黑素瘤中过度表达c-FLIP异常激活β-catenin/TCF路径的研究
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摘要
第一部分c-FLIP、浆／核β-catenin的表达及二者表达的相关性研究
目的观察细胞型FAS结合蛋白样白介素-1β转换酶抑制蛋白(cellular FADD-likeinterleukin -1βconvening enzyme inhibitory protein，c-FLIP)、浆／核β-连环蛋白(β-catenin)在恶性黑素瘤组织、色素痣、银屑病及正常包皮组织中的表达，探讨两者的相关性。
方法以20例色素痣组织及20例正常包皮作为对照，应用免疫组织化学方法检测77例恶性黑素瘤组织，20例银屑病组织中c-FLIP、浆／核β-catenin的表达，对蛋白表达行半定量分析。
结果c-FLIP、浆／核β-catenin、周期素蛋白D1(cyclinD1)在77例恶性黑素瘤组织中的阳性表达率分别为65.0％、32.5％和31.2％，且三者的表达有相关性；c-FLIP的表达在黑素瘤组织中明显增高，且与组织类型相关；在正常色素痣中c-FLIP、浆／核β-catenin及cyclinD1无阳性表达或偶见阳性表达。银屑病组织中c-FLIP的表达增强，与正常对照相比，差异有显著性；几无浆／核β-catenin的表达；在银屑病组织中未观察到三者有MM中的类似关系。
结论c-FLIP高表达可能参与MM的发生、发展；在MM中高表达的c-FLIP可能与β-catenin的浆／核表达有关，从而影响β-连环蛋白／T细胞因子(β-catenin／TCF)下游基因cyclinD1的表达。
第二部分pCMV-Tag2B-FLIP_(L／P43／S)以及c-FLIP-shRNA载体的构建和表达
目的构建细胞FLICE蛋白(c-FLIP)和pCMV-Tag2B融合蛋白基因的重组真核表达载体pCMV-Tag2B-FLIP_L、pCMV-Tag2B-FLIP_S和pCMV-Tag2B-FLIP_(p43)。并转染至A375细胞中进行表达，鉴定融合蛋白的生物学活性。针对c-FLIP各亚型共有靶点设计shRNA分子，连接于真核表达载体pSilencer 3.1-H1 neo，构建pSilencer-c-FLIP质粒，下调c-FLIP的表达。为后续实验奠定基础。
方法从白血病Jurkat细胞中提取总RNA，分离提取mRNA，逆转录获得cDNA。设计三对引物引入酶切位点xhoⅠ和EcoRI，再用PCR的方法，分别扩增FLIP_L、FLIP_(P43)和FLIP_S序列。将获得的基因克隆至pGEM-T载体(FLIP-T)，转化大肠杆菌DH5a，进行蓝白筛选，挑取阳性克隆扩增。提取质粒以xhoⅠ、EcoRI双酶切鉴定，并测序证实正确后，亚克隆至pCMV-Tag 2B，获得pCMV-Tag 2B-FLIP_(L／P43／S)，转染A375细胞，获得稳转细胞系。设计针对c-FLIP的shRNA，酶切pSilencer 3.1-H1 neo后，将合成的shRNA通过BamHⅠ和HindⅢ连接于载体，构建真核载体，表达siRNA。
结果测序结果表明获得了正确的FLIP_L、FLIP_(P43)和FLIP_S基因序列；亚克隆至pGEM-T载体后，xhoⅠ和EcoRI双酶切可见约1.4Kb、1.1Kb和0.6kb的小片段和约4.3Kb的大片段，与理论计算值相符，表明pCMV-Tag 2B-FLIP_L、pCMV-Tag 2B-FLIP_(P43)和pCMV-Tag 2B-FLIP_S真核表达载体构建成功；表达载体转染A375细胞，G418筛选后用westernblot、流式分析鉴定其为稳定转染细胞株。成功pSilencer-c-FLIP构建，转染入黑素瘤细胞中，能够下调c-FLIP各亚型的表达。
结论成功构建pCMV-Tag 2B-FLIP_(L／P43／S)融合蛋白的真核表达载体，融合蛋白可在A375细胞中表达；成功构建pSilencer-c-FLIP载体，在黑素瘤细胞中能够下调c-FLIP各亚型的表达，本实验为进一步研究FLIP对β-catenin／TCF通路的影响奠定基础。
第三部分c-FUP_(L／P43)通过β-catenin／TCF激活cyclinD1的表达
目的观察c-FLIP的改变是否激活β-catenin／TCF通路，引起下游基因的变化；观察c-FLIP各亚型在上述作用中的差异。
方法利用pCMV-Tag-2B-c-FLIP真核表达载体上调低表达c-FLIP的黑素瘤A375细胞系中各亚型c-FLIP的表达水平，用western blot及细胞免疫荧光观察β-catenin定量、定位的改变；用TOP／FOP双荧光报告基因分析观察转录因子TCF的活性，判断是否通过β-catenin／TCF路径启动下游基因的转录；western blot检测β-catenin／TCF路径下游基因cyclinD1蛋白水平的改变；比较c-FLIP各亚型在上述过程中的作用差异；同时利用pSilencer-c-FLIP载体下调c-FLIP各亚型的水平，观察对上述过程的影响，明确c-FLIP各亚型功能特异性。
结果在A375细胞中，高表达c-FLIP_(L／P43)可以增加胞核及胞浆中β-catenin的表达；通过TOP／FOP双荧光报告基因检测，证明确实通过β-catenin／TCF路径启动下游基因的转录；检测下游基因cyclinD1的蛋白水平明显升高。行RNA干扰c-FLIP的表达后，上述结果部分逆转。但在高表达c-FLIP_S的细胞中并未表现出上述现象。
结论过度表达c-FLIP_(L／P43)可以异常激活β-catenin／TCF路径，诱导下游基因的表达。
第四部分c-FLIP对A375细胞生物学特性的影响
目的观察c-FLIP各亚型对A375细胞的生长、增殖、凋亡及转化能力的影响。
方法在pCMV-Tag-2B-c-FLIP_(L／P43／S)稳定转染细胞中，运用绝对细胞计数对c-FLIP各亚型稳定转染细胞绘制生长曲线，比较其与对照组的差异；用MTT法检测各稳定转染细胞增殖能力的差异；用Annexin v／PI双染细胞，检测各组细胞凋亡率的差异；用双层软琼脂克隆形成实验检测各组细胞体外转化能力的差异。同时运用shRNA干扰，下调c-FLIP各亚型的表达，验证c-FLIP所产生的细胞生物学特异改变的特异性。
结果A375／c-FLIP_(L／p43／S)细胞的生长速度均快于A375／Control细胞，其中，A375／c-FLIP_L与A375／c-FLIP_(P43)细胞的生长速度基本一致，A375／c-FLIP_S较二者慢；行MTT法检测细胞增殖能力，与细胞生长曲线结果类似，A375／c-FLIP_(L／p43／S)细胞增殖能力强于A375／Control细胞，其中，A375／c-FLIP_S细胞增殖能力略低于A375／c-FLIP_L及A375／c-FLIP_(P43)细胞；Annexin v／PI双染检测细胞凋亡率，A375／c-FLIP_(L／p43／S)细胞略低于A375／Control细胞；行双层软琼脂克隆形成实验，A375／c-FLIP_(L／p43／S)细胞克隆形成率高于对照细胞，其中，A375／c-FLIP_L与A375／c-FLIP_(P43)细胞克隆形成率高于A375／c-FLIP_S细胞。用shRNA干扰，下调c-FLIP各亚型的表达，上述效应部分得到逆转。
结论c-FLIP各亚型均可以促进细胞生长、增殖及转化，其中c-FLIP_L及c-FLIP_(P43)的能力更强，但在抗凋亡效应中，c-FLIP的三种亚型未表现出显著差异。
PartⅠExpression of c-FLIP and nuclear or cytoplasmicβ-catenin andthe relationship between them
Objective: To study expression of c-FLIP,β-catenin in melanoma, nevus, psoriasis andacrobystia and explore the relationship between them. To investigate the correlationbetween histopathological characteristics and expression of those protein in melanoma.
Methods: The protein of c-FLIP,β-catenin and cyclinD1 were detecded byimmunohistochemistry and quantitative analysis the relationship among c-FLIP, nuclear orcytoplasmicβ-catenin and cyclinD1.
Results: In melanoma, positive expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were observed in 65.0% (50 case), 32.5% (25 case) and 31.2%(24 case), respectively,and the positive relationship were observed among c-FLIP and nuclear or cytoplasmicβ-catenin,cyclinD1. Increased those protein expression in melanoma lesion were significantly associatedwith histological type. In nevus, the expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were lower than that in melanoma. In psoriasis, the expression of c-FLIP was increased,and nearly no expression of nuclear or cytoplasmicβ-catenin.
Conclusions: Our data suggested that increased c-FLIP, nuclear or cytoplasmicβ-catenin and cyclinD1 protein in melanoma may contribute to the development ofmelanoma. The increased expression of c-FLIP maybe have relationship with theexpression of nuclear or cytoplasmicβ-catenin, and then induce the expression ofdownstream genes ofβ-catenin/T Cell Factor(β-catenin/TCF), such as, cyclinD1.
PartⅡConstruction and expression of pCMV-Tag2B-FLIP_(L/P43/S) andc-FLIP-shRNA expression vector
Objective: To construct pCMV-Tag2B-c-FLIP_(L/P43/S) eukaryotic expression vector,transfect into melanoma cells and investigate the expression. Construct c-FLIP-shRNAeukaryotic expression vector, transfect into melanoma cells and explore the effect ofinterference for c-FLIP.
Methods: Extracted total RNA from Jurkat cells and then isolated mRNA, and thenreverse transcripted to obtain cDNA. Three pairs of PCR primers were designed and used toamplify the c-FLIP_L, c-FLIP_(P43) and c-FLIP_S gene respectively. Two restrictionendonuclease sites xho I and EcoRI were introduced. The c-FLIP_L or c-FLIP_(P43) or c-FLIP_Sgene was inserted into the pGME-T vector. The recombinant plasmid was transfomed intoE. coli DH5α. Positive cell clones were indentified by restriction enzyme digestion andsequenced. The c-FLIP_L or c-FLIP_(P43) or c-FLIP_S gene was subcloned into the eukaryoticfusion protein expression vector pCMV-Tag-2B to obtain pCMV-Tag-2B-c-FLIP_(L/P43/S).After identification, pCMV-Tag-2B-c-FLIP_(L/P43/S) plasmid was transfected into A375 cellsand get the stable transfectants of A375/c-FLIP_(L/P43/S). Dectected the expression ofc-FLIP_(L/P43/S) in the A375/c-FLIP_(L/P43/S). To synthesize the sequence target human c-FLIPgene, c-FLIP-shRNA, and insrted into pSilencer 3.1-H1 neo vetor by BamHⅠand HindⅢrestriction endonuclease sites to get pSilencer-c-FLIP. After identification, pSilencer-c-FLIPplasmid was transfected into A375 cells and observe the c-FLIP_(L/P43/S) expression.
Results: The c-FLIP_(L/P43/S) gene was identified by sequencing, and the A375/c-FLIP_(L/P43/S)and pSilencer-c-FLIP were identified by western blot and flow cytometry(FCM).
Conclusions: The pCMV-Tag-2B-c-FLIP_(L/P43/S) and pSilencer-c-FLIP eukaryotic expressionvector were successfully constructed . The expression of c-FLIP_(L/P43/S) protein could beregulated by those vectors. Those vectors could be used in future research for melanoma.
PartⅢc-FLIP_(L/P43) activateβ-catenin/TCF pathway and induce theexpression of cyclinD1
Objective: To observe whether the change of c-FLIP protein expression could lead toaberrant activation of beta-catenin/TCF pathway in melanoma and induce gene expressionof the downstream. Explore the function of c-FLIP_L, c-FLIPP_(43) and c-FLIP_S during theprocess above.
Methods: To detecte the expression of cytoplasm or nuclearβ-catenin protein andcyclinD1 protein in stable clone A375/c-FLIP_(L/P43/S) by western blot. To observe the locationofβ-catenin in stable clone A375/c- FLIP_(L/P43/S) by cell immunofluorescence, and toobserve the activity of transcription factor T cell factor by TOP/FOP Flash. Compare thedifference of function among A375/c- FLIP_(L/P43/S). To reduce the expression of c-FLIP_(L/P43)by pSilencer-c-FLIP vector, and then observe the effect during the process above.
Results: In stable clone A375/c-FLIP_(L/P43), we observed the expression of cytoplasm andnuclearμ-catenin was increased. At the same time, we found in those cells, the expressionof cyclinD1 was increased. By the TOP/FOP Flash, we dectected the activition of TCF wasup-regulated. However, we did not found the same results in the A375/c-FLIPs cells.
Couclusions: Overexpression of c-FLIP_(L/P43) in A375 cell could promote the nulcear/cytoplasm accumulation ofβ-catenin and the increase transcriptional activity ofβ-catenin/TCF pathway and then induce the downstream gene expression, such as,cyclinD1.
PartⅣThe effects of c-FLIP on bionomics of melanoma cell A375
Objective: observe the effects of three type of c-FLIP on bionomics of melanoma cellA375, such as, growth, proliferation, apoptosis and transformation activity.
Methods: To count cells of A375/c-FLIP_(L/P43/S) and obtain the cells' growth curve, andthen to compare the difference among those type of cells. To detecet the proliferationalactivity of A375/ c-FLIP_(L/P43/S) by MTT; to observe the rate of apoptosis by by flowcytometry(FCM) using annexin V and propidium iodide(PI) double staining; to detect thetransformation activity of those cells by soft agar assay. At the same time, suppress theexpression of c-FLIP by RNAi and then observe the effects on A375.
Results: In comparison to control group, c-FLIP_(L/P43/S) over-expression promote thegrowth and proliferation activity of A375 cells, c-FLIP_(L/P43) have more remarkable effect onA375 cells' activity of growth and proliferation than that of c-FLIP_S. Compare to the controlcells, c-FLIP _(L/P43/S) can resist the apoptosis. Sofa agar assay indicate c-FLIP _(L/P43/S)over-expression promote the transformation activity of A375. c-FLIP _(L/P43) have more effectson transformation activity of A375 than that of c-FLIP_S. Using siRNA specifically knockingdown the expression of c-FLIP, those effects above have been reversed partly.
Conclusions: The three subtypes of c-FLIP can promote the activity of growth,proliferation and transformation. As to resist to apoptosis, c-FLIP_(L/P43) have more effectsthan that of c-FLIP_S.
目的观察细胞型FAS结合蛋白样白介素-1β转换酶抑制蛋白(cellular FADD-likeinterleukin -1βconvening enzyme inhibitory protein，c-FLIP)、浆／核β-连环蛋白(β-catenin)在恶性黑素瘤组织、色素痣、银屑病及正常包皮组织中的表达，探讨两者的相关性。
方法以20例色素痣组织及20例正常包皮作为对照，应用免疫组织化学方法检测77例恶性黑素瘤组织，20例银屑病组织中c-FLIP、浆／核β-catenin的表达，对蛋白表达行半定量分析。
结果c-FLIP、浆／核β-catenin、周期素蛋白D1(cyclinD1)在77例恶性黑素瘤组织中的阳性表达率分别为65.0％、32.5％和31.2％，且三者的表达有相关性；c-FLIP的表达在黑素瘤组织中明显增高，且与组织类型相关；在正常色素痣中c-FLIP、浆／核β-catenin及cyclinD1无阳性表达或偶见阳性表达。银屑病组织中c-FLIP的表达增强，与正常对照相比，差异有显著性；几无浆／核β-catenin的表达；在银屑病组织中未观察到三者有MM中的类似关系。
结论c-FLIP高表达可能参与MM的发生、发展；在MM中高表达的c-FLIP可能与β-catenin的浆／核表达有关，从而影响β-连环蛋白／T细胞因子(β-catenin／TCF)下游基因cyclinD1的表达。
第二部分pCMV-Tag2B-FLIP_(L／P43／S)以及c-FLIP-shRNA载体的构建和表达
目的构建细胞FLICE蛋白(c-FLIP)和pCMV-Tag2B融合蛋白基因的重组真核表达载体pCMV-Tag2B-FLIP_L、pCMV-Tag2B-FLIP_S和pCMV-Tag2B-FLIP_(p43)。并转染至A375细胞中进行表达，鉴定融合蛋白的生物学活性。针对c-FLIP各亚型共有靶点设计shRNA分子，连接于真核表达载体pSilencer 3.1-H1 neo，构建pSilencer-c-FLIP质粒，下调c-FLIP的表达。为后续实验奠定基础。
方法从白血病Jurkat细胞中提取总RNA，分离提取mRNA，逆转录获得cDNA。设计三对引物引入酶切位点xhoⅠ和EcoRI，再用PCR的方法，分别扩增FLIP_L、FLIP_(P43)和FLIP_S序列。将获得的基因克隆至pGEM-T载体(FLIP-T)，转化大肠杆菌DH5a，进行蓝白筛选，挑取阳性克隆扩增。提取质粒以xhoⅠ、EcoRI双酶切鉴定，并测序证实正确后，亚克隆至pCMV-Tag 2B，获得pCMV-Tag 2B-FLIP_(L／P43／S)，转染A375细胞，获得稳转细胞系。设计针对c-FLIP的shRNA，酶切pSilencer 3.1-H1 neo后，将合成的shRNA通过BamHⅠ和HindⅢ连接于载体，构建真核载体，表达siRNA。
结果测序结果表明获得了正确的FLIP_L、FLIP_(P43)和FLIP_S基因序列；亚克隆至pGEM-T载体后，xhoⅠ和EcoRI双酶切可见约1.4Kb、1.1Kb和0.6kb的小片段和约4.3Kb的大片段，与理论计算值相符，表明pCMV-Tag 2B-FLIP_L、pCMV-Tag 2B-FLIP_(P43)和pCMV-Tag 2B-FLIP_S真核表达载体构建成功；表达载体转染A375细胞，G418筛选后用westernblot、流式分析鉴定其为稳定转染细胞株。成功pSilencer-c-FLIP构建，转染入黑素瘤细胞中，能够下调c-FLIP各亚型的表达。
结论成功构建pCMV-Tag 2B-FLIP_(L／P43／S)融合蛋白的真核表达载体，融合蛋白可在A375细胞中表达；成功构建pSilencer-c-FLIP载体，在黑素瘤细胞中能够下调c-FLIP各亚型的表达，本实验为进一步研究FLIP对β-catenin／TCF通路的影响奠定基础。
第三部分c-FUP_(L／P43)通过β-catenin／TCF激活cyclinD1的表达
目的观察c-FLIP的改变是否激活β-catenin／TCF通路，引起下游基因的变化；观察c-FLIP各亚型在上述作用中的差异。
方法利用pCMV-Tag-2B-c-FLIP真核表达载体上调低表达c-FLIP的黑素瘤A375细胞系中各亚型c-FLIP的表达水平，用western blot及细胞免疫荧光观察β-catenin定量、定位的改变；用TOP／FOP双荧光报告基因分析观察转录因子TCF的活性，判断是否通过β-catenin／TCF路径启动下游基因的转录；western blot检测β-catenin／TCF路径下游基因cyclinD1蛋白水平的改变；比较c-FLIP各亚型在上述过程中的作用差异；同时利用pSilencer-c-FLIP载体下调c-FLIP各亚型的水平，观察对上述过程的影响，明确c-FLIP各亚型功能特异性。
结果在A375细胞中，高表达c-FLIP_(L／P43)可以增加胞核及胞浆中β-catenin的表达；通过TOP／FOP双荧光报告基因检测，证明确实通过β-catenin／TCF路径启动下游基因的转录；检测下游基因cyclinD1的蛋白水平明显升高。行RNA干扰c-FLIP的表达后，上述结果部分逆转。但在高表达c-FLIP_S的细胞中并未表现出上述现象。
结论过度表达c-FLIP_(L／P43)可以异常激活β-catenin／TCF路径，诱导下游基因的表达。
第四部分c-FLIP对A375细胞生物学特性的影响
目的观察c-FLIP各亚型对A375细胞的生长、增殖、凋亡及转化能力的影响。
方法在pCMV-Tag-2B-c-FLIP_(L／P43／S)稳定转染细胞中，运用绝对细胞计数对c-FLIP各亚型稳定转染细胞绘制生长曲线，比较其与对照组的差异；用MTT法检测各稳定转染细胞增殖能力的差异；用Annexin v／PI双染细胞，检测各组细胞凋亡率的差异；用双层软琼脂克隆形成实验检测各组细胞体外转化能力的差异。同时运用shRNA干扰，下调c-FLIP各亚型的表达，验证c-FLIP所产生的细胞生物学特异改变的特异性。
结果A375／c-FLIP_(L／p43／S)细胞的生长速度均快于A375／Control细胞，其中，A375／c-FLIP_L与A375／c-FLIP_(P43)细胞的生长速度基本一致，A375／c-FLIP_S较二者慢；行MTT法检测细胞增殖能力，与细胞生长曲线结果类似，A375／c-FLIP_(L／p43／S)细胞增殖能力强于A375／Control细胞，其中，A375／c-FLIP_S细胞增殖能力略低于A375／c-FLIP_L及A375／c-FLIP_(P43)细胞；Annexin v／PI双染检测细胞凋亡率，A375／c-FLIP_(L／p43／S)细胞略低于A375／Control细胞；行双层软琼脂克隆形成实验，A375／c-FLIP_(L／p43／S)细胞克隆形成率高于对照细胞，其中，A375／c-FLIP_L与A375／c-FLIP_(P43)细胞克隆形成率高于A375／c-FLIP_S细胞。用shRNA干扰，下调c-FLIP各亚型的表达，上述效应部分得到逆转。
结论c-FLIP各亚型均可以促进细胞生长、增殖及转化，其中c-FLIP_L及c-FLIP_(P43)的能力更强，但在抗凋亡效应中，c-FLIP的三种亚型未表现出显著差异。
PartⅠExpression of c-FLIP and nuclear or cytoplasmicβ-catenin andthe relationship between them
Objective: To study expression of c-FLIP,β-catenin in melanoma, nevus, psoriasis andacrobystia and explore the relationship between them. To investigate the correlationbetween histopathological characteristics and expression of those protein in melanoma.
Methods: The protein of c-FLIP,β-catenin and cyclinD1 were detecded byimmunohistochemistry and quantitative analysis the relationship among c-FLIP, nuclear orcytoplasmicβ-catenin and cyclinD1.
Results: In melanoma, positive expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were observed in 65.0% (50 case), 32.5% (25 case) and 31.2%(24 case), respectively,and the positive relationship were observed among c-FLIP and nuclear or cytoplasmicβ-catenin,cyclinD1. Increased those protein expression in melanoma lesion were significantly associatedwith histological type. In nevus, the expression of c-FLIP, nuclear or cytoplasmicβ-catenin andcyclinD1 were lower than that in melanoma. In psoriasis, the expression of c-FLIP was increased,and nearly no expression of nuclear or cytoplasmicβ-catenin.
Conclusions: Our data suggested that increased c-FLIP, nuclear or cytoplasmicβ-catenin and cyclinD1 protein in melanoma may contribute to the development ofmelanoma. The increased expression of c-FLIP maybe have relationship with theexpression of nuclear or cytoplasmicβ-catenin, and then induce the expression ofdownstream genes ofβ-catenin/T Cell Factor(β-catenin/TCF), such as, cyclinD1.
PartⅡConstruction and expression of pCMV-Tag2B-FLIP_(L/P43/S) andc-FLIP-shRNA expression vector
Objective: To construct pCMV-Tag2B-c-FLIP_(L/P43/S) eukaryotic expression vector,transfect into melanoma cells and investigate the expression. Construct c-FLIP-shRNAeukaryotic expression vector, transfect into melanoma cells and explore the effect ofinterference for c-FLIP.
Methods: Extracted total RNA from Jurkat cells and then isolated mRNA, and thenreverse transcripted to obtain cDNA. Three pairs of PCR primers were designed and used toamplify the c-FLIP_L, c-FLIP_(P43) and c-FLIP_S gene respectively. Two restrictionendonuclease sites xho I and EcoRI were introduced. The c-FLIP_L or c-FLIP_(P43) or c-FLIP_Sgene was inserted into the pGME-T vector. The recombinant plasmid was transfomed intoE. coli DH5α. Positive cell clones were indentified by restriction enzyme digestion andsequenced. The c-FLIP_L or c-FLIP_(P43) or c-FLIP_S gene was subcloned into the eukaryoticfusion protein expression vector pCMV-Tag-2B to obtain pCMV-Tag-2B-c-FLIP_(L/P43/S).After identification, pCMV-Tag-2B-c-FLIP_(L/P43/S) plasmid was transfected into A375 cellsand get the stable transfectants of A375/c-FLIP_(L/P43/S). Dectected the expression ofc-FLIP_(L/P43/S) in the A375/c-FLIP_(L/P43/S). To synthesize the sequence target human c-FLIPgene, c-FLIP-shRNA, and insrted into pSilencer 3.1-H1 neo vetor by BamHⅠand HindⅢrestriction endonuclease sites to get pSilencer-c-FLIP. After identification, pSilencer-c-FLIPplasmid was transfected into A375 cells and observe the c-FLIP_(L/P43/S) expression.
Results: The c-FLIP_(L/P43/S) gene was identified by sequencing, and the A375/c-FLIP_(L/P43/S)and pSilencer-c-FLIP were identified by western blot and flow cytometry(FCM).
Conclusions: The pCMV-Tag-2B-c-FLIP_(L/P43/S) and pSilencer-c-FLIP eukaryotic expressionvector were successfully constructed . The expression of c-FLIP_(L/P43/S) protein could beregulated by those vectors. Those vectors could be used in future research for melanoma.
PartⅢc-FLIP_(L/P43) activateβ-catenin/TCF pathway and induce theexpression of cyclinD1
Objective: To observe whether the change of c-FLIP protein expression could lead toaberrant activation of beta-catenin/TCF pathway in melanoma and induce gene expressionof the downstream. Explore the function of c-FLIP_L, c-FLIPP_(43) and c-FLIP_S during theprocess above.
Methods: To detecte the expression of cytoplasm or nuclearβ-catenin protein andcyclinD1 protein in stable clone A375/c-FLIP_(L/P43/S) by western blot. To observe the locationofβ-catenin in stable clone A375/c- FLIP_(L/P43/S) by cell immunofluorescence, and toobserve the activity of transcription factor T cell factor by TOP/FOP Flash. Compare thedifference of function among A375/c- FLIP_(L/P43/S). To reduce the expression of c-FLIP_(L/P43)by pSilencer-c-FLIP vector, and then observe the effect during the process above.
Results: In stable clone A375/c-FLIP_(L/P43), we observed the expression of cytoplasm andnuclearμ-catenin was increased. At the same time, we found in those cells, the expressionof cyclinD1 was increased. By the TOP/FOP Flash, we dectected the activition of TCF wasup-regulated. However, we did not found the same results in the A375/c-FLIPs cells.
Couclusions: Overexpression of c-FLIP_(L/P43) in A375 cell could promote the nulcear/cytoplasm accumulation ofβ-catenin and the increase transcriptional activity ofβ-catenin/TCF pathway and then induce the downstream gene expression, such as,cyclinD1.
PartⅣThe effects of c-FLIP on bionomics of melanoma cell A375
Objective: observe the effects of three type of c-FLIP on bionomics of melanoma cellA375, such as, growth, proliferation, apoptosis and transformation activity.
Methods: To count cells of A375/c-FLIP_(L/P43/S) and obtain the cells' growth curve, andthen to compare the difference among those type of cells. To detecet the proliferationalactivity of A375/ c-FLIP_(L/P43/S) by MTT; to observe the rate of apoptosis by by flowcytometry(FCM) using annexin V and propidium iodide(PI) double staining; to detect thetransformation activity of those cells by soft agar assay. At the same time, suppress theexpression of c-FLIP by RNAi and then observe the effects on A375.
Results: In comparison to control group, c-FLIP_(L/P43/S) over-expression promote thegrowth and proliferation activity of A375 cells, c-FLIP_(L/P43) have more remarkable effect onA375 cells' activity of growth and proliferation than that of c-FLIP_S. Compare to the controlcells, c-FLIP _(L/P43/S) can resist the apoptosis. Sofa agar assay indicate c-FLIP _(L/P43/S)over-expression promote the transformation activity of A375. c-FLIP _(L/P43) have more effectson transformation activity of A375 than that of c-FLIP_S. Using siRNA specifically knockingdown the expression of c-FLIP, those effects above have been reversed partly.
Conclusions: The three subtypes of c-FLIP can promote the activity of growth,proliferation and transformation. As to resist to apoptosis, c-FLIP_(L/P43) have more effectsthan that of c-FLIP_S.
引文
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