siRNA干扰HPV16E6基因对人鼻咽癌HNE-1细胞株的影响
摘要
背景:鼻咽癌(nasopharyngeal carcinoma,NPC)是我国南方常见的头颈部恶性肿瘤,早期NPC患者对放射治疗敏感,但晚期NPC患者放射治疗疗效欠佳,易出现复发和转移。因此,寻找新的治疗肿瘤的方法迫在眉睫。新的RNA干扰(RNA interference,RNAi)技术,在肿瘤的治疗中显示了极具前景的应用价值。它通过一些小的双链RNA(double-stranded RNA,dsRNA)阻断特定基因的表达,诱导mRNA的降解,达到“基因沉默”(gene silencing)、抑制细胞增殖和侵袭的功效,从而抑制肿瘤的生长和转移。
有研究证实,人类乳头瘤病毒(human papilloma virus,HPV)与头颈部恶性肿瘤有密切关系,主要为高危的HPV16、18型,其中HPV16的E6蛋白表达水平是维持癌症恶性表型的必要条件。已有研究显示,利用RNAi技术干扰HPV16E6基因能抑制多种肿瘤细胞的增殖并诱导凋亡,但有关RNAi技术干扰HPV16E6基因对鼻咽癌细胞株的影响报道甚少。本研究旨在探讨利用RNAi技术干扰HPV16E6基因对鼻咽癌HNE-1细胞株的影响。
目的:本研究以人鼻咽癌HNE-1细胞株为研究对象,用针对HPV16E6的小片段干扰RNA(small interfering RNAs,siRNA)转染细胞,干扰鼻咽癌细胞株中HPV16E6基因转录及蛋白表达,并探讨siRNA对HNE-1细胞增殖、凋亡及侵袭力的影响作用。
方法
1.转染细胞:查找HPV16E6基因序列,设计针对E6基因的4对siRNA。将siRNA转染入HNE-1细胞,并在荧光显微镜下检测转染效率。
2.筛选有效的siRNA。实验分为5组:4对siRNA组及阴性对照组。采用RT-PCR技术检测转染后HPV16E6 mRNA的表达,Western blot及免疫细胞化学法检测转染后HPV16E6蛋白表达情况,筛选出对HPV16E6干扰有效的siRNA。以有效的siRNA进行下一步实验,同时设立细胞空白组(未转染组)及阴性对照组(siRNA-5转染组)。
3.采用MTT法观察各实验组细胞的增殖情况。
4.采用流式细胞仪检测各实验组的细胞周期。
5.流式细胞仪检测各实验组细胞的凋亡。
6.侵袭实验检测各实验组细胞的侵袭力变化。
结果
1.将转染后24h的细胞在荧光显微镜下观察,可见绿色荧光,说明转染成功,计算转染率为60%。
2.转染后48h,RT-PCR检测结果显示,siRNA-3转染组对HPV16E6 mRNA的抑制率为66.3%,与其余4组相比,差异有显著意义(P<0.05);Western blot检测发现siRNA-3明显抑制HPV16E6蛋白表达,抑制率为56.7%,有统计学意义(P<0.05);免疫细胞化学结果显示,阴性对照组中HPV16E6阳性表达细胞数为65.2%,而siRNA-3转染组中HPV16E6阳性表达细胞数为20.1%,提示HPV16E6蛋白表达被抑制。
3.MTT结果显示,在转染24h、48h、72h后,细胞增殖抑制率分别为24%、23%、30%,较阴性对照组明显降低,差异有显著意义(P<0.05)。
4.流式细胞仪检测细胞周期,结果显示转染了siRNA-3后G0-G1期比率为80.46%,而阴性对照组其比率为65.55%,差异具有统计学意义(P<0.05)。
5.流式细胞仪检测发现,转染siRNA-3后HNE-1细胞凋亡率约为16.82%,与阴性对照组相比,有统计学意义(P<0.05)。
6.侵袭实验发现,实验组穿膜细胞数(45.3±4.0)明显少于细胞空白组穿膜细胞数(146.7±5.5)及阴性对照组穿膜细胞数(147.7±4.7),差异有显著的统计学意义(P<0.05)。
结论
1.针对HPV16E6的siRNA可以有效地抑制鼻咽癌HNE-1细胞株中HPV16E6 mRNA及蛋白的表达。
2.siRNA干扰HPV16E6基因能显著地降低人鼻咽癌HNE-1细胞株的增殖能力。
3.siRNA干扰HPV16E6基因能使人鼻咽癌HNE-1细胞株的DNA合成下降(S期细胞分布比例降低),细胞生长停滞于G0-G1期。
4.siRNA干扰HPV16E6基因能诱导人鼻咽癌HNE-1细胞株的凋亡。
5.siRNA干扰HPV16E6基因能降低人鼻咽癌HNE-1细胞株的侵袭能力。
Background
Nasopharyngeal carcinoma(NPC) is a common malignant tumour of head and neck in the Southern China.Radiotherapy is an effective treatment of early NPC patients.But for advanced NPC patients,the effect is poor,and recurrence and metastasis often happen.Therefore, it is extremely urgent to look for a new therapeutic method.New RNA interference (RNAi) is very valuable in the treatment of tumors.It is some small double-stranded RNA(dsRNA) that can be used to block the expression of particular gene, impel the mRNA to degrade and get the result of the‘gene silencing’and inhibiting the cell proliferation and invasion.
It has been confirmed that Human papilloma virus(HPV)has close relation to head and neck malignant tumor,and high-risk type 16/18 plays an important role in the HPV infection.Protein expression levels of HPV16 E6 is the necessary condition of malignant phenotype in cancer.It has been reported that RNAi technology interfering HPV16E6 gene can inhibit the cell proliferation and induce the cell apoptosis.However,little report about nasopharyngeal carcinoma HNE-1 cell line can be read.This research aims to investigate the effect of RNAi technology interfering HPV16E6 gene on nasopharyngeal carcinoma HNE-1 cell line.
Objective
In this study,with the siRNA against HPV16E6 transfected cells,we interfered HPV16E6 gene transcription and protein expression on nasopha- ryngeal carcinoma HNE-1 cell lines,and investigated the cell proliferation, apoptosis and invasion on HNE-1 cells after transfection.
Methords
1.Transfecting siRNA into HNE-1 cell:we designed four pairs of E6 siRNA after looking up HPV16E6 gene sequence.After transfecting siRNA into HNE-1 cell,we observed the transfection efficiency using fluorescence microscopy.
2.Picking out the effective siRNA.Groups in experiment:four pairs of E6 siRNA groups and negative control group.RT-PCR was used to detect the expression of HPV16 E6 and protein level of HPV16 E6 was measured by Immunohistochemical staining and Western blot.Then,the effective siRNA against HPV16E6 was picked out.We selected blank group,the effective siRNA group and negative control group to following experiment.
3.Testing the cell proliferation after transfection using MTT.
4.Measuring the cell cycle by Flow cytometry(FCM).
5.Detecting apoptosis by FCM.
6.Determining invasion by transwell experiment.
Results
1.Under the fluorescence microscope,the green fluorescence with the cells could be seen after transfection,suggesting the transfection was successful.The transfection efficiency was 60 percent.
2.After 48hours of transfection,RT-PCR showed that after transfecting siRNA-3 into HNE-1 cell,the HPV16E6 mRNA inhibition rate was 66.3 percent.Compared with other groups,the difference was statistically significant.Western blot analysis showed that siRNA-3 could significantly inhibit the expression of HPV16E6 protein and the inhibition rate was 56.7 percent(p<0.05).Immunohistochemical assay showed that the number of HPV16E6 positive cells in the negative control group is 65.2 percent, compared with 20.1 percent in the siRNA-3 group,suggesting siRNA-3 could decreased the expression of HPV16E6 protein.
3.MTT showed that the inhibition ratio of cell proliferation were 24%, 23%, 30% in 24h, 48h, 72h respectively after transfection.The result was statistically significant.
4.The cell cycle was detected by FCM.The study showed that the ratio of G0-G1 phase was 80.46 percent after siRNA-3 transfection and negative control group rate of 65.55 percent.The difference was statistically signify- cant.
5.Though FCM,the cell apoptosis rate is about 16.82 percent after siRNA-3 transfection.Compared with negative control group, there was a significant difference.
6.Transwell experiment showed that the number of cells which crossed the membrane was 45.3 in siRNA-3 transfection group,146.7 in blank group and 147.7 in negative control group(p<0.05).
Conclusion
1.siRNA against HPV16E6 could effectively inhibit the expression of HPV16E6 and the protein level of HPV16E6 on nasopharyngeal carcinoma HNE-1 cell line.
2.siRNA interfering HPV16E6 could significantly reduce the cell pro- liferation on nasopharyngeal carcinoma HNE-1 cell line.
3.siRNA interfering HPV16E6 could decrease the DNA synthesis(the proportion of cell in S phase reduced)on nasopharyngeal carcinoma HNE-1 cell line.The cell cycle was blocked in the G0-G1 phase.
4.siRNA interfering HPV16E6 could induce the cell apoptosis on nasopharyngeal carcinoma HNE-1 cell line.
5.siRNA interfering HPV16E6 could downregulate the invasion on nasopharyngeal carcinoma HNE-1 cell line.
有研究证实,人类乳头瘤病毒(human papilloma virus,HPV)与头颈部恶性肿瘤有密切关系,主要为高危的HPV16、18型,其中HPV16的E6蛋白表达水平是维持癌症恶性表型的必要条件。已有研究显示,利用RNAi技术干扰HPV16E6基因能抑制多种肿瘤细胞的增殖并诱导凋亡,但有关RNAi技术干扰HPV16E6基因对鼻咽癌细胞株的影响报道甚少。本研究旨在探讨利用RNAi技术干扰HPV16E6基因对鼻咽癌HNE-1细胞株的影响。
目的:本研究以人鼻咽癌HNE-1细胞株为研究对象,用针对HPV16E6的小片段干扰RNA(small interfering RNAs,siRNA)转染细胞,干扰鼻咽癌细胞株中HPV16E6基因转录及蛋白表达,并探讨siRNA对HNE-1细胞增殖、凋亡及侵袭力的影响作用。
方法
1.转染细胞:查找HPV16E6基因序列,设计针对E6基因的4对siRNA。将siRNA转染入HNE-1细胞,并在荧光显微镜下检测转染效率。
2.筛选有效的siRNA。实验分为5组:4对siRNA组及阴性对照组。采用RT-PCR技术检测转染后HPV16E6 mRNA的表达,Western blot及免疫细胞化学法检测转染后HPV16E6蛋白表达情况,筛选出对HPV16E6干扰有效的siRNA。以有效的siRNA进行下一步实验,同时设立细胞空白组(未转染组)及阴性对照组(siRNA-5转染组)。
3.采用MTT法观察各实验组细胞的增殖情况。
4.采用流式细胞仪检测各实验组的细胞周期。
5.流式细胞仪检测各实验组细胞的凋亡。
6.侵袭实验检测各实验组细胞的侵袭力变化。
结果
1.将转染后24h的细胞在荧光显微镜下观察,可见绿色荧光,说明转染成功,计算转染率为60%。
2.转染后48h,RT-PCR检测结果显示,siRNA-3转染组对HPV16E6 mRNA的抑制率为66.3%,与其余4组相比,差异有显著意义(P<0.05);Western blot检测发现siRNA-3明显抑制HPV16E6蛋白表达,抑制率为56.7%,有统计学意义(P<0.05);免疫细胞化学结果显示,阴性对照组中HPV16E6阳性表达细胞数为65.2%,而siRNA-3转染组中HPV16E6阳性表达细胞数为20.1%,提示HPV16E6蛋白表达被抑制。
3.MTT结果显示,在转染24h、48h、72h后,细胞增殖抑制率分别为24%、23%、30%,较阴性对照组明显降低,差异有显著意义(P<0.05)。
4.流式细胞仪检测细胞周期,结果显示转染了siRNA-3后G0-G1期比率为80.46%,而阴性对照组其比率为65.55%,差异具有统计学意义(P<0.05)。
5.流式细胞仪检测发现,转染siRNA-3后HNE-1细胞凋亡率约为16.82%,与阴性对照组相比,有统计学意义(P<0.05)。
6.侵袭实验发现,实验组穿膜细胞数(45.3±4.0)明显少于细胞空白组穿膜细胞数(146.7±5.5)及阴性对照组穿膜细胞数(147.7±4.7),差异有显著的统计学意义(P<0.05)。
结论
1.针对HPV16E6的siRNA可以有效地抑制鼻咽癌HNE-1细胞株中HPV16E6 mRNA及蛋白的表达。
2.siRNA干扰HPV16E6基因能显著地降低人鼻咽癌HNE-1细胞株的增殖能力。
3.siRNA干扰HPV16E6基因能使人鼻咽癌HNE-1细胞株的DNA合成下降(S期细胞分布比例降低),细胞生长停滞于G0-G1期。
4.siRNA干扰HPV16E6基因能诱导人鼻咽癌HNE-1细胞株的凋亡。
5.siRNA干扰HPV16E6基因能降低人鼻咽癌HNE-1细胞株的侵袭能力。
Background
Nasopharyngeal carcinoma(NPC) is a common malignant tumour of head and neck in the Southern China.Radiotherapy is an effective treatment of early NPC patients.But for advanced NPC patients,the effect is poor,and recurrence and metastasis often happen.Therefore, it is extremely urgent to look for a new therapeutic method.New RNA interference (RNAi) is very valuable in the treatment of tumors.It is some small double-stranded RNA(dsRNA) that can be used to block the expression of particular gene, impel the mRNA to degrade and get the result of the‘gene silencing’and inhibiting the cell proliferation and invasion.
It has been confirmed that Human papilloma virus(HPV)has close relation to head and neck malignant tumor,and high-risk type 16/18 plays an important role in the HPV infection.Protein expression levels of HPV16 E6 is the necessary condition of malignant phenotype in cancer.It has been reported that RNAi technology interfering HPV16E6 gene can inhibit the cell proliferation and induce the cell apoptosis.However,little report about nasopharyngeal carcinoma HNE-1 cell line can be read.This research aims to investigate the effect of RNAi technology interfering HPV16E6 gene on nasopharyngeal carcinoma HNE-1 cell line.
Objective
In this study,with the siRNA against HPV16E6 transfected cells,we interfered HPV16E6 gene transcription and protein expression on nasopha- ryngeal carcinoma HNE-1 cell lines,and investigated the cell proliferation, apoptosis and invasion on HNE-1 cells after transfection.
Methords
1.Transfecting siRNA into HNE-1 cell:we designed four pairs of E6 siRNA after looking up HPV16E6 gene sequence.After transfecting siRNA into HNE-1 cell,we observed the transfection efficiency using fluorescence microscopy.
2.Picking out the effective siRNA.Groups in experiment:four pairs of E6 siRNA groups and negative control group.RT-PCR was used to detect the expression of HPV16 E6 and protein level of HPV16 E6 was measured by Immunohistochemical staining and Western blot.Then,the effective siRNA against HPV16E6 was picked out.We selected blank group,the effective siRNA group and negative control group to following experiment.
3.Testing the cell proliferation after transfection using MTT.
4.Measuring the cell cycle by Flow cytometry(FCM).
5.Detecting apoptosis by FCM.
6.Determining invasion by transwell experiment.
Results
1.Under the fluorescence microscope,the green fluorescence with the cells could be seen after transfection,suggesting the transfection was successful.The transfection efficiency was 60 percent.
2.After 48hours of transfection,RT-PCR showed that after transfecting siRNA-3 into HNE-1 cell,the HPV16E6 mRNA inhibition rate was 66.3 percent.Compared with other groups,the difference was statistically significant.Western blot analysis showed that siRNA-3 could significantly inhibit the expression of HPV16E6 protein and the inhibition rate was 56.7 percent(p<0.05).Immunohistochemical assay showed that the number of HPV16E6 positive cells in the negative control group is 65.2 percent, compared with 20.1 percent in the siRNA-3 group,suggesting siRNA-3 could decreased the expression of HPV16E6 protein.
3.MTT showed that the inhibition ratio of cell proliferation were 24%, 23%, 30% in 24h, 48h, 72h respectively after transfection.The result was statistically significant.
4.The cell cycle was detected by FCM.The study showed that the ratio of G0-G1 phase was 80.46 percent after siRNA-3 transfection and negative control group rate of 65.55 percent.The difference was statistically signify- cant.
5.Though FCM,the cell apoptosis rate is about 16.82 percent after siRNA-3 transfection.Compared with negative control group, there was a significant difference.
6.Transwell experiment showed that the number of cells which crossed the membrane was 45.3 in siRNA-3 transfection group,146.7 in blank group and 147.7 in negative control group(p<0.05).
Conclusion
1.siRNA against HPV16E6 could effectively inhibit the expression of HPV16E6 and the protein level of HPV16E6 on nasopharyngeal carcinoma HNE-1 cell line.
2.siRNA interfering HPV16E6 could significantly reduce the cell pro- liferation on nasopharyngeal carcinoma HNE-1 cell line.
3.siRNA interfering HPV16E6 could decrease the DNA synthesis(the proportion of cell in S phase reduced)on nasopharyngeal carcinoma HNE-1 cell line.The cell cycle was blocked in the G0-G1 phase.
4.siRNA interfering HPV16E6 could induce the cell apoptosis on nasopharyngeal carcinoma HNE-1 cell line.
5.siRNA interfering HPV16E6 could downregulate the invasion on nasopharyngeal carcinoma HNE-1 cell line.
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[1]. Agrawal N,Dasaradhi PV,Mohmmed A,et a1.RNA interference:Biology mechanism and applications[J].Microbiol Mol Biol Rev,2003,67(4):657-685.
[2]. Izant J,Weintranb H.Inhibition thythidine Kinase gene expression by anti-sense RNA:a theleculat approach to genetic analysis[J].Cell,1984,36:1007-1015.
[3]. Jorgenson R.Altered gene expression in plants due to trans inter-actions between homologous genes[J].Trends Biotechnol,1990,8(12):340-344.
[4]. Cogoni C,Romano N,Macino G.Suppression of gene expression by homologo- us transgenes[J].Antonie Van Leeuwenhoek,1994,65:205-209.
[5]. Guo S,Kemphues KJ.Par-1,a gene required for establishing polarity in C. elegans embryos,encodes a putative Ser/Thr kinase that is asymmetrically distributed [J].Cell,l995,8l(4):611-620.
[6]. Fire A,Xu S,Montgomery MK,et e1.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998, 391(6669):806-811.
[7]. Elbashir S M,Harborth J,Lendeckel W,et a1.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured manlmalian cells[J].Nature,2001,411(683 6):494-498.
[8]. Paul C P,Good P D,Winer I,et a1.Effective expression of small interfering RNA in human cells[J].Nat Biotechnol,2002,20(5):505-508.
[9]. Rand T A,Petersen S,Du F H,et e1.Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation[J].Cell,2005,123(4):621-629.
[10]. Meister G,Tuschl T.Mechanisms of gene silencing by double-stranded RNA[J].Nature,2004,431:343-349.
[11]. Sijen T,F1eenor J,Simmer F,et a1.On the role of RNA amplification in dsRNA-triggered gene silencing[J].Cell,2001,107(4):465-476.
[12]. Nishikura K.A short primer on RNAi:RNA-directed RNA polymerase acts as akey catalysL[J].Cell,2001,107(4):4l5-4l8.
[13]. Elbashir S M,Lendecked W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes,2001,15(2):188-200.
[14]. Bernstein E,Caudy A A,Hammond S M,et a1.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
[15]. Kawasaki H,Taira K.Induction of DNA methylation and gene silencing by short interfering RNAs in human cells[J].Nature,2004,431(7005):211-217.
[16]. Morris K V,Chan SW L,Jacobsen SE,et a1.Small interfering RNA-induced transcriptional gene silencing in human cells[J].Science,2004,305(5688): 1289-1292.
[17]. Grishok A,Pasqulnelli AE,Conte JM,et a1.Genes and mechanisms related to RNA interference regulate expression of smal1 temporal RNAs that control C.elegants developmental timing[J].Cell,2001,106(1):23-24.
[18]. Jacque JM,Triques K,Stevenson M.Modulation of HIV-1 replication by RNA interference[J].Nature,2002,418(6896):435-438.
[19]. Davenport RJ.Gene silencing:a faster way to shut down genes[J].Science,2001,292(5521):1469-1471.
[20]. Holen T,Amarzguioui M,Wiiger M T,et a1.Positional effects of short interfer- ing RNAs targeting the human congulation trigger tissue facter[J].Nucleic Acids Res ,2002,30(8):1757-1766.
[21]. 秦玉新,蒙凌华,丁 健.RNA干扰技术的研究进展[J].中国药理学通报,2007,23(4):421-424.
[22]. Hall AH,Alexander KA.RNA interference of human papilloma virus type 18 E6 and E7 induces senescence in Hela cells[J].Journal of virology,2003, 77:6066-6069.
[23]. Nagy P,Arndt-Jovin D J,Jovin T M.Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor(erbB1)and induce apoptosis in erbB1-overexpressing cells[J].Exp CellRes.2003,285(1):39-49.
[24]. 王萍玲,胡丽娜,邓凯贤等.RNA干扰沉默mdrl基因联合阿霉素对卵巢癌耐药细胞株SKOV3/ADM增殖的抑制作用[J].重庆医科大学学报,2005,30 (2):232-235.
[25]. Borkhardt A.Blocking oncogenes in malignant cells by RNA interference new hope for a highly specific cancer treatment[J].Cancer Cell,2002,2:167-168.