可诱导的shRNAs表达系统对HeLa细胞SMYD3基因表达的抑制

Suppression of SMYD3 expression in HeLa cells by an inducible shRNAs expression system

目的:探讨可诱导的shRNAs表达载体介导的RNA干扰技术对HeLa细胞SMYD3基因的抑制作用。方法:设计和构建由强力霉素调控产生针对SMYD3基因的shRNAs表达质粒,转染至HeLa细胞,倒置显微镜下观察细胞形态学变化,RT-PCR检测SMYD3基因的表达,MTT法检测对细胞增殖的影响。结果:酶切鉴定、测序分析证实shRNAs表达质粒构建成功。转染HeLa细胞后,在强力霉素诱导下,受到特异SMYD3 shRNAs干扰的HeLa细胞发生了典型的凋亡形态学改变。同时SMYD3 mRNA表达下调,细胞生长受抑制。而阴性对照组和空白对照组无明显变化。结论:针对SMYD3基因的可诱导的shRNAs表达质粒转染HeLa细胞后,在强力霉素的作用下,可阻断SMYD3基因的表达,抑制HeLa细胞的增殖,并能诱导HeLa细胞凋亡。

Aim：To suppress SMYD3 expression in HeLa cells using an inducible shRNAs expression plasmids mediated RNA interference. Methods： Doxycycline controlled inducible shRNAs expression plasmids for SMYD3 silencing were designed, constructed and transfected to HeLa cells. The cellular morphological changes were then observed under an inverted phase-contrast microscope. The transcription of SMYD3 in the transfected cells was examined by RTPCR. MTF assay was used to evaluate the effects of suppressed SMYD3 on cellular proliferation. Results： Enzyme digestion and sequencing analyses revealed that the inducible shRNAs expression plasmids were constructed successfully. After being transfected to HeLa cells and induced by doxycycline, most of the HeLa cells displayed typical morphological features of apoptosis. SMYD3 mRNAs were knocked down and the cellular growth was inhibited. However, there have been no apparent changes in negative and blank control groups. Conclusion：Mter transfection of the inducible short hairpin RNA expression plasmids targeting for SMYD3, HeLa cells proliferation was inhibited. The expression levels of SMYD3 mRNA were decreased and apoptosis was induced.