Wnt通路抑制分子DKK1在肝细胞癌中异常高表达的分子机制
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摘要
发现肝癌(Human hepatocellular careinoma,HCC)相关基因的重要步骤之一是获得在肝癌组织中异常表达的基因。我们实验室运用cDNA芯片方法筛选到了一批在肝癌中异常表达的已知和未知基因。对这些基因的功能进行进一步研究将有助于对肝癌发病分子机理的深入了解。我们首先挑选了6个在芯片结果显示有差异表达的较有意义的基因,运用Northern blot方法做进一步验证。本课题对其中一个基因Dickkopf1(DKK1)与肝癌的相关性进行较深入的研究。
我们的结果发现在肝癌细胞株中分泌蛋白DKK1对Wnt/β-catenin信号通路具有抑制作用,这种抑制作用由于β-catenin基因的突变而丧失;而在肝癌细胞株中DKK1的表达水平和β-catenin定位相关,部分DKK1表达病例β-catenin存在突变,两套临床病例组化分析显示DKK1高表达与β-catenin胞浆/核累积具有相关性;在肝癌细胞中活化的Wnt信号可以反馈调节DKK1的表达。大样本量肝癌组织(来自314例外科切除和192例肝移植肝癌病人)免疫组化实验显示DKK1高表达肝癌病人,包括AFP阴性和TNM stage I病人,存在较差的预后;DKK1和β-catenin双阳性病人存在更差的预后;并且多因素分析显示DKK1是肝癌病人总体生存和无瘤生存的独立预后指标。我们的分析显示DKK1基因在侵袭性肝癌细胞株和发生复发的肝癌病人中高表达;体外运动侵袭等相关实验和裸鼠体内实验显示干扰DKK1表达后显著抑制肝癌细胞侵袭转移的发生;我们进一步探讨了DKK1促进肝癌细胞侵袭转移过程的具体信号途径和分子机制。上述结果提示在肝癌中DKK1存在高表达并与Wnt信号的活化相关,同时DKK1可以作为肝癌病人的预后指标。
第一部分DKK1在肝癌细胞Wnt通路中作用
【目的】探讨DKK1在Wnt信号途径中的抑制作用是否由于β-catenin基因的突变而丧失和是否由于β-catenin基因的突变引起Wnt信号途径的活化反馈调节DKK1基因的表达。【方法】用Western blot和免疫荧光方法检测肝癌细胞株中DKK1表达和β-catenin定位的情况。选用不同细胞株分别干扰β-catenin基因的表达和转入β-catenin突变体提高β-catenin基因的表达,Western blot方法检测DKK1基因表达水平的改变。分别选用存在β-catenin基因突变和不存在β-catenin基因突变的肝癌细胞株,以TOPflash/FOPflash作为报告质粒,pRL-SV40质粒作为内对照,用双荧光素报告系统、Western blot和免疫荧光方法检测DKK1对Wnt信号变化的影响。用组织芯片免疫组织化学方法分析临床病例中DKK1表达和β-catenin定位的关系。【结果】在肝癌细胞株中DKK1的表达水平和β-catenin定位相关;干扰和提高β-catenin基因的表达可相应降低和上调DKK1基因的表达水平。DKK1能够抑制HuH-7细胞中Wnt-3a活化信号,不抑制β-catenin突变体活化信号,同时DKK1不抑制β-catenin存在突变和胞浆/核累积的肝癌细胞株HepG2、MHCC97L中活化的Wnt信号;在这两类细胞株中DKK1也相应的改变或不改变β-catenin表达和定位的情况。部分DKK1表达病例β-catenin存在突变,大样本量临床病例中组化分析显示DKK1表达与β-catenin胞浆/核累积具有相关性。【结论】在肝癌细胞中,由于β-catenin基因的突变使DKK1对于Wnt信号途径的抑制作用丧失;而Wnt信号途径的活化可以反馈调节DKK1基因的表达。
第二部分DKK1在临床肝癌病人预后分析中的应用
【目的】探讨DKK1表达情况与肝癌病人预后(总体生存和无瘤生存)及肝癌病人临床病理特征间的关系。【方法】应用免疫组织化学染色方法研究肝癌组织芯片中DKK1表达及β-catenin定位的情况。单因素方差分析比较组间差异,卡方检验或Fisher精确概率法比较不同DKK1和β-catenin表达组间阳性率的差别,Kaplan-Meier法计算各组间不同生存率,Log-rank检验比较生存期差别。用Cox比例风险模型进行多因素分析以了解各个相关因素对总体生存率和无瘤生存率的影响。【结果】临床病例中DKK1组化分析显示DKK1表达与肝癌病人总体生存和无瘤生存相关,DKK1高表达肝癌病人,包括AFP阴性和TNMⅠ期病人,存在较差的预后。多因素分析显示DKK1是肝癌病人总体生存和无瘤生存的独立预后指标。DKK1高表达与β-catenin胞浆/核定位显著相关,DKK1/β-catenin双阳性的肝癌病人更倾向于多结节或弥散型复发和肝外转移,而这部分病人也存在更差的预后;DKK1/β-catenin也是影响肝癌病人总体生存率和无瘤生存率的独立因素。【结论】DKK1高表达肝癌病人存在较差的预后,DKK1可以作为肝癌的预后指标。
第三部分DKK1对肝癌细胞侵袭转移的影响及其分子机制
【目的】探讨DKK1基因在肝癌侵袭和转移中的作用和DKK1参与肝癌侵袭转移的分子机制。【方法】用定量PCR和Western blot方法检测肝癌细胞株和临床肝癌标本中DKK1的表达情况。通过化学合成RNA干扰片段瞬时干扰和质粒载体稳定转染干扰HCCLM3细胞DKK1的表达,体外损伤修复实验、侵袭实验和裸鼠体内实验检测下调DKK1表达前后肝癌细胞侵袭转移能力的改变情况。对3种肝癌细胞株DKK1高、低表达情况下的细胞进行表达谱芯片分析,利用配对t检验比较DKK1高表达组和DKK1低表达组之间的差异基因,分析这些差异基因相关的癌相关信号通路,进一步采用定量PCR对芯片结果进行验证,并在临床病例中检测这些分子的表达与DKK1表达的相关性。【结果】DKK1基因在侵袭性肝癌细胞株和发生复发的肝癌病人中高表达。体外损伤修复实验、侵袭实验证实,当DKK1被干扰后,肝癌细胞增殖能力没有发生改变而细胞侵袭被抑制。裸鼠体内实验显示干扰DKK1表达后显著抑制肝癌细胞肺转移的发生。DKK1高表达组对比DKK1低表达组在基因表达谱上存在显著差异,涉及VEGF、NF-κB、P13K和Integrin等癌相关信号通路;MMP16、KDR、PTPRA这3个在两组中差异显著的基因与肿瘤侵袭转移密切相关,定量PCR验证结果与基因芯片结果一致。在66例临床组织标本中MMP16、KDR、PTPRA的mRNA表达水平都与DKK1的表达水平成正相关,因而DKK1可能通过影响MMP16降解细胞外基质和通过KDR促进肿瘤血管形成等途径参与肿瘤侵袭转移过程。【结论】DKK1通过影响细胞外基质的降解和肿瘤血管的形成等促进肝癌侵袭和转移的发生。
In order to identify novel genes related to the carcinogenesis of human hepatocellular carcinoma(HCC),we utilized the eDNA microarray technology to discover differential gene expression profile between HCC tissue and its corresponding non-cancerous liver tissue.We found a cluster of genes with abnormal expression pattern in HCCs.Furthermore,the upregulated expression of 6 genes in HCC samples was confirmed using Northern blot.Among them,we selected Dickkopf-1(DKK1) for further investigation.
Our research showed that DKK1 inhibited Wnt/β-catenin signaling in HCC cell lines withoutβ-catenin mutation,whereas DKK1 in HCC cell lines withβ-catenin mutations had no antagonistic effect on elevated Wnt/β-catenin signaling.The expression level of DKK1 was associated with the staining pattern ofβ-catenin in HCC cell lines,and DKK1 overexpression correlated withβ-catenin cytoplasmic/nuclear accumulation in clinical HCC samples.Meanwhile,the expression of DKK1 is regulated by Wnt/β-eatenin signaling in HCC cell lines.High DKK1 expression predicted unfavorable prognosis in HCC patients,especially in early stage patients and those with normal AFP levels.The HCC patients with high DKK1 expression and cytoplasmic/nuclearβ-catenin accumulation had very poor prognosis.In multivariate analyses,DKK1 was an independent predictor for overall survival(OS) and disease free survival(DFS) of HCC patients.In our studies,we found that DKK1 was preferentially overexpressed in recurrent HCC patients and metastatic HCC cell lines.Depletion of DKK1 caused a notable decrease in cell migration and invasiveness both in vitro and in vivo.We further explored the signal pathway and molecular mechanism triggered by DKK1 for the promotion of cell motility and metastasis.Our data demonstrated that DKK1 expression can be induced by an active Wnt/β-catenin signaling pathway and DKK1 is a novel prognostic predictor for patients with HCC.
SectionⅠRole of DKK1 in the Wnt Pathway in HCC Cells
【Objective】To determine whether extracellular DKK1 exerts a suppressive effect and the relationship between its expression and cytoplasmic/nuclearβ-catenin accumulation in HCC.【Methods】The expression patterns of DKK1 andβ-catenin were assessed by Western blotting and immunofluorescence in HCC cell lines. Regulation of DKK1 expression was also examined by transfectingβ-catenin mutant or small interfering RNAs directed againstβ-catenin into HCC cells.The effect of DKK1 on Wnt signaling was analyzed by TOPflash/FOPflash luciferase reporter assay,Western blotting and immunofluorescence in HCC cell lines with either wild-type or mutantβ-catenin.The expression patterns of DKK1 andβ-catenin were assessed by immunohistochemistry in tissue microarrays.【Results】The level of DKK1 in HCC cell lines is associated with staining pattern ofβ-catenin and its expression is regulated by Wnt/β-catenin signaling.Meanwhile,in HCC cell lines withoutβ-catenin mutation,DKK1 inhibited the Wnt-3a-activated Wnt/β-catenin signaling,whereas DKK1 in HCC cell lines withβ-catenin mutations had no antagonistic effect on elevated Wnt/β-catenin signaling.Immunohistochemical staining using tumor tissue microarrays consisting of 314 HCC patients showed that a high level of DKK1 expression was associated withβ-catenin cytoplasmic/nuclear accumulation.【Conclusion】DKK1 expression can be induced by an active Wnt/β-catenin signaling pathway and the suppressive effect of extracellular DKK1 on Wnt signaling is lost when cytoplasmicβ-catenin mutations occur in HCC cells.
SectionⅡPrognostic Significance of DKK1 for HCC Patients
【Objective】To measure the level of DKK1 expression in patients with HCC to evaluate its correlation with clinicopathological features and significance on prognosis.【Methods】Tissue microarrays containing 314 HCC patients who underwent surgical resection between 1997 and 2000 were used to detect the expression patterns of DKK1 andβ-catenin by immunohistochemistry. Clinicopathologic data for these patients were evaluated.The prognostic significance was assessed by using Kaplan-Meier survival estimates and log-rank tests.【Results】In the large cohort of HCC patients with long-term follow-up,DKK1 expression predicted unfavorable prognosis in HCC patients,especially in normal AFP and early stage HCC patients.In multivariate analyses,DKK1 was an independent predictor for OS and DFS of HCC patients.Furthermore,the high level of DKK1 expression was correlated withβ-catenin cytoplasmic/nuclear staining in clinical HCC samples,and patients with DKK1~+/β-catenin~+ had higher rates of diffused/distant recurrence.HCC patients with high DKK1 expression and cytoplasmic/nuclearβ-catenin staining had very poor prognosis,and the the co-index of DKK1/β-catenin also was an independent prognosticator for both OS and DFS of patients with HCC.【Conclusion】DKK1 expression predicted unfavorable prognosis and is a novel prognostic predictor for HCC patients.
SectionⅢImpact of DKK1 on the Invasion and Metastasis of HCC Cells
【Objective】To explore the role of DKK1 in HCC cell migration and metastasis and investigate the molecular mechanism triggered by DKK1 for the promotion of HCC metastasis.【Methods】DKK1 expression in HCC cell lines and in tumor tissues derived from HCC patients was investigated using quantitative real-time PCR (qRT-PCR) and Western blot analysis.The role of DKK1 in HCC was investigated by DKK1 depletion using transient or stable transfection of small interfering RNAs directed against DKK1.The in vitro and in vivo effects of DKK1 on the cell invasion and metastasis were examined by wound healing assay,cell invasion assay and metastasis model on mice.We examined the expression profile of 3 paired DKK1-high and DKK1-low expression HCC cell lines.Differentially expressed genes were identified based on paired t-test and the involved signaling pathways were analysed among these genes.We further confirmed the data of cDNA microarray by qRT-PCR and verified the correlation among these genes and DKK1 expression in clinical HCC samples.【Results】DKK1 was preferentially overexpressed in recurrent HCC patients and metastatic HCC cell lines.Depletion of DKK1 caused a notable decrease in cell migration and invasiveness both in vitro and in vivo.Differentially expressed genes were identified between DKK1-high and DKK1-low expression group,and these genes were involved in the VEGF,NF-κB,PI3K and Integrin signaling pathways.The expression of 3 selected-genes was analyzed by qRT-PCR,and the results were in agreement with the microarray data.The expression of MMP16,KDR,and PTPRA was positively correlated with DKK1 mRNA expression in 66 HCC samples.
【Conclusion】DKK1 might be critically involved in the metastasis of HCCs through the activation of MMP 16 and promotion of angiogenesis.
我们的结果发现在肝癌细胞株中分泌蛋白DKK1对Wnt/β-catenin信号通路具有抑制作用,这种抑制作用由于β-catenin基因的突变而丧失;而在肝癌细胞株中DKK1的表达水平和β-catenin定位相关,部分DKK1表达病例β-catenin存在突变,两套临床病例组化分析显示DKK1高表达与β-catenin胞浆/核累积具有相关性;在肝癌细胞中活化的Wnt信号可以反馈调节DKK1的表达。大样本量肝癌组织(来自314例外科切除和192例肝移植肝癌病人)免疫组化实验显示DKK1高表达肝癌病人,包括AFP阴性和TNM stage I病人,存在较差的预后;DKK1和β-catenin双阳性病人存在更差的预后;并且多因素分析显示DKK1是肝癌病人总体生存和无瘤生存的独立预后指标。我们的分析显示DKK1基因在侵袭性肝癌细胞株和发生复发的肝癌病人中高表达;体外运动侵袭等相关实验和裸鼠体内实验显示干扰DKK1表达后显著抑制肝癌细胞侵袭转移的发生;我们进一步探讨了DKK1促进肝癌细胞侵袭转移过程的具体信号途径和分子机制。上述结果提示在肝癌中DKK1存在高表达并与Wnt信号的活化相关,同时DKK1可以作为肝癌病人的预后指标。
第一部分DKK1在肝癌细胞Wnt通路中作用
【目的】探讨DKK1在Wnt信号途径中的抑制作用是否由于β-catenin基因的突变而丧失和是否由于β-catenin基因的突变引起Wnt信号途径的活化反馈调节DKK1基因的表达。【方法】用Western blot和免疫荧光方法检测肝癌细胞株中DKK1表达和β-catenin定位的情况。选用不同细胞株分别干扰β-catenin基因的表达和转入β-catenin突变体提高β-catenin基因的表达,Western blot方法检测DKK1基因表达水平的改变。分别选用存在β-catenin基因突变和不存在β-catenin基因突变的肝癌细胞株,以TOPflash/FOPflash作为报告质粒,pRL-SV40质粒作为内对照,用双荧光素报告系统、Western blot和免疫荧光方法检测DKK1对Wnt信号变化的影响。用组织芯片免疫组织化学方法分析临床病例中DKK1表达和β-catenin定位的关系。【结果】在肝癌细胞株中DKK1的表达水平和β-catenin定位相关;干扰和提高β-catenin基因的表达可相应降低和上调DKK1基因的表达水平。DKK1能够抑制HuH-7细胞中Wnt-3a活化信号,不抑制β-catenin突变体活化信号,同时DKK1不抑制β-catenin存在突变和胞浆/核累积的肝癌细胞株HepG2、MHCC97L中活化的Wnt信号;在这两类细胞株中DKK1也相应的改变或不改变β-catenin表达和定位的情况。部分DKK1表达病例β-catenin存在突变,大样本量临床病例中组化分析显示DKK1表达与β-catenin胞浆/核累积具有相关性。【结论】在肝癌细胞中,由于β-catenin基因的突变使DKK1对于Wnt信号途径的抑制作用丧失;而Wnt信号途径的活化可以反馈调节DKK1基因的表达。
第二部分DKK1在临床肝癌病人预后分析中的应用
【目的】探讨DKK1表达情况与肝癌病人预后(总体生存和无瘤生存)及肝癌病人临床病理特征间的关系。【方法】应用免疫组织化学染色方法研究肝癌组织芯片中DKK1表达及β-catenin定位的情况。单因素方差分析比较组间差异,卡方检验或Fisher精确概率法比较不同DKK1和β-catenin表达组间阳性率的差别,Kaplan-Meier法计算各组间不同生存率,Log-rank检验比较生存期差别。用Cox比例风险模型进行多因素分析以了解各个相关因素对总体生存率和无瘤生存率的影响。【结果】临床病例中DKK1组化分析显示DKK1表达与肝癌病人总体生存和无瘤生存相关,DKK1高表达肝癌病人,包括AFP阴性和TNMⅠ期病人,存在较差的预后。多因素分析显示DKK1是肝癌病人总体生存和无瘤生存的独立预后指标。DKK1高表达与β-catenin胞浆/核定位显著相关,DKK1/β-catenin双阳性的肝癌病人更倾向于多结节或弥散型复发和肝外转移,而这部分病人也存在更差的预后;DKK1/β-catenin也是影响肝癌病人总体生存率和无瘤生存率的独立因素。【结论】DKK1高表达肝癌病人存在较差的预后,DKK1可以作为肝癌的预后指标。
第三部分DKK1对肝癌细胞侵袭转移的影响及其分子机制
【目的】探讨DKK1基因在肝癌侵袭和转移中的作用和DKK1参与肝癌侵袭转移的分子机制。【方法】用定量PCR和Western blot方法检测肝癌细胞株和临床肝癌标本中DKK1的表达情况。通过化学合成RNA干扰片段瞬时干扰和质粒载体稳定转染干扰HCCLM3细胞DKK1的表达,体外损伤修复实验、侵袭实验和裸鼠体内实验检测下调DKK1表达前后肝癌细胞侵袭转移能力的改变情况。对3种肝癌细胞株DKK1高、低表达情况下的细胞进行表达谱芯片分析,利用配对t检验比较DKK1高表达组和DKK1低表达组之间的差异基因,分析这些差异基因相关的癌相关信号通路,进一步采用定量PCR对芯片结果进行验证,并在临床病例中检测这些分子的表达与DKK1表达的相关性。【结果】DKK1基因在侵袭性肝癌细胞株和发生复发的肝癌病人中高表达。体外损伤修复实验、侵袭实验证实,当DKK1被干扰后,肝癌细胞增殖能力没有发生改变而细胞侵袭被抑制。裸鼠体内实验显示干扰DKK1表达后显著抑制肝癌细胞肺转移的发生。DKK1高表达组对比DKK1低表达组在基因表达谱上存在显著差异,涉及VEGF、NF-κB、P13K和Integrin等癌相关信号通路;MMP16、KDR、PTPRA这3个在两组中差异显著的基因与肿瘤侵袭转移密切相关,定量PCR验证结果与基因芯片结果一致。在66例临床组织标本中MMP16、KDR、PTPRA的mRNA表达水平都与DKK1的表达水平成正相关,因而DKK1可能通过影响MMP16降解细胞外基质和通过KDR促进肿瘤血管形成等途径参与肿瘤侵袭转移过程。【结论】DKK1通过影响细胞外基质的降解和肿瘤血管的形成等促进肝癌侵袭和转移的发生。
In order to identify novel genes related to the carcinogenesis of human hepatocellular carcinoma(HCC),we utilized the eDNA microarray technology to discover differential gene expression profile between HCC tissue and its corresponding non-cancerous liver tissue.We found a cluster of genes with abnormal expression pattern in HCCs.Furthermore,the upregulated expression of 6 genes in HCC samples was confirmed using Northern blot.Among them,we selected Dickkopf-1(DKK1) for further investigation.
Our research showed that DKK1 inhibited Wnt/β-catenin signaling in HCC cell lines withoutβ-catenin mutation,whereas DKK1 in HCC cell lines withβ-catenin mutations had no antagonistic effect on elevated Wnt/β-catenin signaling.The expression level of DKK1 was associated with the staining pattern ofβ-catenin in HCC cell lines,and DKK1 overexpression correlated withβ-catenin cytoplasmic/nuclear accumulation in clinical HCC samples.Meanwhile,the expression of DKK1 is regulated by Wnt/β-eatenin signaling in HCC cell lines.High DKK1 expression predicted unfavorable prognosis in HCC patients,especially in early stage patients and those with normal AFP levels.The HCC patients with high DKK1 expression and cytoplasmic/nuclearβ-catenin accumulation had very poor prognosis.In multivariate analyses,DKK1 was an independent predictor for overall survival(OS) and disease free survival(DFS) of HCC patients.In our studies,we found that DKK1 was preferentially overexpressed in recurrent HCC patients and metastatic HCC cell lines.Depletion of DKK1 caused a notable decrease in cell migration and invasiveness both in vitro and in vivo.We further explored the signal pathway and molecular mechanism triggered by DKK1 for the promotion of cell motility and metastasis.Our data demonstrated that DKK1 expression can be induced by an active Wnt/β-catenin signaling pathway and DKK1 is a novel prognostic predictor for patients with HCC.
SectionⅠRole of DKK1 in the Wnt Pathway in HCC Cells
【Objective】To determine whether extracellular DKK1 exerts a suppressive effect and the relationship between its expression and cytoplasmic/nuclearβ-catenin accumulation in HCC.【Methods】The expression patterns of DKK1 andβ-catenin were assessed by Western blotting and immunofluorescence in HCC cell lines. Regulation of DKK1 expression was also examined by transfectingβ-catenin mutant or small interfering RNAs directed againstβ-catenin into HCC cells.The effect of DKK1 on Wnt signaling was analyzed by TOPflash/FOPflash luciferase reporter assay,Western blotting and immunofluorescence in HCC cell lines with either wild-type or mutantβ-catenin.The expression patterns of DKK1 andβ-catenin were assessed by immunohistochemistry in tissue microarrays.【Results】The level of DKK1 in HCC cell lines is associated with staining pattern ofβ-catenin and its expression is regulated by Wnt/β-catenin signaling.Meanwhile,in HCC cell lines withoutβ-catenin mutation,DKK1 inhibited the Wnt-3a-activated Wnt/β-catenin signaling,whereas DKK1 in HCC cell lines withβ-catenin mutations had no antagonistic effect on elevated Wnt/β-catenin signaling.Immunohistochemical staining using tumor tissue microarrays consisting of 314 HCC patients showed that a high level of DKK1 expression was associated withβ-catenin cytoplasmic/nuclear accumulation.【Conclusion】DKK1 expression can be induced by an active Wnt/β-catenin signaling pathway and the suppressive effect of extracellular DKK1 on Wnt signaling is lost when cytoplasmicβ-catenin mutations occur in HCC cells.
SectionⅡPrognostic Significance of DKK1 for HCC Patients
【Objective】To measure the level of DKK1 expression in patients with HCC to evaluate its correlation with clinicopathological features and significance on prognosis.【Methods】Tissue microarrays containing 314 HCC patients who underwent surgical resection between 1997 and 2000 were used to detect the expression patterns of DKK1 andβ-catenin by immunohistochemistry. Clinicopathologic data for these patients were evaluated.The prognostic significance was assessed by using Kaplan-Meier survival estimates and log-rank tests.【Results】In the large cohort of HCC patients with long-term follow-up,DKK1 expression predicted unfavorable prognosis in HCC patients,especially in normal AFP and early stage HCC patients.In multivariate analyses,DKK1 was an independent predictor for OS and DFS of HCC patients.Furthermore,the high level of DKK1 expression was correlated withβ-catenin cytoplasmic/nuclear staining in clinical HCC samples,and patients with DKK1~+/β-catenin~+ had higher rates of diffused/distant recurrence.HCC patients with high DKK1 expression and cytoplasmic/nuclearβ-catenin staining had very poor prognosis,and the the co-index of DKK1/β-catenin also was an independent prognosticator for both OS and DFS of patients with HCC.【Conclusion】DKK1 expression predicted unfavorable prognosis and is a novel prognostic predictor for HCC patients.
SectionⅢImpact of DKK1 on the Invasion and Metastasis of HCC Cells
【Objective】To explore the role of DKK1 in HCC cell migration and metastasis and investigate the molecular mechanism triggered by DKK1 for the promotion of HCC metastasis.【Methods】DKK1 expression in HCC cell lines and in tumor tissues derived from HCC patients was investigated using quantitative real-time PCR (qRT-PCR) and Western blot analysis.The role of DKK1 in HCC was investigated by DKK1 depletion using transient or stable transfection of small interfering RNAs directed against DKK1.The in vitro and in vivo effects of DKK1 on the cell invasion and metastasis were examined by wound healing assay,cell invasion assay and metastasis model on mice.We examined the expression profile of 3 paired DKK1-high and DKK1-low expression HCC cell lines.Differentially expressed genes were identified based on paired t-test and the involved signaling pathways were analysed among these genes.We further confirmed the data of cDNA microarray by qRT-PCR and verified the correlation among these genes and DKK1 expression in clinical HCC samples.【Results】DKK1 was preferentially overexpressed in recurrent HCC patients and metastatic HCC cell lines.Depletion of DKK1 caused a notable decrease in cell migration and invasiveness both in vitro and in vivo.Differentially expressed genes were identified between DKK1-high and DKK1-low expression group,and these genes were involved in the VEGF,NF-κB,PI3K and Integrin signaling pathways.The expression of 3 selected-genes was analyzed by qRT-PCR,and the results were in agreement with the microarray data.The expression of MMP16,KDR,and PTPRA was positively correlated with DKK1 mRNA expression in 66 HCC samples.
【Conclusion】DKK1 might be critically involved in the metastasis of HCCs through the activation of MMP 16 and promotion of angiogenesis.
引文
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