Wnt1在卵巢癌SKOV3细胞系干细胞中的作用机制
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摘要
卵巢癌是女性生殖器三大恶性肿瘤之一，发病率呈上升的趋势。虽然诊断方法得到不断的改进，但是卵巢癌易复发、并且伴随着转移和多药耐药性的出现，影响到卵巢癌的治疗效果。因为发病机制到目前为止还不十分清楚，使卵巢癌的预防和诊疗水平的提高受到了限制。所以，进一步研究卵巢癌发生、发展、耐药复发机制，有助于寻找有效的抑制卵巢癌增殖的治疗方法，也是提高卵巢癌患者生存率的关键。
干细胞能较长时间存活，这使得它们更易在增殖和分化的过程中发生基因突变。越来越多的研究表明肿瘤是一种干细胞疾病，肿瘤起源于肿瘤干细胞（tumourstem cell TSCs），在干细胞在自我更新过程中很可能因为出现基因突变，从而使细胞生长失控，导致肿瘤发生。自我复制、较长的寿命、抗凋亡信号通路的激活和端粒酶活性等是肿瘤细胞干细胞的重要特征。“肿瘤干细胞”假说认为：虽然在肿瘤组织中肿瘤干细胞只占有极少的比例，可是这些高致瘤性的细胞却最大可能是肿瘤发生、复发、转移和治疗失败的根源。
Wnt信号通路作用广泛且复杂，一方面负责动物发育模式的调控，另一方面在调控干/祖细胞的正常生长和内外环境稳定方面具有重要作用，在许多实体肿瘤的发生过程中均检测到与这一通路的异常激活有关。Dishevelled (DSH)位于细胞膜上，是相关Wnt受体复合物的主要成分，Wnt蛋白与其结合后被激活，从而抑制下游蛋白质复合物，包括axin、GSK-3与APC蛋白。此三种蛋白形成axin/GSK-3/APC复合体，促进细胞内信号分子β-catenin的降解。而当抑制β-catenin降解复合物，可使细胞胞浆内的β-catenin得以稳定存在，一部分β-catenin进入细胞核，通过TCF/LEF转录因子家族使得某些特定基因得以表达。
有关Wnt信号通路在卵巢发育及卵巢癌发生发展中的确切作用机制还不十分清楚，但现有的研究结果表明Wnt信号通路活化与肿瘤耐药、肿瘤干细胞的维持以及与其他信号通路相互作用等有关，了解Wnt在卵巢肿瘤发生及恶性进展中的作用将为卵巢癌的防治提供新的靶标。
本实验首先从卵巢癌SKOV3细胞系中分选出以CD133+为标记的卵巢癌细胞，检测Wnt1信号通路分子在CD133+细胞和CD133-细胞中的表达情况，了解Wnt信号通路在卵巢癌干细胞中的作用。其次，用表达Wnt1shRNA的真核表达载体pGPU6/GFP/Neo稳定转染SKOV3细胞，诱导Wnt1基因沉默，研究Wnt1基因下调后对不同标记卵巢癌细胞增殖、细胞周期及凋亡的影响，探讨其在卵巢癌发生发展中的作用，从而为卵巢癌的防治提供新的靶标。
第一部分Wnt1在卵巢癌SKOV3细胞系干细胞中的表达及其作用机制
目的：了解Wnt1在卵巢癌干细胞中的表达和作用。
方法：采用免疫磁珠法从无血清培养的卵巢癌悬浮细胞中分选CD133+细胞和CD133-细胞。采用real-time RT-PCR技术检测Wnt1和β-catenin的mRNA和蛋白在CD133+细胞和CD133-细胞中的表达情况。
结果：分选出的CD133+细胞约占细胞总数的7.25%，分选出的CD133+细胞表达干细胞标志蛋白CD133和Oct-4。Wnt1和β-catenin的mRNA及蛋白在CD133+细胞中的表达量与CD133-细胞中的表达量相比,均有显著性增加(P均＜0.05)。
结论：在CD133+细胞中，Wnt1可能处于活化状态，活化的Wnt1可能有助于CD133+细胞形态及功能的维持。
第二部分RNAi沉默Wnt1基因对卵巢癌SKOV3细胞影响的研究
目的：探讨Wnt1基因表达在卵巢癌发生发展中的作用。
方法：设计Wnt-shRNA，用于干扰并下调卵巢癌中目的基因的表达。以脂质体LipofectamineTM2000介导转染卵巢癌SKOV3细胞。转染后48h，采用实时定量RT-PCR技术检测转染细胞中Wnt1基因mRNA的转录水平，筛选有效的Wnt1shRNA质粒，Western blot检测Wnt1蛋白在卵巢癌SKOV3细胞中的表达。
结果：4个Wnt1shRNA表达载体Wnt1-shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3和Wnt1-shRNA-4经测序鉴定证明插入正确。转染48h后，在SKOV3细胞中Wnt1shRNA-1组、Wnt1shRNA-2组、Wnt1shRNA-3组、Wnt1shRNA-4组、空白对照组、阴性对照组的标化Wnt1mRNA水平分别为0.77±0.07、0.98±0.08、0.71±0.05、0.57±0.05、0.96±0.08、1.01±0.08，与Control组相比，Wnt1-shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3、Wnt1-shRNA-4组Wnt1mRNA表达量均下降（p均<0.05），其中转染Wnt-shRNA-4的SKOV3细胞中Wnt1-mRNA表达水平低于Wnt1shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3组（p=0.007,0.002,0.046）。Wnt1-shRNA-4干扰抑制效率为64.3%。Western blot可检测到Wnt1蛋白在SKOV3中的表达，干扰片段Wnt1-shRNA-4的抑制效果较好。
结论：载体构建成功，shRNA可明显下调Wnt1mRNA和蛋白在SKOV3表达。
第三部分Wnt1siRNA抑制卵巢癌SKOV3细胞系干细胞增殖机制的研究
目的：探讨下调Wnt1基因表达后卵巢癌干细胞增殖的机制
方法：通过筛选得到稳定下调Wnt1的卵巢癌SKOV3细胞，克隆形成实验和CCK8实验检测干扰Wnt1表达对卵巢癌的增殖作用，流式细胞仪检测转染前后细胞周期的改变，AV/PI双染检测shRNA干扰对卵巢癌SKOV3细胞诱导的凋亡情况。流式细胞仪法检测shRNA干扰后CD133+卵巢癌干细胞比例，Western blot检测Wnt1、β-catenin、Sox2及CD133蛋白的表达水平。
结果：下调Wnt1表达后，对SKOV3细胞增殖抑制作用明显， G2/M期细胞增多；下调Wnt1的表达后， SKOV3凋亡细胞的比例增加。SKOV3细胞系中CD133+卵巢癌干细胞在卵巢癌细胞中的比例下降，Wnt1，Sox2及CD133蛋白的表达降低。
结论：下调Wnt1可以抑制SKOV3细胞的增殖促进细胞凋亡，使细胞停滞在G2/M期。Sox2可能位于卵巢癌Wnt1信号通路的下游，通过下调Wnt1蛋白的表达，抑制Sox2转录作用，抑制卵巢癌干细胞的增殖。
Ovarian cancer is one of the commonest malignancies of females’ reproductivesystem with high morbidity in the world. Although the diagnostic methods have beenimproved, the recurrence, metastasis and multi-drug resistance still lead to the low5-year survival rate of less than30%. Meanwhile, the pathogenesis of ovarian cancer isunclear which limited the advancement of the treatment and prevention. Therefore, toexplore the mechanisms of the occurrence, recurrence and metastasis of ovarian cancerand to figure out effective pathways to suppress the tumor formation would be of greatimportance in improving the survival rate of ovarian cancer patients.
Stem cells might survive for a long time, which make it possible for the genemutation occurring during their proliferation or differentiation. More and more studieshave shown that the tumors originate from cancer stem cells. Tumor is thought to be akind of disease resulted from gene mutation during the process of the renewal of stemcells. Tumor cells share the similar characteristics with the stem cells: the capacity ofself renewing and proliferating infinitely, longer life, the activation of anti-apoptoticsignal paths and telomerase stimulation."Cancer stem cell" hypothesis deems: the smallamount of stem cells which act as high carcinogenesis subgroups play key roles in thetumor occurrence, recurrence, metastasis as well as the failure of treatments.
Wnt signaling pathway is complicated with double functions as be responsible forthe regulation of animals’ development mode and play a crucial role in the regulation ofnormal growth and homeostasis of stem/progenitor cells. It is reported that manycarcinogenesis exist abnormal activation of this pathway. DSH is a key component ofthe membrane-associated Wnt1receptor complex. It is activated after combined withWnt and then inhibits the downstream protein complexes, including axin, GSK-3andAPC protein. The complex of axin/GSK-3/APC might promote the degradation ofβ-catenin, which are signaling molecules within the cell. When “β-catenin degradation complex” is inhibited, β-catenin in the cytoplasm stably exists. Parts of the β-cateninmight move into the nucleus and interact with the TCF/LEF transcription factor familyto initiate the expression of the genes.
The exact mechanism of Wnt signaling pathway in the development of ovary andthe occurrence of ovarian cancer is still unclear, but the researches have proved that theactivation of Wnt signaling is closely related to drug resistance, maintenance of cancerstem cells and interactions with other signaling pathways. To explore the role of Wnt information and progression of ovarian cancer will provide a new target for the preventionand treatment of ovarian cancer.
In this study, we first selected ovarian cancer cells marked by CD133+fromovarian cancer SKOV3cell lines and then detected the expression of molecules of Wnt1signal pathway in CD133+and CD133-cells. Secondly induced the Wnt1gene silencein SKOV3cell by eukaryotic vector pGPU6/GFP/Neo with Wnt1shRNA stablytransfection. The impacts of Wnt1gene down-regulation on proliferation, cell cycle andapoptosis of ovarian cancer cells were studied to explore the mechanism of drugresistance in ovarian cancer cells and its action in the carcinogenesis of ovarian cancer.
Part I Expression and mechanism of Wnt1Signaling Molecule in ovariancancer SKOV3cell
Objective: To explore the function of Wnt signal pathway in CD133+andCD133-ovarian cancer stem cells.
Methods: CD133+and CD133-cells were selected by magnetic activated cellsorting system (MACS). Real-time RT-PCR to detect the expression of mRNA andprotein of Wnt1signal molecules in the two types of cells, respectively.
Results: CD133+cells accounted for7.25%of the total ovarian cancer cells andCD133and Oct-4expressed. The expression of wnt1mRNA and protein in CD133+cells were significant higher compared with CD133-cells (p＜0.05). The expression ofβ-catenin mRNA and protein in CD133+cells were significant higher compared withCD133-cells(p＜0.05).
Conclusions: Wnt1is activated in ovarian cancer CD133+cells and it may playroles in the maintenance of cellular morphology and function of CD133+cells.
PartⅡ The Effect of RNAi applied to silent Wnt1on Carcinogenesis ofovarian Cancer
Objective: To explore the action of the expression of Wnt gene in carcinogenesisof ovarian cancer.
Methods: The Wnt-shRNA probe was designed to interfere and knockdown thetarget gene expression in SKOV3cells. The RT-PCR and western blot were used todetect transcription level of mRNA and expression of protein of Wnt1in ovarian cancerSKOV3cells after LipofectamineTM2000transfection48hours.
Results: The correct insertion was verified by partial nucleotide sequencing of the4constructed eukaryotic vectors expressing shRNA of Wnt1. In the Wnt1shRNA-1group, Wnt1shRNA-2group, Wnt1shRNA-3group, Wnt1shRNA-4group, blankgroup, negative control group and MOCK group of SKOV3cells, the expression ofWnt1mRNA compared to GAPDH was0.77±0.07,0.98±0.08,0.71±0.05,0.57±0.05,0.96±0.08,1.01±0.08,1.00±0.11, respectively. Contrast to MOCK group, the Wnt1mRNA expression in Wnt1shRNA-1group, Wnt1shRNA-3group and Wnt1shRNA-4group decreased (p=0.007,0.002,0.046), moreover the Wnt1mRNA expressioninWnt1shRNA-4group is significantly lower than the other3groups. The interferingand inhibiting efficiency of Wnt1-shRNA-4was comparatively ideal with the downregulation rate of64.3%. The expression of Wnt1protein was detected in SKOV3byWestern blot, and the protein of Wnt1-shRNA-4showed inhibited.
Conclusions: construction is successful, and Wnt1shRNA significantly inhibitthe expression of Wnt1..
Part Ⅲ Wnt1siRNA inhibit the proliferation of ovarian cancer cells and thes ovarian cancer stem cells
Objective: Explore the impact on proliferation by Wnt1gene down-regulating inovarian cancer cells.
Methods: Wnt1knockdown SKOV3cells were selected and colony formationexperiment and CCK8examination were adopted to check the effect of Wnt1deficiencyon proliferation of ovarian cancer cells. The flow cytometre examined cellular cycle andAV/PI double staining detect apoptosis. The proportion of CD133+stem cells wasdetect by flow cytometry, and Wnt1、β-catenin、CD133and Sox2protein expression were examined by western blot.
Results: Knockdown Wnt1in ovarian cancer SKOV3cells induced significantinhibition on cell proliferation and increased proportion of apoptosis and the G2/Mphase cells. The SKOV3stem cells showed low capacity of self-renew and theproportion of cancer stem cells in ovarian cancer cells declined. The expression of Wnt1,Sox2and CD133protein decreased.
Conclusion: Knockdown Wnt1inhibits the SKOV3cell proliferation, promotesthe apoptosis and maintains the cell in the G2/M stage. The Sox2may be downstreamgene of Wnt1signaling pathway and it can inhibit the proliferation of ovarian cancerstem cells after the transcript inhibition by the expression of Wnt1protein.
干细胞能较长时间存活，这使得它们更易在增殖和分化的过程中发生基因突变。越来越多的研究表明肿瘤是一种干细胞疾病，肿瘤起源于肿瘤干细胞（tumourstem cell TSCs），在干细胞在自我更新过程中很可能因为出现基因突变，从而使细胞生长失控，导致肿瘤发生。自我复制、较长的寿命、抗凋亡信号通路的激活和端粒酶活性等是肿瘤细胞干细胞的重要特征。“肿瘤干细胞”假说认为：虽然在肿瘤组织中肿瘤干细胞只占有极少的比例，可是这些高致瘤性的细胞却最大可能是肿瘤发生、复发、转移和治疗失败的根源。
Wnt信号通路作用广泛且复杂，一方面负责动物发育模式的调控，另一方面在调控干/祖细胞的正常生长和内外环境稳定方面具有重要作用，在许多实体肿瘤的发生过程中均检测到与这一通路的异常激活有关。Dishevelled (DSH)位于细胞膜上，是相关Wnt受体复合物的主要成分，Wnt蛋白与其结合后被激活，从而抑制下游蛋白质复合物，包括axin、GSK-3与APC蛋白。此三种蛋白形成axin/GSK-3/APC复合体，促进细胞内信号分子β-catenin的降解。而当抑制β-catenin降解复合物，可使细胞胞浆内的β-catenin得以稳定存在，一部分β-catenin进入细胞核，通过TCF/LEF转录因子家族使得某些特定基因得以表达。
有关Wnt信号通路在卵巢发育及卵巢癌发生发展中的确切作用机制还不十分清楚，但现有的研究结果表明Wnt信号通路活化与肿瘤耐药、肿瘤干细胞的维持以及与其他信号通路相互作用等有关，了解Wnt在卵巢肿瘤发生及恶性进展中的作用将为卵巢癌的防治提供新的靶标。
本实验首先从卵巢癌SKOV3细胞系中分选出以CD133+为标记的卵巢癌细胞，检测Wnt1信号通路分子在CD133+细胞和CD133-细胞中的表达情况，了解Wnt信号通路在卵巢癌干细胞中的作用。其次，用表达Wnt1shRNA的真核表达载体pGPU6/GFP/Neo稳定转染SKOV3细胞，诱导Wnt1基因沉默，研究Wnt1基因下调后对不同标记卵巢癌细胞增殖、细胞周期及凋亡的影响，探讨其在卵巢癌发生发展中的作用，从而为卵巢癌的防治提供新的靶标。
第一部分Wnt1在卵巢癌SKOV3细胞系干细胞中的表达及其作用机制
目的：了解Wnt1在卵巢癌干细胞中的表达和作用。
方法：采用免疫磁珠法从无血清培养的卵巢癌悬浮细胞中分选CD133+细胞和CD133-细胞。采用real-time RT-PCR技术检测Wnt1和β-catenin的mRNA和蛋白在CD133+细胞和CD133-细胞中的表达情况。
结果：分选出的CD133+细胞约占细胞总数的7.25%，分选出的CD133+细胞表达干细胞标志蛋白CD133和Oct-4。Wnt1和β-catenin的mRNA及蛋白在CD133+细胞中的表达量与CD133-细胞中的表达量相比,均有显著性增加(P均＜0.05)。
结论：在CD133+细胞中，Wnt1可能处于活化状态，活化的Wnt1可能有助于CD133+细胞形态及功能的维持。
第二部分RNAi沉默Wnt1基因对卵巢癌SKOV3细胞影响的研究
目的：探讨Wnt1基因表达在卵巢癌发生发展中的作用。
方法：设计Wnt-shRNA，用于干扰并下调卵巢癌中目的基因的表达。以脂质体LipofectamineTM2000介导转染卵巢癌SKOV3细胞。转染后48h，采用实时定量RT-PCR技术检测转染细胞中Wnt1基因mRNA的转录水平，筛选有效的Wnt1shRNA质粒，Western blot检测Wnt1蛋白在卵巢癌SKOV3细胞中的表达。
结果：4个Wnt1shRNA表达载体Wnt1-shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3和Wnt1-shRNA-4经测序鉴定证明插入正确。转染48h后，在SKOV3细胞中Wnt1shRNA-1组、Wnt1shRNA-2组、Wnt1shRNA-3组、Wnt1shRNA-4组、空白对照组、阴性对照组的标化Wnt1mRNA水平分别为0.77±0.07、0.98±0.08、0.71±0.05、0.57±0.05、0.96±0.08、1.01±0.08，与Control组相比，Wnt1-shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3、Wnt1-shRNA-4组Wnt1mRNA表达量均下降（p均<0.05），其中转染Wnt-shRNA-4的SKOV3细胞中Wnt1-mRNA表达水平低于Wnt1shRNA-1、Wnt1-shRNA-2、Wnt1-shRNA-3组（p=0.007,0.002,0.046）。Wnt1-shRNA-4干扰抑制效率为64.3%。Western blot可检测到Wnt1蛋白在SKOV3中的表达，干扰片段Wnt1-shRNA-4的抑制效果较好。
结论：载体构建成功，shRNA可明显下调Wnt1mRNA和蛋白在SKOV3表达。
第三部分Wnt1siRNA抑制卵巢癌SKOV3细胞系干细胞增殖机制的研究
目的：探讨下调Wnt1基因表达后卵巢癌干细胞增殖的机制
方法：通过筛选得到稳定下调Wnt1的卵巢癌SKOV3细胞，克隆形成实验和CCK8实验检测干扰Wnt1表达对卵巢癌的增殖作用，流式细胞仪检测转染前后细胞周期的改变，AV/PI双染检测shRNA干扰对卵巢癌SKOV3细胞诱导的凋亡情况。流式细胞仪法检测shRNA干扰后CD133+卵巢癌干细胞比例，Western blot检测Wnt1、β-catenin、Sox2及CD133蛋白的表达水平。
结果：下调Wnt1表达后，对SKOV3细胞增殖抑制作用明显， G2/M期细胞增多；下调Wnt1的表达后， SKOV3凋亡细胞的比例增加。SKOV3细胞系中CD133+卵巢癌干细胞在卵巢癌细胞中的比例下降，Wnt1，Sox2及CD133蛋白的表达降低。
结论：下调Wnt1可以抑制SKOV3细胞的增殖促进细胞凋亡，使细胞停滞在G2/M期。Sox2可能位于卵巢癌Wnt1信号通路的下游，通过下调Wnt1蛋白的表达，抑制Sox2转录作用，抑制卵巢癌干细胞的增殖。
Ovarian cancer is one of the commonest malignancies of females’ reproductivesystem with high morbidity in the world. Although the diagnostic methods have beenimproved, the recurrence, metastasis and multi-drug resistance still lead to the low5-year survival rate of less than30%. Meanwhile, the pathogenesis of ovarian cancer isunclear which limited the advancement of the treatment and prevention. Therefore, toexplore the mechanisms of the occurrence, recurrence and metastasis of ovarian cancerand to figure out effective pathways to suppress the tumor formation would be of greatimportance in improving the survival rate of ovarian cancer patients.
Stem cells might survive for a long time, which make it possible for the genemutation occurring during their proliferation or differentiation. More and more studieshave shown that the tumors originate from cancer stem cells. Tumor is thought to be akind of disease resulted from gene mutation during the process of the renewal of stemcells. Tumor cells share the similar characteristics with the stem cells: the capacity ofself renewing and proliferating infinitely, longer life, the activation of anti-apoptoticsignal paths and telomerase stimulation."Cancer stem cell" hypothesis deems: the smallamount of stem cells which act as high carcinogenesis subgroups play key roles in thetumor occurrence, recurrence, metastasis as well as the failure of treatments.
Wnt signaling pathway is complicated with double functions as be responsible forthe regulation of animals’ development mode and play a crucial role in the regulation ofnormal growth and homeostasis of stem/progenitor cells. It is reported that manycarcinogenesis exist abnormal activation of this pathway. DSH is a key component ofthe membrane-associated Wnt1receptor complex. It is activated after combined withWnt and then inhibits the downstream protein complexes, including axin, GSK-3andAPC protein. The complex of axin/GSK-3/APC might promote the degradation ofβ-catenin, which are signaling molecules within the cell. When “β-catenin degradation complex” is inhibited, β-catenin in the cytoplasm stably exists. Parts of the β-cateninmight move into the nucleus and interact with the TCF/LEF transcription factor familyto initiate the expression of the genes.
The exact mechanism of Wnt signaling pathway in the development of ovary andthe occurrence of ovarian cancer is still unclear, but the researches have proved that theactivation of Wnt signaling is closely related to drug resistance, maintenance of cancerstem cells and interactions with other signaling pathways. To explore the role of Wnt information and progression of ovarian cancer will provide a new target for the preventionand treatment of ovarian cancer.
In this study, we first selected ovarian cancer cells marked by CD133+fromovarian cancer SKOV3cell lines and then detected the expression of molecules of Wnt1signal pathway in CD133+and CD133-cells. Secondly induced the Wnt1gene silencein SKOV3cell by eukaryotic vector pGPU6/GFP/Neo with Wnt1shRNA stablytransfection. The impacts of Wnt1gene down-regulation on proliferation, cell cycle andapoptosis of ovarian cancer cells were studied to explore the mechanism of drugresistance in ovarian cancer cells and its action in the carcinogenesis of ovarian cancer.
Part I Expression and mechanism of Wnt1Signaling Molecule in ovariancancer SKOV3cell
Objective: To explore the function of Wnt signal pathway in CD133+andCD133-ovarian cancer stem cells.
Methods: CD133+and CD133-cells were selected by magnetic activated cellsorting system (MACS). Real-time RT-PCR to detect the expression of mRNA andprotein of Wnt1signal molecules in the two types of cells, respectively.
Results: CD133+cells accounted for7.25%of the total ovarian cancer cells andCD133and Oct-4expressed. The expression of wnt1mRNA and protein in CD133+cells were significant higher compared with CD133-cells (p＜0.05). The expression ofβ-catenin mRNA and protein in CD133+cells were significant higher compared withCD133-cells(p＜0.05).
Conclusions: Wnt1is activated in ovarian cancer CD133+cells and it may playroles in the maintenance of cellular morphology and function of CD133+cells.
PartⅡ The Effect of RNAi applied to silent Wnt1on Carcinogenesis ofovarian Cancer
Objective: To explore the action of the expression of Wnt gene in carcinogenesisof ovarian cancer.
Methods: The Wnt-shRNA probe was designed to interfere and knockdown thetarget gene expression in SKOV3cells. The RT-PCR and western blot were used todetect transcription level of mRNA and expression of protein of Wnt1in ovarian cancerSKOV3cells after LipofectamineTM2000transfection48hours.
Results: The correct insertion was verified by partial nucleotide sequencing of the4constructed eukaryotic vectors expressing shRNA of Wnt1. In the Wnt1shRNA-1group, Wnt1shRNA-2group, Wnt1shRNA-3group, Wnt1shRNA-4group, blankgroup, negative control group and MOCK group of SKOV3cells, the expression ofWnt1mRNA compared to GAPDH was0.77±0.07,0.98±0.08,0.71±0.05,0.57±0.05,0.96±0.08,1.01±0.08,1.00±0.11, respectively. Contrast to MOCK group, the Wnt1mRNA expression in Wnt1shRNA-1group, Wnt1shRNA-3group and Wnt1shRNA-4group decreased (p=0.007,0.002,0.046), moreover the Wnt1mRNA expressioninWnt1shRNA-4group is significantly lower than the other3groups. The interferingand inhibiting efficiency of Wnt1-shRNA-4was comparatively ideal with the downregulation rate of64.3%. The expression of Wnt1protein was detected in SKOV3byWestern blot, and the protein of Wnt1-shRNA-4showed inhibited.
Conclusions: construction is successful, and Wnt1shRNA significantly inhibitthe expression of Wnt1..
Part Ⅲ Wnt1siRNA inhibit the proliferation of ovarian cancer cells and thes ovarian cancer stem cells
Objective: Explore the impact on proliferation by Wnt1gene down-regulating inovarian cancer cells.
Methods: Wnt1knockdown SKOV3cells were selected and colony formationexperiment and CCK8examination were adopted to check the effect of Wnt1deficiencyon proliferation of ovarian cancer cells. The flow cytometre examined cellular cycle andAV/PI double staining detect apoptosis. The proportion of CD133+stem cells wasdetect by flow cytometry, and Wnt1、β-catenin、CD133and Sox2protein expression were examined by western blot.
Results: Knockdown Wnt1in ovarian cancer SKOV3cells induced significantinhibition on cell proliferation and increased proportion of apoptosis and the G2/Mphase cells. The SKOV3stem cells showed low capacity of self-renew and theproportion of cancer stem cells in ovarian cancer cells declined. The expression of Wnt1,Sox2and CD133protein decreased.
Conclusion: Knockdown Wnt1inhibits the SKOV3cell proliferation, promotesthe apoptosis and maintains the cell in the G2/M stage. The Sox2may be downstreamgene of Wnt1signaling pathway and it can inhibit the proliferation of ovarian cancerstem cells after the transcript inhibition by the expression of Wnt1protein.
引文
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