shRNA抑制小鼠肝癌细胞株JNK表达及与淋巴道转移关系的研究
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摘要
背景:恶性肿瘤区别于良性肿瘤的根本特征是其转移潜能。转移是导致恶性肿瘤患者死亡率高、预后差的根本原因,目前其发生机制尚不清楚,已成为当今研究的重要课题。对于上皮来源的恶性肿瘤,其早期转移的主要方式是淋巴道转移,是影响患者预后的重要因素。因此,对于淋巴道转移机制进行研究,并在此基础上建立起相应的干预手段,将对恶性肿瘤患者具有重要的现实意义。原发性肝细胞癌( primary hepatocellular carcinoma )是常见的恶性肿瘤之一,在我国具有较高的发病率。尽管进行了大量的研究与探讨,但是肝癌转移的分子基础和机制尚不清楚。
Hca-F和Hca-P是一对来源于同一亲本细胞,而且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株。其中Hca-F是具有高转移潜能的细胞株,淋巴结转移率高于70%,Hca-P是低转移潜能的细胞株,淋巴结转移率低于30%。这为我们研究淋巴道转移机制提供了较为理想的细胞模型。
本实验组前期致力于小鼠肝癌淋巴道转移机制的研究。在对Hca-F细胞和Hca-P细胞的一系列研究中,利用抑制性消减杂交技术及高通量基因芯片技术筛选出了在Hca-F细胞和Hca-P细胞的差异表达基因;并采用定量蛋白质组学技术建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱。其中在基因水平上,JNK在Hca-F细胞中的表达显著高于Hca-P细胞,提示JNK可能与小鼠肝癌的淋巴道转移潜能相关。由于在人类组织细胞中具有JNK同源蛋白,因此,研究小鼠肝癌细胞株JNK与淋巴道转移的关系,对人类肝癌淋巴道转移的的防治具有重要意义。
c-Jun N端激酶( c-Jun N-terminal kinase, JNK )是一种能够特异性磷酸化c-Jun的蛋白激酶,位于重要的细胞信号传导通路-粘着斑(focal adhesion)通路上,是丝裂原活化蛋白激酶( mitogen-activated proteinkinase pathway,MAPK )家族的主要成员之一。MAPK是广泛存在于哺乳动物细胞内的一类丝氨酸/苏氨酸蛋白激酶,对于调节蛋白质的功能起着非常重要的作用,其本身及其介导的信号转导通路与肿瘤有着密切的关系。JNK对于细胞发育,形态形成和分化是必需的。JNK信号通路参与了多种生理过程,同时近来的研究表明JNK信号途径也参与某些疾病和肿瘤的发生发展过程,使JNK在临床上可做为一个潜在的分子治疗靶点。本研究以JNK为研究对象,探索其在Hca-F细胞株淋巴道转移过程中的作用。经检索,目前国内外尚无JNK与肝癌淋巴道转移关系的研究报道。
目的:1.从蛋白质水平上比较JNK在淋巴道转移潜能不同小鼠肝癌细胞系中的表达,为进一步研究肝癌淋巴道转移的机制奠定基础。2.构建pSilencer -shRNA表达载体,稳定转染Hca-F细胞获得JNK的表达水平显著下调的Hca-F细胞株。3.研究JNK表达水平的下降对小鼠肝癌细胞株Hca-F在细胞增殖、细胞迁移和侵袭能力等方面的影响,由此确定JNK表达水平与小鼠肝癌细胞淋巴道转移潜能间的关系。
方法:1.采用蛋白免疫印迹方法检测磷酸化JNK(p-JNK)在Hca-F、Hca-P细胞株中表达有无差异。2.由上海之江生物公司依据Gene Bank中小鼠JNK基因序列(序列登录号:NM_016700.3)设计shRNA靶序列,合成3条shRNA的DNA模板的两条单链,分别命名为shRNA-1、shRNA-2、shRNA-3,同时设计shRNA-无关序列。获取pSilencer? 3.1-H1 neo质粒,然后对质粒进行双酶切,shRNA合成序列退火,T4 DNA ligase连接质粒与三条shRNA的DNA模板和无关序列的退火后产物,连接产物分别命名为pSilencer-shRNA-1、pSilencer-shRNA-2、pSilencer- shRNA-3、pSilencer-shRNA-无关序列。pSilencer -shRNA质粒抽提获得表达载体,对三条pSilencer-shRNA表达载体经测序鉴定(引物为5'-GTTTTCCCA GTCACGAC-3'),结果与我们设计的三条shRNA靶序列和Gene Bank中序列比对。确定序列无误后,将三条pSilencer-shRNA表达载体和无关序列分别以脂质体法稳定转染Hca-F细胞,用含400ug/ml G418的完全培养基筛选,获得pSilencer- shRNA表达载体稳定转染的Hca-F细胞和无关序列对照组细胞。在mRNA和蛋白质水平检测三条shRNA对JNK的表达的抑制作用,同时设RNAi阴性对照组(稳定转染pSilencer- shRNA-无关序列的Hca-F细胞株)以及阳性对照组(正常的Hca-F细胞株),以筛选出RNAi效果最好的shRNA,用于后续实验。3.以三组细胞(分别为Hca-F细胞株、稳定转染pSilencer- shRNA-无关序列的Hca-F细胞株、经筛选的稳定转染pSilencer-shRNA表达载体的Hca-F细胞株)为实验对象。以CCK-8法检测三组细胞的细胞活力和分裂增殖能力;Transwell实验检测pSilencer-shRNA转染Hca-F细胞后对细胞迁移能力和侵袭能力的影响。
结果:1. p-JNK在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,在Hca-F细胞株中的表达显著高于Hca-P,二者间有显著性差异(P<0.01)。2.成功构建pSilencer-shRNA表达载体并稳定转染Hca-F细胞,3个表达载体中所含3条shRNA序列被正确检测出,靶序列与GenBank中序列一致。经筛选pSilencer-shRNA-2对JNK表达抑制的效果最好,转染后细胞株中JNK在mRNA和蛋白质水平的表达远低于其他2组及无关序列组和Hca-F细胞组( P<0.01,P<0.05 ), pSilencer -shRNA-2被选择作为后续实验的JNK稳定下调的细胞株。3. CCK-8实验结果表明JNK有促进细胞增殖的作用;体外迁移实验结果表明,JNK下调组的穿膜细胞数明显低于阳性和阴性对照组(17.89±1.65 vs 35.10±1.27, 31.83±1.69, P<0.05);体外侵袭实验结果表明,JNK下调组穿过人工基底膜的细胞数明显低于阳性和阴性对照组( 10.03±2.81vs19.73±1.60,19.23±1.24 ,P<0.05)。说明在Hca-F中下调JNK表达之后,其迁移能力、侵袭能力均显著下降。
结论:1. p-JNK在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,在Hca-F细胞株中的表达显著高于Hca-P,二者间有显著性差异。2.利用pSilencer? 3.1-H1 neo质粒和shRNA成功构建了三个pSilencer -shRNA表达载体,并稳定转染Hca-F细胞株,获得JNK表达稳定下调的Hca-F细胞株,经过筛选pSilencer -shRNA-2对Hca-F细胞中JNK抑制效果最明显,可作为后续研究的细胞株,为通过下调JNK在Hca-F的表达以研究JNK与肝癌淋巴道转移的关系打下了基础。3. JNK作为小鼠肝癌Hca-F和Hca-P细胞株的差异表达蛋白质,降低其表达水平可抑制小鼠肝癌细胞的增殖,降低小鼠肝癌细胞的迁移和侵袭能力,提示JNK可能是决定Hca-F淋巴道转移潜能的重要基因之一,在肝癌淋巴道转移中起着重要作用,其可能机制是JNK在粘着斑通路内通过调节paxillin、c-Jun、AP-1及由AP-1所调节的各种与肿瘤转移有关的基因所致。
Background: The most important difference between malignant and benign tumors is metastasis. Metastasis is the fundamental cause of high mortality and poor prognosis of the patients who suffer from malignant tumors. The mechanism is not clear until now and it is an important project for discussion. To epithelial malignant tumors, the early metastatic mode is lymphatic metastasis which is an important factor which affects prognosis of the patients. Therefore, it is very important to investigate the molecular mechanism of lymphatic metastasis and find new approaches of intervention . Primary hepatocellular carcinoma is one of the most common malignant tumor with high incidence of a disease in our country. Although a mass of researches have been done, its mechanism is not clear.
Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines which has the different rates of lymphatic metastasis . Hca-F is the cell lines whose metastatic rate is more than 70% and Hca-P less than 30%. They are the ideal models for the researches of mechanism of lymphatic metastasis .
Our experimental group has engaged in the research work for the mechanism of lymphatic metastasis. We have screened out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines by using suppressive subtractive hybridization and gene chip assays respectively and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. The expressing level of JNK was much higher in Hca-F than that in Hca-P cell lines in both gene and protein levels which showed that JNK maybe play an important role in lymphatic metastasis of mouse hepatocarcinoma. In the tissue of human being, there is JNK protein of the same origin with mouse.So it is very important to study the relationship between JNK and lymphatic metastasis of mouse hepatocarcinoma.
c-Jun N-terminal kinase ( JNK )is the kinase which can phosphates c-Jun in a specific way. INK is an important member of mitogen-activated protein kinase pathway( MAPK ) and is located in focal adhesion which is the important cellular signal transduction. MAPK is a Ser/Thr kinase which is widely present in mammal cells. It has a close relation with tumors. JNK is necessary to growth, morphogenesis and differentiation of cells. JNK signal transduction participates in many physiological process. The recent studies showed that JNK signal transduction also has a close relation with certain diseases and tumors. And it is regarded as a potential target for treatment in clinical practice.
In this study, JNK was being as the subject to discuss the role it played in process of lymphatic metastasis of Hca-F celllines. There was not such reports home and abroad by retrieval.
Objective: 1.To observe the expression levels of p-JNK in mouse hepatocarcinoma ascites cell lines Hca-F and Hca-P for further study of its function and mechanism in lymphatic metastasis of hepatocarcinoma. 2.To build expression vector of pSilencer-shRNA and obtain cellline Hca-F of markedly decreased expression of JNK by stable transfection. 3. To study the influence on proliferation, migration and invasion of mouse hepatocellular carcinoma Hca-F celllines after inhibition of JNK expression by shRNA and discuss the correlation between expressing level of JNK and lymphatic metastasis of mouse hepatocarcinoma.
Methods: 1. The expression of p-JNK protein was detected in Hca-F and Hca-P cells by Western Blot. 2. Three shRNAs (shRNA-1、shRNA-2、shRNA-3) and unrelated sequence were designed according to the gene sequence of JNK ( NM_016700.3 ). pSilencer? 3.1-H1 neo vector was obtained and BamH I and EcoR I enzymes were used to digest.The annealing of shRNA compositive sequence was performed and T4 DNA ligase was used to connect the pSilencer vector with three shRNAs and unrelated sequence after annealing. The connecting products was named pSilencer-shRNA-1,pSilencer-shRNA-2, pSilencer-shRNA-3 and pSilencer- shRNA-unrelated sequence. The expressing vector was obtained by extraction of vectors. The sequence of expressing vectors was identified by sequencing and the result was compared to that in gene bank. The three pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively and the most effective pSilencer-shRNA vector was selected according to the results of RT-PCR and Western blotting. The unrelated shRNA transfected Hca-F celllines and normal Hca-F celllines were negative control group and positive control group respectively. 3. The cell viability was evaluated by CCK-8. The cell migration and invasion capability was evaluated by transwell assays.
Results: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P (P<0.01). 2. The expression vector of pSilencer-shRNA was built and transfected to Hca-F cells successfully. The sequence of three shRNAs in expressing vectors was identified by sequencing and the result was identical to that in gene bank.The most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. The expressions of mRNA and protein of JNK in Hca-F cells after transfection of shRNA-2 were markedly decreased compared with the other groups ( P<0.01, P<0.05 ). pSilencer-shRNA-2 was chosen as the cellline for further study. 3. The result of CCK-8 showed that JNK could promote cell proliferation. Ttranswell assays showed that migration capability was decreased after knock-down of JNK expression in Hca-F cell lines (17.89±1.65 vs 35.10±1.27, 31.83±1.69, P<0.05 ). Invasion capability was decreased after knock-down of JNK expression in Hca-F cell lines(10.03±2.81 vs19.73±1.60 ,19.23±1.24,P<0.05 ).
Conclusions: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P . 2. The Hca-F cell lines of markedly knock-down of JNK expression by stable transfection was obtained and the most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. And pSilencer-shRNA-2 was chosen as the cellline for further study which provided a solid foundation for further studies of relationship between JNK and lymphatic metastasis of hepatocellular carcinoma. 3. Being as the different protein between Hca-F and Hca-P cell lines, the knock-down of JNK expression could inhibit proliferation, decrease migration and invasion capability of mouse hepatocellular carcinoma celllines. JNK maybe play an important role in lymphatic metastasis of hepatocellular carcinoma.The possible mechanism is that JNK located in focal adhesion can regulate paxillin, c-Jun, AP-1 and the genes in connection with tumor metastasis regulated by AP-1.
Hca-F和Hca-P是一对来源于同一亲本细胞,而且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株。其中Hca-F是具有高转移潜能的细胞株,淋巴结转移率高于70%,Hca-P是低转移潜能的细胞株,淋巴结转移率低于30%。这为我们研究淋巴道转移机制提供了较为理想的细胞模型。
本实验组前期致力于小鼠肝癌淋巴道转移机制的研究。在对Hca-F细胞和Hca-P细胞的一系列研究中,利用抑制性消减杂交技术及高通量基因芯片技术筛选出了在Hca-F细胞和Hca-P细胞的差异表达基因;并采用定量蛋白质组学技术建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱。其中在基因水平上,JNK在Hca-F细胞中的表达显著高于Hca-P细胞,提示JNK可能与小鼠肝癌的淋巴道转移潜能相关。由于在人类组织细胞中具有JNK同源蛋白,因此,研究小鼠肝癌细胞株JNK与淋巴道转移的关系,对人类肝癌淋巴道转移的的防治具有重要意义。
c-Jun N端激酶( c-Jun N-terminal kinase, JNK )是一种能够特异性磷酸化c-Jun的蛋白激酶,位于重要的细胞信号传导通路-粘着斑(focal adhesion)通路上,是丝裂原活化蛋白激酶( mitogen-activated proteinkinase pathway,MAPK )家族的主要成员之一。MAPK是广泛存在于哺乳动物细胞内的一类丝氨酸/苏氨酸蛋白激酶,对于调节蛋白质的功能起着非常重要的作用,其本身及其介导的信号转导通路与肿瘤有着密切的关系。JNK对于细胞发育,形态形成和分化是必需的。JNK信号通路参与了多种生理过程,同时近来的研究表明JNK信号途径也参与某些疾病和肿瘤的发生发展过程,使JNK在临床上可做为一个潜在的分子治疗靶点。本研究以JNK为研究对象,探索其在Hca-F细胞株淋巴道转移过程中的作用。经检索,目前国内外尚无JNK与肝癌淋巴道转移关系的研究报道。
目的:1.从蛋白质水平上比较JNK在淋巴道转移潜能不同小鼠肝癌细胞系中的表达,为进一步研究肝癌淋巴道转移的机制奠定基础。2.构建pSilencer -shRNA表达载体,稳定转染Hca-F细胞获得JNK的表达水平显著下调的Hca-F细胞株。3.研究JNK表达水平的下降对小鼠肝癌细胞株Hca-F在细胞增殖、细胞迁移和侵袭能力等方面的影响,由此确定JNK表达水平与小鼠肝癌细胞淋巴道转移潜能间的关系。
方法:1.采用蛋白免疫印迹方法检测磷酸化JNK(p-JNK)在Hca-F、Hca-P细胞株中表达有无差异。2.由上海之江生物公司依据Gene Bank中小鼠JNK基因序列(序列登录号:NM_016700.3)设计shRNA靶序列,合成3条shRNA的DNA模板的两条单链,分别命名为shRNA-1、shRNA-2、shRNA-3,同时设计shRNA-无关序列。获取pSilencer? 3.1-H1 neo质粒,然后对质粒进行双酶切,shRNA合成序列退火,T4 DNA ligase连接质粒与三条shRNA的DNA模板和无关序列的退火后产物,连接产物分别命名为pSilencer-shRNA-1、pSilencer-shRNA-2、pSilencer- shRNA-3、pSilencer-shRNA-无关序列。pSilencer -shRNA质粒抽提获得表达载体,对三条pSilencer-shRNA表达载体经测序鉴定(引物为5'-GTTTTCCCA GTCACGAC-3'),结果与我们设计的三条shRNA靶序列和Gene Bank中序列比对。确定序列无误后,将三条pSilencer-shRNA表达载体和无关序列分别以脂质体法稳定转染Hca-F细胞,用含400ug/ml G418的完全培养基筛选,获得pSilencer- shRNA表达载体稳定转染的Hca-F细胞和无关序列对照组细胞。在mRNA和蛋白质水平检测三条shRNA对JNK的表达的抑制作用,同时设RNAi阴性对照组(稳定转染pSilencer- shRNA-无关序列的Hca-F细胞株)以及阳性对照组(正常的Hca-F细胞株),以筛选出RNAi效果最好的shRNA,用于后续实验。3.以三组细胞(分别为Hca-F细胞株、稳定转染pSilencer- shRNA-无关序列的Hca-F细胞株、经筛选的稳定转染pSilencer-shRNA表达载体的Hca-F细胞株)为实验对象。以CCK-8法检测三组细胞的细胞活力和分裂增殖能力;Transwell实验检测pSilencer-shRNA转染Hca-F细胞后对细胞迁移能力和侵袭能力的影响。
结果:1. p-JNK在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,在Hca-F细胞株中的表达显著高于Hca-P,二者间有显著性差异(P<0.01)。2.成功构建pSilencer-shRNA表达载体并稳定转染Hca-F细胞,3个表达载体中所含3条shRNA序列被正确检测出,靶序列与GenBank中序列一致。经筛选pSilencer-shRNA-2对JNK表达抑制的效果最好,转染后细胞株中JNK在mRNA和蛋白质水平的表达远低于其他2组及无关序列组和Hca-F细胞组( P<0.01,P<0.05 ), pSilencer -shRNA-2被选择作为后续实验的JNK稳定下调的细胞株。3. CCK-8实验结果表明JNK有促进细胞增殖的作用;体外迁移实验结果表明,JNK下调组的穿膜细胞数明显低于阳性和阴性对照组(17.89±1.65 vs 35.10±1.27, 31.83±1.69, P<0.05);体外侵袭实验结果表明,JNK下调组穿过人工基底膜的细胞数明显低于阳性和阴性对照组( 10.03±2.81vs19.73±1.60,19.23±1.24 ,P<0.05)。说明在Hca-F中下调JNK表达之后,其迁移能力、侵袭能力均显著下降。
结论:1. p-JNK在小鼠腹水型肝癌细胞系Hca-F和Hca-P均有表达,在Hca-F细胞株中的表达显著高于Hca-P,二者间有显著性差异。2.利用pSilencer? 3.1-H1 neo质粒和shRNA成功构建了三个pSilencer -shRNA表达载体,并稳定转染Hca-F细胞株,获得JNK表达稳定下调的Hca-F细胞株,经过筛选pSilencer -shRNA-2对Hca-F细胞中JNK抑制效果最明显,可作为后续研究的细胞株,为通过下调JNK在Hca-F的表达以研究JNK与肝癌淋巴道转移的关系打下了基础。3. JNK作为小鼠肝癌Hca-F和Hca-P细胞株的差异表达蛋白质,降低其表达水平可抑制小鼠肝癌细胞的增殖,降低小鼠肝癌细胞的迁移和侵袭能力,提示JNK可能是决定Hca-F淋巴道转移潜能的重要基因之一,在肝癌淋巴道转移中起着重要作用,其可能机制是JNK在粘着斑通路内通过调节paxillin、c-Jun、AP-1及由AP-1所调节的各种与肿瘤转移有关的基因所致。
Background: The most important difference between malignant and benign tumors is metastasis. Metastasis is the fundamental cause of high mortality and poor prognosis of the patients who suffer from malignant tumors. The mechanism is not clear until now and it is an important project for discussion. To epithelial malignant tumors, the early metastatic mode is lymphatic metastasis which is an important factor which affects prognosis of the patients. Therefore, it is very important to investigate the molecular mechanism of lymphatic metastasis and find new approaches of intervention . Primary hepatocellular carcinoma is one of the most common malignant tumor with high incidence of a disease in our country. Although a mass of researches have been done, its mechanism is not clear.
Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines which has the different rates of lymphatic metastasis . Hca-F is the cell lines whose metastatic rate is more than 70% and Hca-P less than 30%. They are the ideal models for the researches of mechanism of lymphatic metastasis .
Our experimental group has engaged in the research work for the mechanism of lymphatic metastasis. We have screened out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines by using suppressive subtractive hybridization and gene chip assays respectively and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. The expressing level of JNK was much higher in Hca-F than that in Hca-P cell lines in both gene and protein levels which showed that JNK maybe play an important role in lymphatic metastasis of mouse hepatocarcinoma. In the tissue of human being, there is JNK protein of the same origin with mouse.So it is very important to study the relationship between JNK and lymphatic metastasis of mouse hepatocarcinoma.
c-Jun N-terminal kinase ( JNK )is the kinase which can phosphates c-Jun in a specific way. INK is an important member of mitogen-activated protein kinase pathway( MAPK ) and is located in focal adhesion which is the important cellular signal transduction. MAPK is a Ser/Thr kinase which is widely present in mammal cells. It has a close relation with tumors. JNK is necessary to growth, morphogenesis and differentiation of cells. JNK signal transduction participates in many physiological process. The recent studies showed that JNK signal transduction also has a close relation with certain diseases and tumors. And it is regarded as a potential target for treatment in clinical practice.
In this study, JNK was being as the subject to discuss the role it played in process of lymphatic metastasis of Hca-F celllines. There was not such reports home and abroad by retrieval.
Objective: 1.To observe the expression levels of p-JNK in mouse hepatocarcinoma ascites cell lines Hca-F and Hca-P for further study of its function and mechanism in lymphatic metastasis of hepatocarcinoma. 2.To build expression vector of pSilencer-shRNA and obtain cellline Hca-F of markedly decreased expression of JNK by stable transfection. 3. To study the influence on proliferation, migration and invasion of mouse hepatocellular carcinoma Hca-F celllines after inhibition of JNK expression by shRNA and discuss the correlation between expressing level of JNK and lymphatic metastasis of mouse hepatocarcinoma.
Methods: 1. The expression of p-JNK protein was detected in Hca-F and Hca-P cells by Western Blot. 2. Three shRNAs (shRNA-1、shRNA-2、shRNA-3) and unrelated sequence were designed according to the gene sequence of JNK ( NM_016700.3 ). pSilencer? 3.1-H1 neo vector was obtained and BamH I and EcoR I enzymes were used to digest.The annealing of shRNA compositive sequence was performed and T4 DNA ligase was used to connect the pSilencer vector with three shRNAs and unrelated sequence after annealing. The connecting products was named pSilencer-shRNA-1,pSilencer-shRNA-2, pSilencer-shRNA-3 and pSilencer- shRNA-unrelated sequence. The expressing vector was obtained by extraction of vectors. The sequence of expressing vectors was identified by sequencing and the result was compared to that in gene bank. The three pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively and the most effective pSilencer-shRNA vector was selected according to the results of RT-PCR and Western blotting. The unrelated shRNA transfected Hca-F celllines and normal Hca-F celllines were negative control group and positive control group respectively. 3. The cell viability was evaluated by CCK-8. The cell migration and invasion capability was evaluated by transwell assays.
Results: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P (P<0.01). 2. The expression vector of pSilencer-shRNA was built and transfected to Hca-F cells successfully. The sequence of three shRNAs in expressing vectors was identified by sequencing and the result was identical to that in gene bank.The most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. The expressions of mRNA and protein of JNK in Hca-F cells after transfection of shRNA-2 were markedly decreased compared with the other groups ( P<0.01, P<0.05 ). pSilencer-shRNA-2 was chosen as the cellline for further study. 3. The result of CCK-8 showed that JNK could promote cell proliferation. Ttranswell assays showed that migration capability was decreased after knock-down of JNK expression in Hca-F cell lines (17.89±1.65 vs 35.10±1.27, 31.83±1.69, P<0.05 ). Invasion capability was decreased after knock-down of JNK expression in Hca-F cell lines(10.03±2.81 vs19.73±1.60 ,19.23±1.24,P<0.05 ).
Conclusions: 1. The expreesion of p-JNK in Hca-F was significantly higher than that in Hca-P . 2. The Hca-F cell lines of markedly knock-down of JNK expression by stable transfection was obtained and the most effective shRNA was pSilencer-shRNA-2 which inhibited JNK expression. And pSilencer-shRNA-2 was chosen as the cellline for further study which provided a solid foundation for further studies of relationship between JNK and lymphatic metastasis of hepatocellular carcinoma. 3. Being as the different protein between Hca-F and Hca-P cell lines, the knock-down of JNK expression could inhibit proliferation, decrease migration and invasion capability of mouse hepatocellular carcinoma celllines. JNK maybe play an important role in lymphatic metastasis of hepatocellular carcinoma.The possible mechanism is that JNK located in focal adhesion can regulate paxillin, c-Jun, AP-1 and the genes in connection with tumor metastasis regulated by AP-1.
引文
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