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【摘要】 RNA干扰是由双链RNA引起的序列特异的基因沉默现象，其作用机制包括转录抑制和目标基因降解。它可以特异、高效的抑制某一基因的表达，达到抗肿瘤的目的，可以用于抑制肿瘤的生长和转移。鼻咽癌是华南地区的高发性恶性肿瘤，具有基因遗传性［1，2］。RNA干扰技术可以用于阻断鼻咽癌基因的表达。
【关键词】 RNA干扰；RNA诱导的沉默复合体；鼻咽癌；基因治疗
Progress of RNA interference on nasopharyngeal carcinoma
CUI Guo-hui，HUANG Xiu-lan，ZHOU Ke-yuan.Institute of Biochemistry and Molecular Biology,Guangdong Medical College,Zhanjiang 524023,China
［Abstract］ RNA interference （RNAi） is the sequence-specific silencing of gene expression induced by dsRNA.Two mechanisms,including translational repression and target degradation are mentioned in suppression of target-gene expression by RNAi.It can silence genes with a high degree of specificity and potentially represents a general approach for molecular targeted anticancer therapy.Now we can use this technology to silence gene expression and to assess the effect of this approach on tumor growth and metastasis.Nasopharyngeal carcinoma （NPC）,a high metastic disease,is most commonly seen in the south-east Asia,especial in China,and it appears to be a genetically determined risk［1，2］.RNAi can be used to interrupt the expression of the cancer's gene.
［Key words］ RNA interference；RNA-induced silencing complex；nasopharyngeal carcinoma；gene therapy
RNAi is now widely used in studies of gene function,signal transduction pathway,and gene therapy because it can effectively and specifically inhibit gene expression.Nasopharyngeal carcinoma （NPC）,a highly metastic disease,is most commonly seen in the south-east Asia,especially in China,and it appears to be a genetically determined risk［1，2］.NPC is a rare tumor arising from the epithelium of the nasopharynx.Now the treatment of NPC requires highly intensive chemotherapy along with surgery and/or radiation for successful treatment［3］.But significant short- and long-term side effects were found during these therapies,so RNAi is used as a wonderful approach.
This review is based on a review of the literature on RNAi and PTGS appearing in the PubMed database,along with personal experiences of working on RNAi studied in NPC,and focused on the experimentalresults that have led to conceptual advancements in dealing with this disease.
1 The mechanism of gene silencing by RNAi
In the past years,studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena.However,its detailed molecular mechanism remains unclear.Much insight into the mechanism of RNAi has come from biochemical analysis within Drosophila cells and extracts［4］.A key finding provided the clue of the mechanisms,in the year 2000,researchers adapted Drosophila in vitro system,found that RNAi is ATP dependent yet uncoupled from mRNA translation.And both strands of the dsRNA are processed to RNA segments 21~23 nucleotides in length.They demonstrated that the 21~23 nucleotide fragments are guiding mRNA cleavage［5］.Rapidly,the mechanism of RNAi was becoming clear.
Both biochemical and genetic approaches have led to the current models of the RNAi mechanism.In which,RNAi includes two steps:the initiation step and the effector step.It would be prudent to overview the process.
1.1 In the initiation step
The pathway of RNA interference starts when Dicer cuts dsRNA into small interfering RNAs （siRNAs） that subsequently target homologous mRNAs for destruction.Two C.elegans genes,rde-1 and rde-4 （“rde” stands for “RNAi deficient”）,are believed to be involved in the initiation step of RNAi.RNAi can be triggered by various types of molecule,it can be actived by long dsRNAs,short hairpin RNAs （shRNAs） or micro RNAs （miRNAs）.siRNAs are produced when the evolutionarily conserved Dicer enzyme Dicer,which is a member of the RNase III family of dsRNA-specific ribonucleases,cleaves dsRNA in an ATP-dependent,processive manner.Successive cleavage events degrade the RNA to 19~21 bp duplexes （siRNAs）,each with 2-nucleotide 3' overhangs［6，7］.
The two RNase III proteins,Drosha and Dicer,may collaborate in the stepwise processing of miRNAs,and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation［8］.
1.2 In the effector step
These siRNAs are binded to a nuclease complex,known as the RNA-induced silencing complex （RISC）,which in turn uses an ATP-dependent RNA-helicase activity to unwind the duplex siRNA into single-stranded siRNA.But ATP is not required for miRNA processing,RISC assembly,or multiple rounds of target-RNA cleavage［9］.RISC is composed of Dicer,the double-stranded RNA binding protein TRBP,and Argonaute 2.This complex can cleave target RNA using precursor microRNA （pre-miRNA） hairpin as the source of siRNA.
The TRBP （HIV-1 TAR RNA-binding protein） ［10，11］ has been studied to have important role in gene scilencing,the interaction with Dicer involves the third dsRNA-binding domain （dsRBD） of PACT and the N-terminal region of Dicer containing the helicase motif.the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference［12］.The active RISC then targets the homologous transcript by base pairing interactions,the RISC-associated endoribonuclease cleaves the target mRNA ~12 nucleotides from the 3' terminus of the siRNA.The Argonaute 2 protein （Ago2） is a critical component of RISC.Both Argonaute and Dicer family proteins contain a common PAZ domain.In the year 2003,researchers［13］ presented the three-dimensional nuclear magnetic resonance structure of the Drosophila melanogaster Ago2 PAZ domain,and proved it played an important role in gene silencing.
In Drosophila,Argonaute 2 is an essential component for RNAi response and is required for the unwinding of siRNA duplex.And Argonaute 1,is required for mature miRNA production that impacts on miRNA-directed RNA cleavage.In the year 2004,researchers［13］found that distinct Argonaute proteins had different roles,indicated that siRNA-initiated RISCs and miRNA-initiated RISCs might be different in Drosophila.
In addition,an amplification step within the RNAi pathway has also been proposed.Amplification could occur by copying of the input dsRNAs,or by replication of the siRNAs themselves.
2 The development of RNAi on NPC
Although the history and mechanism of RNAi and PTGS are fascinating,many researchers are most excited about RNAi's potential use as a functional genomics tool to deal with diseases,especially malignancy.In this article,I want to introduce the application of RNA interference on NPC.
NPC is a rare tumor arising from the epithelium of the nasopharynx.they have powerful abilities of proliferation,migration and invasion.Now the majority of treatments of this carcinoma are surgical methods and chemotherapeutic agents.But they offer little survival benefit and have no significance on improving the quality of life.RNAi appears to play important roles in many aspects of cellular biology.We hypothesized that this approach could be applied as a target-specific treatment of NPC.
cDNA microarray is a powerful tool to analyze simultaneously the expression levels of tens of thousands of genes.Compared with normal nasopharynx （NP） tissues,2210 genes were highly differentially expressed in nasopharyngeal carcinoma （NPC） tissues detected by cDNA microarray［14］.So we can interrupt some special genes with the help of RNAi.
2.1 Association with Epstein-Barr virus
The link between NPC and the Epstein-Barr virus （EBV） was discovered in 1966,we can inhabit this virus to suppress the expressions of NPC,RNAi is used to interrupt the expression of the gene.
Epstein-Barr virus -encoded latent membrane protein （LMP） 1 is vulnerable to RNAi and so it becomes a potential target for immunotherapy of some proportion of NPC,RNAi is used to interrupt its expression.The effect of silencing LMP-1 on cell cycle distribution and chemosensitivity in C666-1 cells was investigated［15］.Silencing of LMP-1 by specific siRNA induced G1 arrest.Phosphorylation of AKT and its downstream targets I kappaB,FKHR was inhibited by LMP-1 siRNA.The chemosensitivity of C666-1 cells to bleomycin and cisplatin was enhanced by siRNA targeting LMP-1.The similar results were found in many other experiments［16,17］.
In some experiments,the vector is used to enhance the efficiency of RNAi.In 2006,a recombinant adeno-associated virus type 2 vector （rAAV-2） was used to deliver small hairpin RNA targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells,which had a good result.It demonstrated that rAAV-shRNA-LMP-1 could effectively infect C666-1 cells and suppress LMP-1 expression［18］.
These results provided solid testimony that EBV-encoded latent membrane protein-1 was vulnerable to RNAi,and selective inhibition of LMP-1 had anti-proliferation effect on NPC.And RNAi could be a powerful tool in further investigations of LMP-1 and a novel therapeutic strategy for EBV-related NPC patients.
2.2 Inhabiting the important gene bcl family to suppress the expressions
Bcl-2 gene encodes an anti-apoptotic protein which can suppress the programmed cell death.To study the inhibition of the expression of bcl-xL gene induced by RNAi in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction.In the year 2005,Li JX and his colleagues［19］ successfully used transient transfected bcl-xL siRNA to inhibit the growth of the NPC and effectively induce its apoptosis.And in other tests,the similar results were observed ［20］,these effects are associated with impaired cellular proliferation,increased apoptosis,and lower levels of tumor angiogenesis.He CW,et al［21］.treated NPC with pmU6-RNAi,cell shrinkage,chromatin condensation,and nuclear fragmentation were observed in most cells.The mRNA levels of bcl-x（L）,bcl-2,and caspase-3 were down-regulated,protein level of p53 was increased.In the year 2004,Yin ZH,et al［22］.demonstrated that stable transfection of CNE1 cells with vectors expressing shRNAs against bcl-2 decreased the expression of bcl-2 protein;suppression of bcl-2 expression did not affect cell proliferation but could increase the chemosensitivity to cisplatin in CNE1 cells.This will help physicians to make some clinical trials of gene therapy on NPC by RNAi.
2.3 Others genetic sites
Weng DS,et al［23］.demonstrated that effect of silencing epidermal growth factor receptor （EGFR） expression by RNAi on the growth of NPC cell 5-8F can reduce the proliferation of NPC cells and induce cell cycle arrest at G1 phase,which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of NPC.
By suppressing human homologue MAD2B expression using RNAi in NPC cells.The frequencies of spontaneous and drug-induced mutations were reduced,phosphorylation of histone H2AX elevated,and chromosomal aberrations in response to DNA damage markedly increased［24］.These results indicate that MAD2B may be a key factor in regulating cellular response to DNA damage in NPC cells.
CD44 has been observed to be involved in many tumor-supporting functions,it was found to be significantly inhibited in the human nasopharyngeal carcinoma cell line CNE-2L2 caused by inhibition of alpha-mannosidase Man2c1 gene expression.Suppressing of CD44 gene expression by RNA silencing technique resulted in profound reduction of malignant potential of CNE-2L2 in nude mice,and direct injection of the adenoviruses harboring and producing siRNA to CD44 into the tumor inoculated with CNE-2L2 cells in nude mice caused inhibition of tumor growth［25］.
Wang Y,et al［26］.found that short hairpin RNA directed against human telomerase reverse transcriptase inhibits NPC viability by regulating telomerase activity and the expression of c-myc and proliferating cell nuclear antigen were both inhibited,while the expression of caspase-3 was up-regulated in NPC.
Shi W,et al［27］.revealed upregulation of NF kappaB2 and survivin playing critical roles in increasing resistance to apoptosis,and changes in integrin and WNT/beta-catenin signaling leading to uncontrolled proliferation.The results provide novel insights into the development and progression of NPC,and suggest survivin as a novel therapeutic target for NPC.
The in vivo studies referred to above demonstrate the potential for the use of RNAi in the therapy.In the coming years there will be an increase in animal studies that use this technology to explore pathogenic mechanisms,and which will suggest potential therapeutic targets.To date there are no studies using RNAi in humans,although this will surely come about soon.
3 Shortcoming and perspective
RNAi is a normal biological process,and an emerging technique for inhibition of gene expression in vitro.Many practical obstacles need to be overcome before it becomes viable in mammalian systems.Greater specificity and efficiency of RNAi in mammals is being achieved by improving the design and selection of siRNAs［28］,by increasing the efficacy of their delivery to cells［29~32］,by improving the accuracy with which susceptible sites in the target RNA molecules can be identified,by reducing the non-specific gene silencing and off-target effects［33~37］,and by evaluating the safe guards［38，39］ in order to engineer their conditional expression.
RNAi is used in many fields today,the research is becoming more profound and mysterious,in the coming years,we should be able to dissect specific pathways by treating cells sequentially with siRNAs.An important direction for future research will be to integrate the data that are emerging from large-scale RNAi screens with that obtained from other approaches.
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作者单位：524023 广东湛江，广东医学院生物化学与分子生物学研究所（*通讯作者）