SPINK1通过EGFR通路促进前列腺癌细胞上皮间质转化的研究
发布时间：2018-08-17 19:18
【摘要】：最近有关西方国家前列腺癌的研究证实,SPINK1过表达为ETS基因融合阴性的前列腺癌病例中侵袭性较强的一种分子亚型,然而SPINK1对前列腺癌肿瘤细胞侵袭转移发挥作用的具体机制尚未明确。我们收集了224例前列腺癌病例,证实在中国前列腺癌患者中SPINK1过表达与不良预后具有显著的相关性(P=0.025),且与EGFR具有共同过表达的特征(P=0.003)。本研究中的体外实验证实SPINK1可促进前列腺癌细胞发生上皮间质转化(epithelial-mesenchymal transition, EMT),从而增强前列腺癌细胞的侵袭转移能力。进一步的研究证实SPINK1主要通过EGFR/MEK/ERK通路发挥促进EMT的作用,CTGF可能为该通路下游的关键作用分子。实验结果为SPINK1在中国前列腺癌中作为诊断指标及治疗靶点提供了更深入的实验及理论依据。 第一部分SPINK1在前列腺癌组织中的表达、临床病理学特征及与预后的关系 目的： 1.研究中国前列腺癌组织中SPINK1过表达的频度及其与患者不良预后的相关性。 2.探究在中国前列腺癌组织中SPINK1过表达与其他分子指标的相关性。 结果： 1.中国前列腺癌中SPINK1过表达的发生频率为7.6%,与患者不良预后相关。 2. SPINK1过表达与TMPRSS2-ERG基因融合存在排他性。 3. EGFR过表达的发生率在SPINK1阳性的前列腺癌病例中比在SPINK1阴性的前列腺癌病例中高。 4. SPINK1阳性病例中E-Cadherin表达相对较低,vimentin表达相对较高。 结论： 在中国前列腺癌病例中SPINK1过表达的发生频率为7.6%,与西方国家接近。与关于SPINK1在西方国家的相关报道一致,SPINK1过表达与TMPRSS2-ERG基因融合呈现排他性存在,SPINK过表达与不良预后具有显著相关性。另外,我们首次证实在前列腺癌组织中SPINK1与EGFR存在共过表达的趋势。SPINK1阳性病例中E-Cadherin表达相对较低,vimentin表达相对较高,暗示SPINK1可能与前列腺癌细胞发生上皮间质转化过程有关。 第二部分SPINK1促进前列腺癌细胞上皮间质转化 目的： 1.明确SPINK1是否可诱导前列腺癌细胞由上皮细胞表型向间质细胞表型转变。 2.探究SPINK1是否可引起前列腺癌细胞上皮指标及间质指标蛋白表达量的改变。 3.验证SPINK1对前列腺癌细胞侵袭迁移能力的影响。 结果： 1. rSPINK1持续刺激6天后,RWPE、LnCap及VCap细胞中均含有一定比例的细胞变为长梭形,细胞失去极性,细胞分布较松散。 2.western blot及免疫荧光技术均证明rSPINK1可引起RWPE细胞中E-Cadherin表达量增多、vimentin表达量减少,而转染SPINK1SiRNA则可引起22RV1细胞中E-Cadherin表达量上调、vimentin表达量下调。 3.细胞划痕实验及Transwell实验证实rSPINK1可促进RWPE细胞迁移侵袭能力增强,SPINK1SiRNA可抑制22RV1细胞侵袭转移。 结论： 我们首次证实SPINK1在体外可促进前列腺癌细胞发生EMT。 第三部分SPINK1促进上皮间质转化的机制研究 目的： 1.明确SPINK1是否通过EGFR通路促进EMT及具体通过哪条下游通路促进EMT。 2.明确SPINK1通过EGFR通路调控的促进EMT的下游关键作用分子。 结果： 1. rSPINKl作用于RWPE细胞,可使其p-EGFR、p-STAT3、p-Akt、p-ERK水平升高。转染SPINK1SiRNA的22RV1细胞,相对于其对照组,EGFR、STAT3、 Akt、ERK磷酸化水平均显著降低。 2.AG1478和U0126可几乎完全阻断rSPINKl对EMT分子指标的调节作用。而LY294002和AG490只可较低程度地抑制rSPINKl对EMT分子指标的调节作用。 3.细胞划痕实验结果示AG1478、U0126、LY294002、AG490均可完全阻断rSPINK对细胞迁移能力的提高作用。侵袭实验结果示只有AG1478、U0126可阻断rSPINK对细胞侵袭的促进作用。 4. rSPINK1处理RWPE细胞,SPINK1SiRNA转染22RV1细胞,snail slug、twist、 β-catenin、zeb1的mRNA表达量及核内蛋白表达量均无明显变化。 5. rSPINKl处理RWPE细胞,CTGF表达量升高；SPINK1SiRNA转染22RV1细胞,CTGF表达量下降。 6. CTGF SiRNA转染RWPE细胞可部分逆转rSPINKl促进EMT的作用。 7.AG1478和U0126可阻断rSPINKl对CTGF的上调作用,而LY294002和AG490无此作用。 结论： 1.SPINK1主要通过EGFR/MEK/ERK通路发挥促进EMT的作用。 2.CTGF可能为SPINK1/EGFR/MEK/ERK促进EMT的下游关键作用分子。
[Abstract]:Recent studies on prostate cancer in western countries have confirmed that SPINK1 overexpression is a more aggressive molecular subtype in prostate cancer patients with negative ETS fusion. However, the specific mechanisms underlying the role of SPINK1 in the invasion and metastasis of prostate cancer cells remain unclear. The overexpression of SPINK1 was significantly associated with poor prognosis (P = 0.025) and co-overexpression with EGFR (P = 0.003). In vitro experiments in this study confirmed that SPINK1 could promote epithelial-mesenchymal transition (EMT) of prostate cancer cells, thereby enhancing prostate cancer cells. Further studies have confirmed that SPINK1 promotes EMT mainly through the EGFR/MEK/ERK pathway, and CTGF may be a key downstream molecule of this pathway. The results provide further experimental and theoretical basis for SPINK1 as a diagnostic indicator and therapeutic target for prostate cancer in China.
Part I SPINK1 expression in prostate cancer, its clinicopathological features and prognosis
Objective:
1. to study the frequency of SPINK1 overexpression in Chinese prostate cancer and its correlation with poor prognosis.
2. to explore the correlation between SPINK1 overexpression and other molecular markers in Chinese prostate cancer tissues.
Result:
1. the frequency of SPINK1 overexpression in prostate cancer is 7.6%, which is associated with poor prognosis.
2. SPINK1 overexpression and TMPRSS2-ERG gene fusion exist exclusiveness.
3. The incidence of EGFR overexpression in SPINK1-positive prostate cancer was higher than that in SPINK1-negative prostate cancer.
4. in SPINK1 positive cases, E-Cadherin expression was relatively low and vimentin expression was relatively high.
Conclusion:
The frequency of overexpression of SPINK1 was 7.6% in Chinese prostate cancer patients, which was similar to that in Western countries. The overexpression of SPINK1 was exclusive with the fusion of TMPRSS2-ERG gene. The overexpression of SPINK1 was significantly associated with poor prognosis in prostate cancer patients for the first time. In SPINK1 positive cases, E-Cadherin expression was relatively low and vimentin expression was relatively high, suggesting that SPINK1 may be involved in the epithelial-mesenchymal transition of prostate cancer cells.
The second part of SPINK1 promotes epithelial mesenchymal transition in prostate cancer cells.
Objective:
1. whether SPINK1 can induce prostate cancer cells to change from epithelial phenotype to mesenchymal phenotype.
2. to explore whether SPINK1 can cause changes in the expression of epithelial markers and interstitial protein in prostate cancer cells.
3. verify the effect of SPINK1 on invasion and migration of prostate cancer cells.
Result:
1. After 6 days of continuous stimulation with rSPINK1, a certain proportion of cells in RWPE, LnCap and VCap cells became spindle-shaped. The cells lost polarity and distributed loosely.
2. Western blot and immunofluorescence assay showed that rSPINK1 could increase the expression of E-Cadherin and decrease the expression of vimentin in RWPE cells, while transfection of SPINK1 SiRNA could increase the expression of E-Cadherin and decrease the expression of vimentin in 22RV1 cells.
3. Scratch test and Transwell test showed that rSPINK1 could promote the migration and invasion of RWPE cells, and SPINK1SiRNA could inhibit the invasion and metastasis of 22RV1 cells.
Conclusion:
We confirmed for the first time that SPINK1 could promote EMT. in prostate cancer cells in vitro.
The third part is about the mechanism of SPINK1 promoting epithelial mesenchymal transition.
Objective:
1. clear whether SPINK1 promotes EMT through the EGFR pathway, and which downstream channel promotes EMT..
2. identify the key downstream molecules that promote SPINK1 through the EGFR pathway, which promotes EMT.
Result:
1. The phosphorylation levels of p-EGFR, p-STAT3, p-Akt and p-ERK in RWPE cells transfected with rSPINK1 SiRNA were significantly lower than those in the control group.
2. AG1478 and U0126 almost completely blocked the regulation of rSPINKl on EMT markers, while LY29400 2 and AG490 only inhibited the regulation of rSPINKl on EMT markers to a lesser extent.
3. Scratch test showed that AG1478, U0126, LY294002 and AG490 could completely block the enhancement of cell migration by rSPINK. Invasion test showed that only AG1478 and U0126 could block the promotion of rSPINK on cell invasion.
4. The expression of snail slug, twist, beta catenin, ZEB1 mRNA and nuclear protein in RWPE cells treated with rSPINK1 and transfected with SPINK1 SiRNA did not change significantly.
5. rSPINKl treatment of RWPE cells, CTGF expression increased; SPINK1SiRNA transfected 22RV1 cells, CTGF expression decreased.
The transfection of RWPE cells by 6. CTGF SiRNA partially reversed the role of rSPINKl in promoting EMT.
7.AG1478 and U0126 could block the up regulation effect of rSPINKl on CTGF, but LY294002 and AG490 had no effect.
Conclusion:
1.SPINK1 mainly promotes the role of EMT through the EGFR/MEK/ERK pathway.
2.CTGF may promote the downstream key molecules of EMT for SPINK1/EGFR/MEK/ERK.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2188631
[Abstract]:Recent studies on prostate cancer in western countries have confirmed that SPINK1 overexpression is a more aggressive molecular subtype in prostate cancer patients with negative ETS fusion. However, the specific mechanisms underlying the role of SPINK1 in the invasion and metastasis of prostate cancer cells remain unclear. The overexpression of SPINK1 was significantly associated with poor prognosis (P = 0.025) and co-overexpression with EGFR (P = 0.003). In vitro experiments in this study confirmed that SPINK1 could promote epithelial-mesenchymal transition (EMT) of prostate cancer cells, thereby enhancing prostate cancer cells. Further studies have confirmed that SPINK1 promotes EMT mainly through the EGFR/MEK/ERK pathway, and CTGF may be a key downstream molecule of this pathway. The results provide further experimental and theoretical basis for SPINK1 as a diagnostic indicator and therapeutic target for prostate cancer in China.
Part I SPINK1 expression in prostate cancer, its clinicopathological features and prognosis
Objective:
1. to study the frequency of SPINK1 overexpression in Chinese prostate cancer and its correlation with poor prognosis.
2. to explore the correlation between SPINK1 overexpression and other molecular markers in Chinese prostate cancer tissues.
Result:
1. the frequency of SPINK1 overexpression in prostate cancer is 7.6%, which is associated with poor prognosis.
2. SPINK1 overexpression and TMPRSS2-ERG gene fusion exist exclusiveness.
3. The incidence of EGFR overexpression in SPINK1-positive prostate cancer was higher than that in SPINK1-negative prostate cancer.
4. in SPINK1 positive cases, E-Cadherin expression was relatively low and vimentin expression was relatively high.
Conclusion:
The frequency of overexpression of SPINK1 was 7.6% in Chinese prostate cancer patients, which was similar to that in Western countries. The overexpression of SPINK1 was exclusive with the fusion of TMPRSS2-ERG gene. The overexpression of SPINK1 was significantly associated with poor prognosis in prostate cancer patients for the first time. In SPINK1 positive cases, E-Cadherin expression was relatively low and vimentin expression was relatively high, suggesting that SPINK1 may be involved in the epithelial-mesenchymal transition of prostate cancer cells.
The second part of SPINK1 promotes epithelial mesenchymal transition in prostate cancer cells.
Objective:
1. whether SPINK1 can induce prostate cancer cells to change from epithelial phenotype to mesenchymal phenotype.
2. to explore whether SPINK1 can cause changes in the expression of epithelial markers and interstitial protein in prostate cancer cells.
3. verify the effect of SPINK1 on invasion and migration of prostate cancer cells.
Result:
1. After 6 days of continuous stimulation with rSPINK1, a certain proportion of cells in RWPE, LnCap and VCap cells became spindle-shaped. The cells lost polarity and distributed loosely.
2. Western blot and immunofluorescence assay showed that rSPINK1 could increase the expression of E-Cadherin and decrease the expression of vimentin in RWPE cells, while transfection of SPINK1 SiRNA could increase the expression of E-Cadherin and decrease the expression of vimentin in 22RV1 cells.
3. Scratch test and Transwell test showed that rSPINK1 could promote the migration and invasion of RWPE cells, and SPINK1SiRNA could inhibit the invasion and metastasis of 22RV1 cells.
Conclusion:
We confirmed for the first time that SPINK1 could promote EMT. in prostate cancer cells in vitro.
The third part is about the mechanism of SPINK1 promoting epithelial mesenchymal transition.
Objective:
1. clear whether SPINK1 promotes EMT through the EGFR pathway, and which downstream channel promotes EMT..
2. identify the key downstream molecules that promote SPINK1 through the EGFR pathway, which promotes EMT.
Result:
1. The phosphorylation levels of p-EGFR, p-STAT3, p-Akt and p-ERK in RWPE cells transfected with rSPINK1 SiRNA were significantly lower than those in the control group.
2. AG1478 and U0126 almost completely blocked the regulation of rSPINKl on EMT markers, while LY29400 2 and AG490 only inhibited the regulation of rSPINKl on EMT markers to a lesser extent.
3. Scratch test showed that AG1478, U0126, LY294002 and AG490 could completely block the enhancement of cell migration by rSPINK. Invasion test showed that only AG1478 and U0126 could block the promotion of rSPINK on cell invasion.
4. The expression of snail slug, twist, beta catenin, ZEB1 mRNA and nuclear protein in RWPE cells treated with rSPINK1 and transfected with SPINK1 SiRNA did not change significantly.
5. rSPINKl treatment of RWPE cells, CTGF expression increased; SPINK1SiRNA transfected 22RV1 cells, CTGF expression decreased.
The transfection of RWPE cells by 6. CTGF SiRNA partially reversed the role of rSPINKl in promoting EMT.
7.AG1478 and U0126 could block the up regulation effect of rSPINKl on CTGF, but LY294002 and AG490 had no effect.
Conclusion:
1.SPINK1 mainly promotes the role of EMT through the EGFR/MEK/ERK pathway.
2.CTGF may promote the downstream key molecules of EMT for SPINK1/EGFR/MEK/ERK.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 戚美;杨晓庆;王林;张娟;王妍;韩博;;ETS基因重排在前列腺癌中的特征分析[J];山东大学学报(医学版);2013年06期
，本文编号：2188631
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