摘要
目的探究Spink3在小鼠肝癌组织中的表达调控机制。方法采用RT-qPCR方法检测SPINK1在人肝癌及Spink3在H-ras12V转基因肝癌中的mRNA表达水平。采用Western blot和RT-q PCR法检测原代培养小鼠肝癌组织和肝癌旁组织中的ERK和mTOR信号通路的变化及Spink3的mRNA表达水平。体内抑制ERK和mTOR信号分子活性,检测其对Spink3 mRNA表达水平的影响。结果与肝癌旁组织相比,SPINK1和Spink3的mRNA水平分别在人肝癌组织和小鼠肝癌组织中显著升高(P<0.05)。在原代培养的小鼠肝癌和肝癌旁组织中,与0 h相比,随着体外培养时间的延长(6、12、24、48 h),p-ERK和p-mTOR信号分子水平明显下降;与此相一致,Spink3的mRNA的表达水平也显著降低(P<0.01)。体内抑制ERK信号分子的活性,显著下调了Spink3的mRNA表达水平(P<0.05)。但体内抑制mTOR信号分子的活性,对Spink3的mRNA表达没有显著影响。结论 SPINK1/Spink3在肝癌组织中过表达,有望成为肝癌的临床诊断指标。ERK通路可能是上调Spink3表达的主要信号通路。
Objective To investigate the molecular mechanisms of the regulation to Spink3 expression in mouse hepatic tumors. Method The mRNA levels of SPINK1 in human hepatocellular carcinoma and Spink3 in hepatic tumors of H-rasl2V transgenic mice were detected by RT-qPCR. The protein levels of p-ERK and p-mTOR and mRNA levels of Spink3 in primary cultured hepatic tumor tissues and tumor-adjacent normal tissues of H-rasl2V transgenic mice were detected by Western blot and RT-qPCR. In vivo inhibition of the activities of p-ERK and p-mTOR was performed to investigate their influence on the mRNA levels of Spink3. Result Compared with tumor-adjacent normal tissues, the mRNA levels of SPINK1 and Spink3 were significantly higher expressed in hepatic tumor tissues （P 〈 0.05）. In the primary cultured tumor and normal liver tissues of transgenic mice, along with the cultured time （6, 12. 24. 48 h）, the protein levels of p-ERK and p-mTOR are significantly decreased compared to the 0 h. Consistently, the mRNA levels of Spink3 were also significantly decreased （P 〈 0.01 ）. Inhibition of the activities of p-ERK significantly reduced the mRNA levels of Spink3 （P 〈 0.05 ）. However, Inhibition of the activities of p-mTOR had no influence on the mRNA levels of Spink3. Conclusion SPINK1 and Spink3 are significantly higher expressed in hepatic tumor tissues and could be used as potential biomarkers for clinical diagnosis. The ERK pathway may play important roles in up-regulating the expression of Spink3.
出处
《实验动物科学》
2017年第2期1-6,共6页
Laboratory Animal Science
基金
国家自然科学基金(No.30872950)
