前列腺癌患者血清microRNA表达特点及miR-21在前列腺癌细胞中的功能研究
摘要
研究目的：
一、microRNA广泛表达于多种肿瘤组织内,血清microRNA的研究报道不多,在前列腺癌中更是罕有报道。本研究根据已有的microRNA差异表达谱筛选在前列腺癌中高表达的microRNA(主要有miR-20a, miR-21, miR-125b, miR-141),在前列腺癌患者血清中检测这些microRNA的表达情况,比较不同microRNA在前列腺癌的不同疾病阶段的表达情况及其与骨转移、预后和治疗有效性的相关性。
二、根据第一部分的研究结果,通过检测血清microRNA的表达情况,发现miR-21在激素抵抗性前列腺癌患血清者中的表达显著高于激素敏感性前列腺癌患者,而且miR-21随着血清PSA的升高而升高,提示miR-21可能对前列腺细胞的生长增殖有一定作用,而抑制细胞内miR-21表达有望抑制前列腺癌细胞的生长,甚至于逆转雄激素非依赖状态。为了探讨其中产生差异的机制,本课题进一步研究miR-21反义寡核苷酸对LNCaP前列腺癌细胞的生长的抑制作用及其内在分子机制。
材料和方法：
一、采用特异性TaqMan探针的实时定量PCR方法检测56例患者(包括局限性前列腺癌20例,激素敏感性骨转移前列腺癌20例,激素抵抗性骨转移前列腺癌10例,良性前列腺增生6例)血清中的miR-20a, miR-21, miR-125b, miR-141, U6 snRNA作为内参。方差检验比较以上4类患者血清中miR-20a, miR-21, miR-125b, miR-141表达差异,探寻诊治方案选择及预后判断的新指标。
二、将特异性miR-21反义寡核苷酸,通过脂质体2000转染LNCaP前列腺癌细胞,观察前列腺癌细胞在转染后的增殖情况,绘制肿瘤细胞增殖曲线；转染48小时后分别提取肿瘤细胞胞浆和胞核内的蛋白,采用western-blotting方法分别检测胞浆、胞核内PTEN、mTOR、AKT、AR、PSA等蛋白的表达情况。比较转染特异性miR-21寡核苷酸对LNCaP前列腺癌细胞增殖的影响和肿瘤细胞内蛋白的影响。
结果：
一、miR-21在激素抵抗性骨转移前列腺癌患血清者中的表达显著高于激素敏感性骨转移前列腺癌患者,p=0.016。分层分析显示,激素敏感性前列腺癌患者中,PSA小于4ng/ml的患者的血清miR-21表达显著低于PSA大于4ng/ml的患者；对多西他赛化疗抵抗的前列腺癌患者的血清miR-21表达明显高于对多西他赛化疗敏感的患者,p=0.032。而miR-20a和miR-125b在4组患者中未显示存在差异表达。miR-141在骨转移前列腺癌患者血清中的表达显著高于无骨转移的前列腺癌和良性前列腺增生患者,p=0.006；并且miR-141的表达水平与骨转移灶个数呈正相关性,p<0.001。血清miR-21和miR141与前列腺癌Gleason评分呈正相关,Gleason评分>7分的患者的血清microRNA表达水平显著高于Gleason评分<=7分的患者,p值分别为0.001和<0.001。
二、细胞生长曲线显示,在特异性miR-21反义寡核苷酸转染后,前列腺癌LNCaP细胞的增殖受到明显抑制。转染后,细胞核内的雄激素受体(AR)蛋白表达水平明显下调,细胞浆中无AR蛋白表达；胞浆内的前列腺特异抗原(PSA)蛋白的表达也明显下降,细胞核中无PSA蛋白的表达。此外,在转染后,细胞核内的AKT蛋白表达水平明显下调,而细胞浆中的AKT蛋白表达变化不明显；mTOR蛋白在细胞核内的表达也明显下降,但细胞核中的mTOR蛋白的表达反而升高。
结论：
在前列腺癌患者血清中检测microRNA是可行的,miR-21高表达于雄激素非依赖的骨转移前列腺癌患者,并对多西他赛化疗有效性具有预测价值；miR-141高表达于骨转移前列腺癌患者。miR-21可以作为雄激素非依赖前列腺癌的分子标记物,而miR-141可作为骨转移性前列腺癌的分子标记物。
抑制LNCaP前列腺癌细胞内的miR-21表达对细胞的增殖有明显抑制作用,其内在机制可能是miR-21有上调AKT表达的作用,进而使mTOR发生从胞浆到胞核的转运重分布；同时miR-21还可能具有上调AR的作用。而抑制细胞内的miR-21表达,胞核内的AR、mTOR和AKT蛋白的表达均下降,最终导致细胞增殖的抑制。因此,miR-21是一个潜在的基因治疗新靶点。
Purpose:
(1) McroRNAs are widely expressed in various tumor tissues, nevertheless serum microRNA is seldom reported, especially in prostate cancer. According to the existing expression profile of microRNA, miR-20a, miR-21, miR-125b, and miR-141 have been documented to overexpress in prostate cancer. We designed to detect these four microRNAs in the serum of patients with prostate cancer, and to compare different expression pattern in different stage of prostate cancer, to achieve an overview of the expression profile of these microRNAs and validate their role in predicting treatment efficacy and prognosis of patients.
(2) According to the findings in the first part of our study, the levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than those in hormone-sensitive prostate cancer patients, and also correlating to serum PSA levels, which indicated that miR-21 might play an important role in the proliferation of prostate cancer cells. Inhibiting the expression of miR-21 might slow down the growth of tumor cells, even reversing the androgen-independent status. In order to investigate the intrinsic molecular mechanism, we further studied the effect of specific miR-21 antisense oligonucleotides on the growth of LNCap prostate cancer cells. MiR-21 is a potentially novel target of gene therapy.
Materials and methods:
(1) Specific TaqMan probes for real-time quantitative PCR were employed to detect serum miR-20a, miR-21, miR-125b, and miR-141 in 56 patients (including 20 cases of localized prostate cancer,20 cases of hormone sensitive prostate cancer with bone metastasis,10 cases of hormone refractory prostate cancer with bone metastasis, and 6 patients of benign prostatic hyperplasia). U6 snRNA was used as an inner standard. One-Way ANOVA test was applied to compare the differences of serum miR-20a, miR-21, miR-125b, miR-141 in the above 4 groups of patients, to find out the novel predictors for the diagnosis, treatment and prognosis of patients with prostate cancer.
(2) The specific miR-21 antisense oligonucleotides were transfected to LNCaP prostate cancer cells through Lipofectamine 2000. Growth curves were drawn to observe effect of miR-21 antisense oligonucleotides on the proliferation of tumor cells. Forty-eight hours after transfection, proteins were extracted from both the cytoplasm and nucleus of tumor cells. Protein levels of PTEN, mTOR, AKT, AR, and PSA were detected by western-blotting method in the cytoplasm and nuclear proteins, seperately.
Results:
(1) The levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than in hormone-sensitive prostate cancer patients, p =0.016. Stratified analysis in patients with hormone sensitive prostate cancer showed that levels of serum miR-21 were lower in patients with PSA levels of less than 4ng/ml than in those with PSA levels of more than 4ng/ml. Furthermore, Serum miR-21 levels were higher in patients who were resistant to docetaxel-based chemotherapy when compared to those sensitive to chemotherapy, p=0.032. The expressions of serum miR-20a and miR-125b showed no statistical differences between the 4 groups of patients. Serum miR-141 expressed higher in patients with bone metastases than in those with localized prostate cancer and those with benign prostatic hyperplasia, p=0.006, and its level even correlated to the number of bone lesions, p<0.001. What's more, serum miR-21 and miR-141 were also found to be positively related to Gleason Score, and patients with higher Gleason Score had higher serum miR-21 and miR-141 levels, p=0.001and p<0.001, respectively.
(2) The cell growth curve showed that the specific miR-21 antisense oligonucleotides could inhibit the growth of LNCaP prostate cancer cells. After transfection, the AR in nucleus was down regulated, which induced the reduction of cytoplasm PSA expression. Furthermore, the nuclear AKT level also decreased after the transfection of specific miR-21 antisense oligonucleotides, which might cause the mTOR protein traslocating from nucleus to cytoplasm.
Conclusion:
(1) It is feasible to detect MicroRNAs in the serum of prostate cancer patients. Serum miR-21 was highly expressed in hormone refractory prostate cancer patients with bone metastases, and it might have predictive value for the efficacy of docetaxel-based chemotherapy. Serum miR-141 overexpressed in patients with bone metastasis. Thus, serum miR-21 can be a potential predictor for hormone-refractory prostate cancer, and serum miR-141 be used as a biomarker for bone metastasis.
(2) The proliferation of LNCaP prostate cancer cells can be significantly inhibited by down regulating the expression of miR-21 in tumor cells. MiR-21 may have the function of up-regulating AR and AKT in prostate cancer cells, and the expression of mTOR protein might traslocate from the cytoplasm to the nucleus. Therefore, after inhibition of miR-21, expressions AR, mTOR and AKT proteins in the nucleus decreased, leading to the restriction of cell proliferation.
一、microRNA广泛表达于多种肿瘤组织内,血清microRNA的研究报道不多,在前列腺癌中更是罕有报道。本研究根据已有的microRNA差异表达谱筛选在前列腺癌中高表达的microRNA(主要有miR-20a, miR-21, miR-125b, miR-141),在前列腺癌患者血清中检测这些microRNA的表达情况,比较不同microRNA在前列腺癌的不同疾病阶段的表达情况及其与骨转移、预后和治疗有效性的相关性。
二、根据第一部分的研究结果,通过检测血清microRNA的表达情况,发现miR-21在激素抵抗性前列腺癌患血清者中的表达显著高于激素敏感性前列腺癌患者,而且miR-21随着血清PSA的升高而升高,提示miR-21可能对前列腺细胞的生长增殖有一定作用,而抑制细胞内miR-21表达有望抑制前列腺癌细胞的生长,甚至于逆转雄激素非依赖状态。为了探讨其中产生差异的机制,本课题进一步研究miR-21反义寡核苷酸对LNCaP前列腺癌细胞的生长的抑制作用及其内在分子机制。
材料和方法：
一、采用特异性TaqMan探针的实时定量PCR方法检测56例患者(包括局限性前列腺癌20例,激素敏感性骨转移前列腺癌20例,激素抵抗性骨转移前列腺癌10例,良性前列腺增生6例)血清中的miR-20a, miR-21, miR-125b, miR-141, U6 snRNA作为内参。方差检验比较以上4类患者血清中miR-20a, miR-21, miR-125b, miR-141表达差异,探寻诊治方案选择及预后判断的新指标。
二、将特异性miR-21反义寡核苷酸,通过脂质体2000转染LNCaP前列腺癌细胞,观察前列腺癌细胞在转染后的增殖情况,绘制肿瘤细胞增殖曲线；转染48小时后分别提取肿瘤细胞胞浆和胞核内的蛋白,采用western-blotting方法分别检测胞浆、胞核内PTEN、mTOR、AKT、AR、PSA等蛋白的表达情况。比较转染特异性miR-21寡核苷酸对LNCaP前列腺癌细胞增殖的影响和肿瘤细胞内蛋白的影响。
结果：
一、miR-21在激素抵抗性骨转移前列腺癌患血清者中的表达显著高于激素敏感性骨转移前列腺癌患者,p=0.016。分层分析显示,激素敏感性前列腺癌患者中,PSA小于4ng/ml的患者的血清miR-21表达显著低于PSA大于4ng/ml的患者；对多西他赛化疗抵抗的前列腺癌患者的血清miR-21表达明显高于对多西他赛化疗敏感的患者,p=0.032。而miR-20a和miR-125b在4组患者中未显示存在差异表达。miR-141在骨转移前列腺癌患者血清中的表达显著高于无骨转移的前列腺癌和良性前列腺增生患者,p=0.006；并且miR-141的表达水平与骨转移灶个数呈正相关性,p<0.001。血清miR-21和miR141与前列腺癌Gleason评分呈正相关,Gleason评分>7分的患者的血清microRNA表达水平显著高于Gleason评分<=7分的患者,p值分别为0.001和<0.001。
二、细胞生长曲线显示,在特异性miR-21反义寡核苷酸转染后,前列腺癌LNCaP细胞的增殖受到明显抑制。转染后,细胞核内的雄激素受体(AR)蛋白表达水平明显下调,细胞浆中无AR蛋白表达；胞浆内的前列腺特异抗原(PSA)蛋白的表达也明显下降,细胞核中无PSA蛋白的表达。此外,在转染后,细胞核内的AKT蛋白表达水平明显下调,而细胞浆中的AKT蛋白表达变化不明显；mTOR蛋白在细胞核内的表达也明显下降,但细胞核中的mTOR蛋白的表达反而升高。
结论：
在前列腺癌患者血清中检测microRNA是可行的,miR-21高表达于雄激素非依赖的骨转移前列腺癌患者,并对多西他赛化疗有效性具有预测价值；miR-141高表达于骨转移前列腺癌患者。miR-21可以作为雄激素非依赖前列腺癌的分子标记物,而miR-141可作为骨转移性前列腺癌的分子标记物。
抑制LNCaP前列腺癌细胞内的miR-21表达对细胞的增殖有明显抑制作用,其内在机制可能是miR-21有上调AKT表达的作用,进而使mTOR发生从胞浆到胞核的转运重分布；同时miR-21还可能具有上调AR的作用。而抑制细胞内的miR-21表达,胞核内的AR、mTOR和AKT蛋白的表达均下降,最终导致细胞增殖的抑制。因此,miR-21是一个潜在的基因治疗新靶点。
Purpose:
(1) McroRNAs are widely expressed in various tumor tissues, nevertheless serum microRNA is seldom reported, especially in prostate cancer. According to the existing expression profile of microRNA, miR-20a, miR-21, miR-125b, and miR-141 have been documented to overexpress in prostate cancer. We designed to detect these four microRNAs in the serum of patients with prostate cancer, and to compare different expression pattern in different stage of prostate cancer, to achieve an overview of the expression profile of these microRNAs and validate their role in predicting treatment efficacy and prognosis of patients.
(2) According to the findings in the first part of our study, the levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than those in hormone-sensitive prostate cancer patients, and also correlating to serum PSA levels, which indicated that miR-21 might play an important role in the proliferation of prostate cancer cells. Inhibiting the expression of miR-21 might slow down the growth of tumor cells, even reversing the androgen-independent status. In order to investigate the intrinsic molecular mechanism, we further studied the effect of specific miR-21 antisense oligonucleotides on the growth of LNCap prostate cancer cells. MiR-21 is a potentially novel target of gene therapy.
Materials and methods:
(1) Specific TaqMan probes for real-time quantitative PCR were employed to detect serum miR-20a, miR-21, miR-125b, and miR-141 in 56 patients (including 20 cases of localized prostate cancer,20 cases of hormone sensitive prostate cancer with bone metastasis,10 cases of hormone refractory prostate cancer with bone metastasis, and 6 patients of benign prostatic hyperplasia). U6 snRNA was used as an inner standard. One-Way ANOVA test was applied to compare the differences of serum miR-20a, miR-21, miR-125b, miR-141 in the above 4 groups of patients, to find out the novel predictors for the diagnosis, treatment and prognosis of patients with prostate cancer.
(2) The specific miR-21 antisense oligonucleotides were transfected to LNCaP prostate cancer cells through Lipofectamine 2000. Growth curves were drawn to observe effect of miR-21 antisense oligonucleotides on the proliferation of tumor cells. Forty-eight hours after transfection, proteins were extracted from both the cytoplasm and nucleus of tumor cells. Protein levels of PTEN, mTOR, AKT, AR, and PSA were detected by western-blotting method in the cytoplasm and nuclear proteins, seperately.
Results:
(1) The levels of serum miR-21 were significantly higher in hormone refractory prostate cancer patients than in hormone-sensitive prostate cancer patients, p =0.016. Stratified analysis in patients with hormone sensitive prostate cancer showed that levels of serum miR-21 were lower in patients with PSA levels of less than 4ng/ml than in those with PSA levels of more than 4ng/ml. Furthermore, Serum miR-21 levels were higher in patients who were resistant to docetaxel-based chemotherapy when compared to those sensitive to chemotherapy, p=0.032. The expressions of serum miR-20a and miR-125b showed no statistical differences between the 4 groups of patients. Serum miR-141 expressed higher in patients with bone metastases than in those with localized prostate cancer and those with benign prostatic hyperplasia, p=0.006, and its level even correlated to the number of bone lesions, p<0.001. What's more, serum miR-21 and miR-141 were also found to be positively related to Gleason Score, and patients with higher Gleason Score had higher serum miR-21 and miR-141 levels, p=0.001and p<0.001, respectively.
(2) The cell growth curve showed that the specific miR-21 antisense oligonucleotides could inhibit the growth of LNCaP prostate cancer cells. After transfection, the AR in nucleus was down regulated, which induced the reduction of cytoplasm PSA expression. Furthermore, the nuclear AKT level also decreased after the transfection of specific miR-21 antisense oligonucleotides, which might cause the mTOR protein traslocating from nucleus to cytoplasm.
Conclusion:
(1) It is feasible to detect MicroRNAs in the serum of prostate cancer patients. Serum miR-21 was highly expressed in hormone refractory prostate cancer patients with bone metastases, and it might have predictive value for the efficacy of docetaxel-based chemotherapy. Serum miR-141 overexpressed in patients with bone metastasis. Thus, serum miR-21 can be a potential predictor for hormone-refractory prostate cancer, and serum miR-141 be used as a biomarker for bone metastasis.
(2) The proliferation of LNCaP prostate cancer cells can be significantly inhibited by down regulating the expression of miR-21 in tumor cells. MiR-21 may have the function of up-regulating AR and AKT in prostate cancer cells, and the expression of mTOR protein might traslocate from the cytoplasm to the nucleus. Therefore, after inhibition of miR-21, expressions AR, mTOR and AKT proteins in the nucleus decreased, leading to the restriction of cell proliferation.
引文
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