miR-181a的表达载体构建及其在胰腺癌细胞中抑制作用的初步探讨
摘要
背景:胰腺癌是发病率高、预后差、死亡率高的恶性肿瘤,它发生转移早,手术切除困难,对放化疗不敏感。因此,寻找新的肿瘤标记和基因治疗方法有十分重要的意义。miRNA是一种长度为21—25bp的单链非编码RNA,不编码蛋白质,功能为负调控转录后水平的基因表达,进而调节细胞的代谢、增殖、分化和凋亡等过程。K-RAS是一原癌基因,在胰腺癌中90%以上的病例中有突变,已有证据表明在肺癌中受miRNA作用,如let-7。近年的研究发现胰腺癌中miRNA表达谱与慢性胰腺炎和正常胰腺中有很大差异,并确定了某些miRNA分子在胰腺癌中的靶基因和具体作用。
目标:构建重组MiR-181a载体,表达miR-181a,并进一步研究MiR-181a对胰腺癌细胞的生长、凋亡、迁移作用的影响及其对癌基因K-RAS的作用。
方法:1、以pcDNA4.0为载体骨架,用限制性内切酶和PCR方法构建pcDNA4.0-EGFP载体和pcDNA4.0-EGFP-MiR-181a载体,作为标记空载体和表达MiR-181a载体。
2、转染胰腺癌细胞系panc-1和mia paca-2,检测处理组和非处理组生长、凋亡、迁移情况和K-RAS蛋白的表达。
结果:1、转染重组MiR-181a载体后可降低panc-1细胞系中K-RAS蛋白表达;
2、转染重组MiR-181a载体减缓panc-1和mia paca-2细胞生长;
3、转染重组MiR-181a载体增加panc-1和mia paca-2细胞凋亡;
4、转染重组MiR-181a载体降低panc-1和mia paca-2细胞迁移能力;
结论:重组MiR-181a载体对胰腺癌细胞系有生长抑制、增加凋亡、减少迁移的作用,并降低癌基因K-RAS蛋白的表达。
BACKGROUND:Pancreatic cancer is a lethal disease with poor survival and high mortality,and up to now no effective therapeutic approaches have been found in blame of hardly early diagnosis,early metastasis,and resistance to chemotherapy and radiation. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs(miRNAs) are small noncoding RNAs form 19 to 25 nucleotides,which negatively regulate post-transcriptional gene expression.Disturbance of microRNA expression may play a role in the metabolism,proliferation,differentiation,apoptosis of cells.K-RAS is an oncogene expressed nearly 100%in pancreatic ductal adenocarcinoma.It is the target gene of some accurancy miRNAs such as let-7 in lung cancers.MicroRNA expression patterns have been made to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis.And the target gene of certain aberrantly expressed miRNAs and the role of certain miRNAs in pancreatic cancer cell has been identified.
Objective:
Construct the recombinant MiR-181a vector to express miR-181 a in pancreatic cancer cell lines.Transduct the recombinant vector,comfirm whether miR-181a targeting at K-RAS oncogene and its expression has effect in inhibiting pancreatic cancer cell growth.
Methords:
Construct the recombinant vector pcDNA4.0-EGFP-MiR-181a to express miR-181a and vector pcDNA4.0-EGFP as control.
Transfect the recombinant vector in pancreatic cancer cell lines,comfirm whether miR-181a targeting at K-RAS oncogene and its expression has effect in inhibiting pancreatic cancer cell growth.
Result:
Expression of miR-181a has effect in inhibiting the K-RAS protein in pancreatic cancer cells.
Expression of miR-181a has effect in inhibiting pancreatic cancer cell growth in vitro;
Expression of miR-181a has effect in promoting pancreatic cancer cell apoptosis in vitro;
Expression of miR-181a has effect in inhibiting pancreatic cancer cell migration in vitro;
Conclusion:
Expression of miR-181a has effects in inhibiting pancreatic cancer cell growth and inducing cell apoptosis in vitro and inhibits K-RAS expression in pancreatic cancer cell lines.
目标:构建重组MiR-181a载体,表达miR-181a,并进一步研究MiR-181a对胰腺癌细胞的生长、凋亡、迁移作用的影响及其对癌基因K-RAS的作用。
方法:1、以pcDNA4.0为载体骨架,用限制性内切酶和PCR方法构建pcDNA4.0-EGFP载体和pcDNA4.0-EGFP-MiR-181a载体,作为标记空载体和表达MiR-181a载体。
2、转染胰腺癌细胞系panc-1和mia paca-2,检测处理组和非处理组生长、凋亡、迁移情况和K-RAS蛋白的表达。
结果:1、转染重组MiR-181a载体后可降低panc-1细胞系中K-RAS蛋白表达;
2、转染重组MiR-181a载体减缓panc-1和mia paca-2细胞生长;
3、转染重组MiR-181a载体增加panc-1和mia paca-2细胞凋亡;
4、转染重组MiR-181a载体降低panc-1和mia paca-2细胞迁移能力;
结论:重组MiR-181a载体对胰腺癌细胞系有生长抑制、增加凋亡、减少迁移的作用,并降低癌基因K-RAS蛋白的表达。
BACKGROUND:Pancreatic cancer is a lethal disease with poor survival and high mortality,and up to now no effective therapeutic approaches have been found in blame of hardly early diagnosis,early metastasis,and resistance to chemotherapy and radiation. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs(miRNAs) are small noncoding RNAs form 19 to 25 nucleotides,which negatively regulate post-transcriptional gene expression.Disturbance of microRNA expression may play a role in the metabolism,proliferation,differentiation,apoptosis of cells.K-RAS is an oncogene expressed nearly 100%in pancreatic ductal adenocarcinoma.It is the target gene of some accurancy miRNAs such as let-7 in lung cancers.MicroRNA expression patterns have been made to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis.And the target gene of certain aberrantly expressed miRNAs and the role of certain miRNAs in pancreatic cancer cell has been identified.
Objective:
Construct the recombinant MiR-181a vector to express miR-181 a in pancreatic cancer cell lines.Transduct the recombinant vector,comfirm whether miR-181a targeting at K-RAS oncogene and its expression has effect in inhibiting pancreatic cancer cell growth.
Methords:
Construct the recombinant vector pcDNA4.0-EGFP-MiR-181a to express miR-181a and vector pcDNA4.0-EGFP as control.
Transfect the recombinant vector in pancreatic cancer cell lines,comfirm whether miR-181a targeting at K-RAS oncogene and its expression has effect in inhibiting pancreatic cancer cell growth.
Result:
Expression of miR-181a has effect in inhibiting the K-RAS protein in pancreatic cancer cells.
Expression of miR-181a has effect in inhibiting pancreatic cancer cell growth in vitro;
Expression of miR-181a has effect in promoting pancreatic cancer cell apoptosis in vitro;
Expression of miR-181a has effect in inhibiting pancreatic cancer cell migration in vitro;
Conclusion:
Expression of miR-181a has effects in inhibiting pancreatic cancer cell growth and inducing cell apoptosis in vitro and inhibits K-RAS expression in pancreatic cancer cell lines.
引文
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