摘要
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 eukaryotic expression vectors of RNA interference of STAT3 gene and to investigate the effects of STAT3 expressing interference on proliferation of human colorectal cancer HT-29 cells.Methods Three sequences of siRNA targeting STAT3 gene were found from GenBank.The complementary DNA containing both sene and antisene Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pRNAT-U6.1/Neo vector.The recombinant plasmids were conformed by PCR and the DNA sequence ana-lysis.HT-29 cells were transfected with the recombinant plasmids,and Real time quantitative PCR、Western-blot were used to certify the expression of STAT3 gene.In the transfected group with the best recombinant plasmid,the cell growth was assessed by MTT assay and the cell cycle distribution was detected by flow cytometry.Results PCR and the DNA sequence analysis demonstrated that the eukaryotic expression vectors of RNA interference of STAT3 gene were constructed successfully.STAT3 shRNA could inhibit the expression of STAT3 gene in HT-29 cells.In the transfected group with the best recombinant plasmid,MTT assay showed that the proliferation of HT-29 cells was suppressed and FCM assay indicated the proportion of cells at G0/G1 and S phase was （74.80±1.85）% and （15.72±2.26）% respectively,which was great different from that in the control group（P0.01）.Conclusion STAT3 targeting shRNA can silence the STAT3 gene remarkably and induce HT-29 cell growth inhibition.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2010年第2期252-256,共5页
Suzhou University Journal of Medical Science
基金
苏州市社会发展计划项目(SS08033)
关键词
RNA干扰
SHRNA
STAT3
大肠癌
RNA interfering
short hairin RNA
signal transducers and activators of transcription 3
colorectal cancer
