组蛋白赖氨酸转移酶KAT6B基因shRNA慢病毒载体的构建与鉴定

doi:10.11904/j.issn.1002-3070.2018.01.001

Abstract

Abstract:

Objective The aims of this study were to construct short hairpin RNA(shRNA)lentiviral vector in breast cancer T47D cells,to carry out RNA interference on lysine acetyltransferase 6B(KAT6B/MORF)gene,to down-regulate its expression and to explore its function. Methods Two pairs of single-stranded short hairpin RNA(shRNA5 and shRNA8)and the corresponding control sequences(Scramble5 and Scramble8)were synthesized based on the CDS of KAT6B gene.Polymerase chain reaction(PCR)was used to amplify double-stranded and ligated with the entry vector(pENTR/pSM2(CMV)GFP),which were subjected to a double digestion(EcoRl and Xhol)linearization and homologous recombination with the entry vector(pENTR/pSM₂(CMV)GFP)to obtain an entry clone containing the desired fragment.The target fragment was recombined onto the target vector(pLenti x1 puro DEST)via the LR cloning reaction of the Gateway system.The lentiviral packaging plasmids were co-transfected into HEK-293T cells with two pairs of target plasmids.The supernatant of HEK-293T cells was collected and transformed into T47D cells.The expression of KAT6B protein was detected in T47D cells by Western blot. Results The single colony obtained from the transformation was identified by sequencing,which was consistent with the target sequence,indicating that the lentiviral vector had been successfully constructed.The expression of KAT6B protein was significantly lower in the shRNA KAT6B group than that in the control group,which indicated that the constructed gene silencing vector could play a role in the KAT6B gene in T47D cells. Conclusion The shRNA lentiviral gene silencing vectors of KAT6B were constructed and identified in T47D cells,which indicated that the foundation for further study KAT6B gene plays an inhibitory effect on breast cancer.

Key words: Vector construction, Gene silence, RNA interference, KAT6B, Lentiviral vector

CLC Number:

R737.9

JIN Junjie, AN Jing, CAO Difei, SONG Aili, ZHAO Lili, LIU Zhaoliang. Construction and identification of shRNA lentiviral vectors targeting histone lysine transferase KAT6B gene[J]. PRACTICAL ONCOLOGY JOURNAL, 2018, 32(1): 1-6.

References

1 Huang F,Abmayr SM,Workman JL.Regulation of KAT6 acetyltransferases and their roles in cell cycle progression,stem cell maintenance,and human disease[J].Mol Cell Biol,2016,36(14):1900-1907.
2 Simó-Riudalbas L,Pérez-Salvia M,Setien F,et al.KAT6B is a tumor suppressor histone H3 lysine 23 acetyltransferase undergoing genomic loss in small cell lung cancer[J].Cancer Res,2015,75(18):3936-3945.
3 Mattioli F,Schaefer E,Magee A,et al.Mutations in histone acetylase modifier BRPF1 cause an autosomal-dominant form of intellectual disability with associated aptosis[J].Am J Hum Genet,2017,100(1):105-116.
4 Li M,Yuan L,Jing A,et al.Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer[J].Tumour Biol,2016,37(11):14803-14812.
5 Lundsgaard M,Le VQ,Ernst A,et al.De novo KAT6B mutation identified with whole-exome sequencing in a girl with say-barber/biesecker/young-simpson syndrome[J].Mol Syndromol,2017,8(1):24-29.
6 Szakszon K,Salpietro C,Kakar N,et al.De novo mutations of the gene encoding the histone acetyltransferase KAT6B in two patients with say-barber/biesecker/young-simpson syndrome[J].Am J Med Genet A,2013,161(4):884-888.
7 Desh H,Gray SL,Horton MJ,et al.Molecular motor MYO1C,acetyltransferase KAT6B,and osteogenetic transcription factor RUNX2,expression in human masseter muscle contributes to development of malocclusion[J].Arch Oral Biol,2014,59(6):601-607.
8 Campeau PM,Kim JC,Lu JT,et al.Mutations in KAT6B,encoding a histone acetyltransferase,cause genitopatellar syndrome[J].Am J Hum Genet,2012,90(2):282-289.
9 Clayton-Smith J,O′Sullivan J,Daly S,et al.Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the say-barber-biesecker variant of ohdo syndrome[J].Am J Hum Genet,2011,89(5):675-681.
10 He W,Zhang MG,Wang XJ,et al.KAT5 and KAT6B are in positive regulation on cell proliferation of prostate cancer through PI3K-AKT signaling[J].Int J Clin Exp Pathol,2013,6(12):2864-2871.
11 Du P,Zhao J,Wang J,et al.Sine oculis homeobox homolog 1 regulates mitochondrial apoptosis pathway via caspase-7 in gastric cancer cells[J].J Cancer,2017,8(4):636-645.
12 Panagopoulos I,Gorunova L,Bjerkehagen B,et al.Novel KAT6B-KANSL1 fusion gene identified by RNA sequencing in retroperitoneal leiomyoma with t(10;17)(q22;q21)[J].PLoS One,2015,10(1):e0117010.
13 You L,Yan K,Zou J,et al.The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors[J].PLoS Genet,2015,11(3):e1005034.
14 Yang H,Pinello CE,Luo J,et al.Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents[J].Mol Cancer Ther,2013,12(5):610-620.
15 Campeau E,Ruhl VE,Rodier F,et al.A versatile viral system for expression and depletion of proteins in mammalian cells[J].PLoS One,2009,4(8):e6529.
16 Fussenegger M,Moser S,Mazur X,et al.Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells[J].Biotechnol Prog,2010,13(6):733-740.
17 Novina CD,Sharp PA.The RNAi revolution[J].Nature,2004,430(6996):161-164.
18 Hannon GJ.RNA interference[J].Nature,2002,418(6894):244-251.
19 Ni JQ,Zhou R,Czech B,et al.A genome-scale shRNA resource for transgenic RNAi in Drosophila[J].Nat Methods,2011,8(5):405-407.
20 Meter TEV,Khan A,Fitzmartin K,et al.Eg-16aberrant myst4/brpf1 signaling misdirects regional neurogenesis programs,sustaining expression of self-renewal genes in pediatric brain cancers[J]. Neuro Oncology,2014,16(5):78.

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Construction and identification of shRNA lentiviral vectors targeting histone lysine transferase KAT6B gene

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