摘要
目的研究泛素连接酶E3的环状RNA(circHERC4/hsa_circ_0007113)与人胚肺成纤维细胞(IMR-90)的衰老的关系。方法从circBase数据库上获得环状RNA circHERC4的全基因序列,将其克隆到环状RNA表达载体pLC5-ciR中,体外构建重组体pLC5-ciR(+)hsa_circ_0007113,经PCR、酶切、测序等对重组质粒进行鉴定。利用脂质体2000转染试剂将重组质粒转染到HEK293T和IMR90细胞中,并利用RT-PCR检测正常细胞组、空载体组及重组载体组中环状RNA circHERC4的表达情况;SA-β-gal染色检测各组细胞衰老情况;CCK-8法检测各组细胞增殖情况。结果测序正确,成功构建了环状RNA circHERC4的过表达载体,circHERC4可在HEK293T细胞中高效表达。与对照组相比,重组载体组的SA-β-gal染色阳性率降低(P<0.05),细胞增殖率升高(P<0.05)。过表达circHERC4后能提高IMR90细胞的增殖,抑制细胞的衰老。结论circHERC4具有潜在的抗衰老作用,为深入研究circHERC4的功能奠定了基础。
Objective To investigate the relationship between the ubiquitin ligase E3 cirRNA(circHERC4/hsa_circ_0007113) and the senescence in Human embryonic lung fibroblasts(IMR-90)cells. Methods The whole gene sequence of circHERC4 was obtained from circBase database and cloned into cirRNA expression vector pLC5-ciR. The recombinant pLC5-ciR(+) hsa_circ_0007113 was constructed in vitro. The recombinant plasmid was identified by PCR, enzyme digestion and sequencing. The recombinant plasmid was transfected into HEK293 T and IMR90 cells with liposome 2000 transfection reagent. The expression of circHERC4 in normal cells, empty vector group and recombinant vector group was detected by RT-PCR, and the senescence of cells was detected by SA-β-Gal staining. Cell proliferation was detected by CCK-8 method. Results The overexpression vector of circHERC4 was successfully constructed by correct sequencing, and circHERC4 could be efficiently transfected in HEK293 T cells. Compared with the control group, the positive rate of SA-β-Gal staining in the recombinant vector group decreased(P<0.05), and the proliferation rate of cells increased(P<0.05). Overexpression of circHERC4 could improve the proliferation and inhibit the senescence in IMR90 cells. Conclusion It suggests that circHERC4 has potential function of anti-senescence, which lays a foundation for further study on the function of circHERC4.
作者
李华玲
杨迪
韦俊杰
陈章悦
陈菊萍
Li Hualing;Yang Di;Wei Junjie;Chen Zhangyue;Chen Juping(Dept of Biochemistry and molecular biology,Medical College,Yangzhou University,Yangzhou 225001;Dept of Dermatology,Affiliated Hospital of Yangzhou University,Yangzhou 225001)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第7期1100-1105,共6页
Acta Universitatis Medicinalis Anhui
基金
江苏省高等学校自然科学研究重大项目(编号:18KJA320014)。
