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目的:通过成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats/Cas9,CRISPR/Cas9)系统在SK-Hep1肝癌细胞系中构建AT富集作用域2(AT-rich interaction domain 2,ARID2)基因敲除模型,并初步观察ARID2敲除对肝癌细胞增殖和迁移能力的影响。方法:设计并合成靶向ARID2的向导RNA(single guide RNA,sg RNA)寡核苷酸序列,长度为20 bp,构建到CRISPR表达载体上,转染HEK293T细胞包装慢病毒并筛选稳定细胞株,采用克隆形成、Transwell和划痕实验分别检测细胞增殖和迁移能力。结果:目的 sg RNA寡核苷酸双链成功插入酶切后的Lenti CRISPER-V2质粒载体中且测序正确;经Western blot鉴定ARID2敲除的细胞株筛选成功;ARID2基因敲除后,与亲本细胞系相比,其蛋白表达缺失;克隆形成实验结果示ARID2敲除细胞株(1#6)培养至10 d时克隆形成数为(125.600±5.131)个,亲本细胞株克隆形成数为(69.000±5.567)个(t=12.962,P=0.000);ARID2敲除细胞株(2#1)培养至8 d时克隆形成数为(94.000±8.737)个,亲本细胞株克隆形成数为(67.000±5.292)个(t=4.635,P=0.010),差异有统计学意义。Transwell结果示ARID2敲除细胞株(1#6)在24 h的迁移细胞数为(180.000±5.542)个,亲本细胞组的迁移细胞数为(90.400±5.031)个(t=20.731,P=0.000);ARID2敲除细胞株(2#1)在24 h的迁移细胞数为(138.600±5.749)个,亲本细胞组的迁移细胞数为(78.400±5.703)个(t=12.891,P=0.000),差异有统计学意义。划痕实验结果示ARID2敲除细胞株(1#6)16 h划痕愈合度为(88.000±0.011)%,亲本细胞组划痕愈合度为(52.500±0.047)%(t=12.490,P=0.000);ARID2敲除细胞株(2#1)16 h划痕愈合度为(71.900±0.020)%,亲本细胞组划痕愈合度为(55.200±0.024)%(t=9.190,P=0.001),差异有统计学意义。结论:ARID2敲除可明显促进肝癌细胞的增殖和迁移能力,证实ARID2作为抑癌基因参与肝癌的发生和进程。ARID2敲除细胞模型为肝癌的深入研究提供新的手段。
OBJECTIVE: To construct AT-rich interaction domain 2 (SK-Hep1) cell line by clustered regularly interspaced short palindromic repeats / Cas9 (CRISPR / Cas9) 2, ARID2) knockout model, and preliminary observation of ARID2 knockdown on the proliferation and migration of liver cancer cells. Methods: The oligonucleotide sequence of single guide RNA (sg RNA) targeting ARID2 was designed and synthesized. The length of the oligonucleotide was 20 bp. The oligonucleotide sequence was constructed on the CRISPR expression vector, transfected into HEK293T cells and packaged with lentivirus, Clonal formation, Transwell and scratch assays were used to detect cell proliferation and migration ability respectively. Results: The target sg RNA oligonucleotide double strand was successfully inserted into Lenti CRISPER-V2 plasmid vector and sequenced correctly. Western blot was used to identify ARID2 knockout cell lines. After ARID2 knockout, The results of colony formation assay showed that the clonogenicity of ARID2 knockout cell line (1 # 6) was (125.600 ± 5.131) and the number of clone formation of the parental cell line was (69.000 ± 5.567) (P = 0.000). The number of clone formation in ARID2 knockout cell line (2 # 1) was (94.000 ± 8.737) and the number of clone formation in the parental cell line was (67.000 ± 5.292 ) (T = 4.635, P = 0.010), the difference was statistically significant. Transwell results showed that the number of migrating cells in the ARID2 knockout cell line (1 # 6) at 24 h was (180.000 ± 5.542) and the number of migrating cells in the parental cell group was (90.400 ± 5.031) (t = 20.731, P = 0.000 ). The number of migrating cells in the ARID2 knockdown cell line (2 # 1) at 24 h was (138.600 ± 5.749) cells, and the number of migrating cells in the parent cell group was (78.400 ± 5.703) (t = 12.891, ,The difference was statistically significant. Scratch test results showed that the scratch healing degree of ARID2 knock-out cell line (1 # 6) for 16 h was (88.000 ± 0.011)% and that of the parental cell group was (52.500 ± 0.047)% (t = 12.490, P = 0.000). The wound healing degree of ARID2 knock-out cell line (2 # 1) after scratching for 16 h was (71.900 ± 0.020)% and that of the parental cell group was (55.200 ± 0.024)% (t = 9.190, ,The difference was statistically significant. Conclusion: ARID2 knockdown can significantly promote the proliferation and migration of hepatocellular carcinoma cells, and confirm ARID2 as a tumor suppressor gene involved in the occurrence and progression of hepatocellular carcinoma. ARID2 knock-out cell model provides a new method for the further study of liver cancer.