目的构建携带信号传导和转录激活子3(STAT3)基因短发夹状干扰RNA(shRNA)的真核表达载体,观察干扰STAT3表达对大肠癌细胞HT-29细胞增殖的影响。方法从GenBank STAT3mRNA上寻找到3条符合特征的靶序列,合成靶序列的DNA寡核苷酸链,合成双链DNA,并和BamHⅠ和HindⅢ酶切后的pRNAT-U6.1/Neo载体质粒连接产生重组质粒,行PCR和测序鉴定。重组质粒瞬时转染大肠癌HT-29细胞,实时定量PCR、Western blot检测STAT3的表达。筛选出最佳重组质粒,转染大肠癌细胞后,MTT检测细胞的增殖情况,流式细胞仪检测细胞周期变化。结果成功构建了携带有STAT3基因的shRNA的重组质粒,PCR和测序证实重组质粒构建正确。3种重组质粒对大肠癌HT-29细胞STAT3的表达有明显的抑制作用。筛选出的最佳重组质粒转染大肠癌细胞后,细胞增殖能力明显减弱;细胞周期分析显示,G0/G1期细胞占(74.80±1.85)%,S期细胞占(15.72±2.26)%,与对照组差异显著(P<0.01)。结论干扰STAT3基因的表达可以有效抑制大肠癌HT-29细胞的生长。 Objective To construct an eukaryotic expression vector carrying short hairpin RNA (shRNA) of STAT3 gene and observe its effect on the proliferation of HT-29 cells. Methods Three target sequences were found from GenBank STAT3 mRNA. The DNA oligonucleotide of the target sequence was synthesized and double-stranded DNA was synthesized. The double-stranded DNA was ligated with plasmid pRNAT-U6.1 / Neo vector digested with BamHⅠ and HindⅢ Produce recombinant plasmids, PCR and sequencing identification. The recombinant plasmids were transiently transfected into HT-29 cells. Real-time quantitative PCR and Western blot were used to detect STAT3 expression. The best recombinant plasmid was screened and transfected into colorectal cancer cells. MTT assay was used to detect the proliferation of cells. Flow cytometry was used to detect cell cycle changes. Results The recombinant plasmid carrying shRNA of STAT3 gene was successfully constructed. PCR and sequencing confirmed that the recombinant plasmid was constructed correctly. The three recombinant plasmids could obviously inhibit the STAT3 expression in HT-29 cells. The results of cell cycle analysis showed that cells in G0 / G1 phase accounted for (74.80 ± 1.85)% and cells in S phase accounted for (15.72 ± 2.26)%, which was significantly higher than that in cells transfected with the optimal recombinant plasmid The difference between the control group was significant (P <0.01). Conclusion Interfere with STAT3 gene expression can effectively inhibit the growth of HT-29 cells.