利用CRISPR/Cas9技术敲除长非编码RNA SNHG16基因促进K562细胞CD235a的表达上调
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摘要
为了探究敲除长非编码RNA SNHG16对人慢性髓系白血病K562细胞的影响,我们利用CRISPR/Cas9技术在K562细胞中敲除SNHG16基因,通过流式分选获得单细胞,经扩增培养、基因组PCR鉴定、测序鉴定后,获得SNHG16杂合和纯合敲除株;通过Wright-Giemsa染色、MTS检测、流式分析和qRT-PCR分别检测了SNHG16敲除后对K562细胞形态、增殖、细胞表面标志蛋白及红系分化调控因子的影响。实验结果显示,敲除SNHG16后不影响K562细胞的形态和增殖,显著促进了K562细胞表面标志蛋白CD235a和红系分化调控因子的表达水平。该研究表明,长非编码RNA SNHG16不影响K562细胞的增殖,但SNHG16对K562细胞表面标志蛋白CD235a的表达水平有一定的调控作用。
The aim of this work was to investigate the effect of long noncoding RNA SNHG16 knock-out on human chronic myeloid leukemia cells(K562 cells). The SNHG16 gene in K562 cells was knocked out by using CRISPR/Cas9 system. Single cells were gained by flow cytometric sorting technique. SNHG16+/– and SNHG16~(–/–) cell lines were gained after culture, identification of PCR and sequencing. The effects of SNHG16 depletion on morphology, proliferation, cell surface marker and erythroid transcription factors of K562 cells were detected by Wright-Giemsa staining, MTS assay, flow cytometry, and qRT-PCR. The results indicated that SNHG16 depletion did not affect the morphology and proliferation of K562 cells, but promoted the expression level of K562 cell surface marker CD235 a and erythroid transcription factors. In conclusion, long noncoding RNA SNHG16 does not affect the proliferation of K562 cells, but SNHG16 plays a role in the regulation of K562 cell surface marker protein CD235 a.
The aim of this work was to investigate the effect of long noncoding RNA SNHG16 knock-out on human chronic myeloid leukemia cells(K562 cells). The SNHG16 gene in K562 cells was knocked out by using CRISPR/Cas9 system. Single cells were gained by flow cytometric sorting technique. SNHG16+/– and SNHG16~(–/–) cell lines were gained after culture, identification of PCR and sequencing. The effects of SNHG16 depletion on morphology, proliferation, cell surface marker and erythroid transcription factors of K562 cells were detected by Wright-Giemsa staining, MTS assay, flow cytometry, and qRT-PCR. The results indicated that SNHG16 depletion did not affect the morphology and proliferation of K562 cells, but promoted the expression level of K562 cell surface marker CD235 a and erythroid transcription factors. In conclusion, long noncoding RNA SNHG16 does not affect the proliferation of K562 cells, but SNHG16 plays a role in the regulation of K562 cell surface marker protein CD235 a.
引文
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18 Ye J,Zhang R,Du X,Chai W,Zhou Q.Long noncoding RNASNHG16 induces sorafenib resistance in hepatocellular carcinoma cells through sponging miR-140-5p.Onco Targets Ther 2019;12:415-22.
19 Xu F,Zha G,Wu Y,Cai W,Ao J.Overexpressing lncRNASNHG16 inhibited HCC proliferation and chemoresistance by functionally sponging hsa-miR-93.Onco Targets Ther 2018;11:8855-63.
20 Kholghi OV,Geranpayeh L,Omrani MD,Ghafouri-Fard S.Assessment of functional variants and expression of long noncoding RNAs in vitamin D receptor signaling in breast cancer.Cancer Manag Res 2018;10:3451-62.
21 Cai C,Huo Q,Wang X,Chen B,Yang Q.SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5.Biochem Biophys Res Commun 2017;485(2):272-8.
22 Li J,Gao C,Liu C,Zhou C,Ma X,Li H,et al.Four lncRNAs associated with breast cancer prognosis identified by coexpression network analysis.J Cell Physiol 2019;234(8):14019-30.
2 Li W,Ren Y,Si Y,Wang F,Yu J.Long non-coding RNAs in hematopoietic regulation.Cell Regen(Lond)2018;7(2):27-32.
3 Lee Y,Park C,Lee S,Lee D,Kim J.Isolation and Functional Examination of the Long Non-Coding RNA Redrum.Mol Cells2018;41(2):134-9.
4 Zhu H,Zeng Y,Zhou CC,Ye W.SNHG16/miR-216-5p/ZEB1signal pathway contributes to the tumorigenesis of cervical cancer cells.Arch Biochem Biophys 2018;637:1-8.
5 Christensen LL,True K,Hamilton MP,Nielsen MM,Damas ND,Damgaard CK,et al.SNHG16 is regulated by the Wnt pathway in colorectal cancer and affects genes involved in lipid metabolism.Mol Oncol 2016;10(8):1266-82.
6 Cao X,Xu J,Yue D.LncRNA-SNHG16 predicts poor prognosis and promotes tumor proliferation through epigenetically silencing p21 in bladder cancer.Cancer Gene Ther 2018;25(1/2):10-7.
7 Feng F,Chen A,Huang J,Xia Q,Chen Y,Jin X.Long noncoding RNA SNHG16 contributes to the development of bladder cancer via regulating miR-98/STAT3/Wnt/beta-catenin pathway axis.JCell Biochem 2018;119(11):9408-18.
8 Zhang S,Du L,Wang L,Jiang X,Zhan Y,Li J,et al.Evaluation of serum exosomal LncRNA-based biomarker panel for diagnosis and recurrence prediction of bladder cancer.J Cell Mol Med2019;23(2):1396-405.
9 Duan W,Du L,Jiang X,Wang R,Yan S,Xie Y,et al.Identification of a serum circulating lncRNA panel for the diagnosis and recurrence prediction of bladder cancer.Oncotarget 2016;7(48):78850-8.
10 Yang T,Jin X,Lan J,Wang W.Long non-coding RNA SNHG16has Tumor suppressing effect in acute lymphoblastic leukemia by inverse interaction on hsa-miR-124-3p.IUBMB Life 2019;71(1):134-42.
11 Zhang F,Wen Y,Guo X.CRISPR/Cas9 for genome editing:progress,implications and challenges.Hum Mol Genet 2014;23(R1):R40-6.
12 Guo Z,Zhang J,Fan L,Liu J,Yu H,Li X,et al.Long Noncoding RNA(lncRNA)small nucleolar RNA host gene 16(SNHG16)predicts poor prognosis and sorafenib resistance in hepatocellular carcinoma.Med Sci Monit 2019;25:2079-86.
13 Kong Q,Qiu M.Long noncoding RNA SNHG15 promotes human breast cancer proliferation,migration and invasion by sponging miR-211-3p.Biochem Biophys Res Commun 2018;495(2):1594-600.
14 Aalijahan H,Ghorbian S.Long non-coding RNAs and cervical cancer.Exp Mol Pathol 2019;106:7-16.
15 Lu YF,Cai XL,Li ZZ,Lv J,Xiang YA,Chen JJ,et al.LncRNASNHG16 functions as an oncogene by sponging MiR-4518 and up-regulating PRMT5 expression in glioma.Cell Physiol Biochem 2018;45(5):1975-85.
16 Li W,Xu W,Song JS,Wu T,Wang WX.LncRNA SNHG16promotes cell proliferation through miR-302a-3p/FGF19 axis in hepatocellular carcinoma.Neoplasma 2019.
17 Lin Q,Zheng H,Xu J,Zhang F,Pan H.LncRNA SNHG16aggravates tumorigenesis and development of hepatocellular carcinoma by sponging miR-4500 and targeting STAT3.J Cell Biochem 2019;120(7):11604-15.
18 Ye J,Zhang R,Du X,Chai W,Zhou Q.Long noncoding RNASNHG16 induces sorafenib resistance in hepatocellular carcinoma cells through sponging miR-140-5p.Onco Targets Ther 2019;12:415-22.
19 Xu F,Zha G,Wu Y,Cai W,Ao J.Overexpressing lncRNASNHG16 inhibited HCC proliferation and chemoresistance by functionally sponging hsa-miR-93.Onco Targets Ther 2018;11:8855-63.
20 Kholghi OV,Geranpayeh L,Omrani MD,Ghafouri-Fard S.Assessment of functional variants and expression of long noncoding RNAs in vitamin D receptor signaling in breast cancer.Cancer Manag Res 2018;10:3451-62.
21 Cai C,Huo Q,Wang X,Chen B,Yang Q.SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5.Biochem Biophys Res Commun 2017;485(2):272-8.
22 Li J,Gao C,Liu C,Zhou C,Ma X,Li H,et al.Four lncRNAs associated with breast cancer prognosis identified by coexpression network analysis.J Cell Physiol 2019;234(8):14019-30.