SPINK1通過EGFR通路促進前列腺癌細胞上皮間質(zhì)轉(zhuǎn)化的研究
發(fā)布時間：2018-08-17 19:18
【摘要】：最近有關(guān)西方國家前列腺癌的研究證實,SPINK1過表達為ETS基因融合陰性的前列腺癌病例中侵襲性較強的一種分子亞型,然而SPINK1對前列腺癌腫瘤細胞侵襲轉(zhuǎn)移發(fā)揮作用的具體機制尚未明確。我們收集了224例前列腺癌病例,證實在中國前列腺癌患者中SPINK1過表達與不良預(yù)后具有顯著的相關(guān)性(P=0.025),且與EGFR具有共同過表達的特征(P=0.003)。本研究中的體外實驗證實SPINK1可促進前列腺癌細胞發(fā)生上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition, EMT),從而增強前列腺癌細胞的侵襲轉(zhuǎn)移能力。進一步的研究證實SPINK1主要通過EGFR/MEK/ERK通路發(fā)揮促進EMT的作用,CTGF可能為該通路下游的關(guān)鍵作用分子。實驗結(jié)果為SPINK1在中國前列腺癌中作為診斷指標及治療靶點提供了更深入的實驗及理論依據(jù)。 第一部分SPINK1在前列腺癌組織中的表達、臨床病理學(xué)特征及與預(yù)后的關(guān)系 目的： 1.研究中國前列腺癌組織中SPINK1過表達的頻度及其與患者不良預(yù)后的相關(guān)性。 2.探究在中國前列腺癌組織中SPINK1過表達與其他分子指標的相關(guān)性。 結(jié)果： 1.中國前列腺癌中SPINK1過表達的發(fā)生頻率為7.6%,與患者不良預(yù)后相關(guān)。 2. SPINK1過表達與TMPRSS2-ERG基因融合存在排他性。 3. EGFR過表達的發(fā)生率在SPINK1陽性的前列腺癌病例中比在SPINK1陰性的前列腺癌病例中高。 4. SPINK1陽性病例中E-Cadherin表達相對較低,vimentin表達相對較高。 結(jié)論： 在中國前列腺癌病例中SPINK1過表達的發(fā)生頻率為7.6%,與西方國家接近。與關(guān)于SPINK1在西方國家的相關(guān)報道一致,SPINK1過表達與TMPRSS2-ERG基因融合呈現(xiàn)排他性存在,SPINK過表達與不良預(yù)后具有顯著相關(guān)性。另外,我們首次證實在前列腺癌組織中SPINK1與EGFR存在共過表達的趨勢。SPINK1陽性病例中E-Cadherin表達相對較低,vimentin表達相對較高,暗示SPINK1可能與前列腺癌細胞發(fā)生上皮間質(zhì)轉(zhuǎn)化過程有關(guān)。 第二部分SPINK1促進前列腺癌細胞上皮間質(zhì)轉(zhuǎn)化 目的： 1.明確SPINK1是否可誘導(dǎo)前列腺癌細胞由上皮細胞表型向間質(zhì)細胞表型轉(zhuǎn)變。 2.探究SPINK1是否可引起前列腺癌細胞上皮指標及間質(zhì)指標蛋白表達量的改變。 3.驗證SPINK1對前列腺癌細胞侵襲遷移能力的影響。 結(jié)果： 1. rSPINK1持續(xù)刺激6天后,RWPE、LnCap及VCap細胞中均含有一定比例的細胞變?yōu)殚L梭形,細胞失去極性,細胞分布較松散。 2.western blot及免疫熒光技術(shù)均證明rSPINK1可引起RWPE細胞中E-Cadherin表達量增多、vimentin表達量減少,而轉(zhuǎn)染SPINK1SiRNA則可引起22RV1細胞中E-Cadherin表達量上調(diào)、vimentin表達量下調(diào)。 3.細胞劃痕實驗及Transwell實驗證實rSPINK1可促進RWPE細胞遷移侵襲能力增強,SPINK1SiRNA可抑制22RV1細胞侵襲轉(zhuǎn)移。 結(jié)論： 我們首次證實SPINK1在體外可促進前列腺癌細胞發(fā)生EMT。 第三部分SPINK1促進上皮間質(zhì)轉(zhuǎn)化的機制研究 目的： 1.明確SPINK1是否通過EGFR通路促進EMT及具體通過哪條下游通路促進EMT。 2.明確SPINK1通過EGFR通路調(diào)控的促進EMT的下游關(guān)鍵作用分子。 結(jié)果： 1. rSPINKl作用于RWPE細胞,可使其p-EGFR、p-STAT3、p-Akt、p-ERK水平升高。轉(zhuǎn)染SPINK1SiRNA的22RV1細胞,相對于其對照組,EGFR、STAT3、 Akt、ERK磷酸化水平均顯著降低。 2.AG1478和U0126可幾乎完全阻斷rSPINKl對EMT分子指標的調(diào)節(jié)作用。而LY294002和AG490只可較低程度地抑制rSPINKl對EMT分子指標的調(diào)節(jié)作用。 3.細胞劃痕實驗結(jié)果示AG1478、U0126、LY294002、AG490均可完全阻斷rSPINK對細胞遷移能力的提高作用。侵襲實驗結(jié)果示只有AG1478、U0126可阻斷rSPINK對細胞侵襲的促進作用。 4. rSPINK1處理RWPE細胞,SPINK1SiRNA轉(zhuǎn)染22RV1細胞,snail slug、twist、 β-catenin、zeb1的mRNA表達量及核內(nèi)蛋白表達量均無明顯變化。 5. rSPINKl處理RWPE細胞,CTGF表達量升高；SPINK1SiRNA轉(zhuǎn)染22RV1細胞,CTGF表達量下降。 6. CTGF SiRNA轉(zhuǎn)染RWPE細胞可部分逆轉(zhuǎn)rSPINKl促進EMT的作用。 7.AG1478和U0126可阻斷rSPINKl對CTGF的上調(diào)作用,而LY294002和AG490無此作用。 結(jié)論： 1.SPINK1主要通過EGFR/MEK/ERK通路發(fā)揮促進EMT的作用。 2.CTGF可能為SPINK1/EGFR/MEK/ERK促進EMT的下游關(guān)鍵作用分子。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.25
本文編號：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.25
【參考文獻】
相關(guān)期刊論文 前1條
1 戚美;楊曉慶;王林;張娟;王妍;韓博;;ETS基因重排在前列腺癌中的特征分析[J];山東大學(xué)學(xué)報(醫(yī)學(xué)版);2013年06期
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