechnol, 2020, 8:940.
[17]	齐兴财, 秦晓东, 甘晓丽, 等. 慢病毒介导siRNA抑制O型口蹄疫病毒ON株病毒复制[J]. 畜牧兽医学报, 2018, 49(2):360-367.QI X C, QIN X D, GAN X L, et al. Inhibition of type O foot-and-mouth disease virus ON strain replication by lentivirus-mediated siRNA[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(2):360-367. (in Chinese)
[18]	高爱君, 司维柯, 赵宸, 等. 聚凝胺增强Ror2重组腺病毒感染K562细胞效应的研究[J]. 解放军医学杂志, 2015, 40(12):976-980.GAO A J, SI W K, ZHAO C, et al. Polybrene enhances infection effect of Ror2 recombinant adenovirus on K562 cells[J]. Medical Journal of Chinese People's Liberation Army, 2015, 40(12):976-980. (in Chinese)
[19]	HILL S M, WROBEL L, RUBINSZTEIN D C. Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation[J]. Cell Death Differ, 2019, 26(4):617-629.
[20]	LIU Y R, SONG A W, WU H F, et al. Paeonol inhibits apoptosis of vascular smooth muscle cells via up-regulation of autophagy by activating class III PI3K/Beclin-1 signaling pathway[J]. Life Sci, 2021, 264:118714.
[21]	张松. Beclin-1基因沉默对前列腺增生上皮细胞BPH-1自噬及凋亡作用机制的研究[D]. 福州:福建医科大学, 2018.ZHANG S. The study of Beclin-1 gene silencing on the autophagy and apoptosis of the Prostatic hyperplasia epithelial cells[D]. Fuzhou:Fujian Medical University, 2018. (in Chinese)
[22]	应杰, 王梦, 张蒙, 等. Beclin-1非自噬依赖性通路在肿瘤中的研究进展[J]. 生命科学, 2019, 31(8):795-801.YING J, WANG M, ZHANG M, et al. Advances in the research of autophagy-independent pathways of Beclin-1 in tumors[J]. Chinese Bulletin of Life Sciences, 2019, 31(8):795-801. (in Chinese)
[23]	YE C L, YU X L, LIU X Q, et al. Beclin-1 knockdown decreases proliferation, invasion and migration of Ewing sarcoma SK-ES-1 cells via inhibition of MMP-9[J]. Oncol Lett, 2018, 15(3):3221-3225.
[24]	SUN H Y, YU J, WEN Z R, et al. Decreased expression of Beclin-1 in patients with hepatocellular carcinoma[J]. J Buon, 2019, 24(2):634-641.

Construction and Influence of Beclin-1 Gene shRNA Lentiviral Vector on Autophagy and Virability of B16F10 Cell

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