作者：张红玲 陈爱平 杨蕊蕊
【摘要】 目的 探讨RNA干扰（RNAi）对卵巢癌细胞表皮生长因子受体（EGFR）基因表达的影响及其效应。方法 体外合成EGFR的DNA模板序列和pSilencer 2.1-U6neo质粒构建编码shRNA的表达载体，应用脂质体Lipofectamine 2000将其转染到人卵巢癌Skov-3细胞，采用半定量RT-PCR、免疫细胞化学技术检测转染前后Skov-3细胞EGFR基因的变化；采用AnnexinV/PI双染色标记的流式细胞仪检测siRNA诱导的细胞凋亡，PI染色检测细胞周期阻滞。结果 成功构建pSilencer-EGFR短发卡状siRNA真核表达载体；靶向EGFR的序列特异性的siRNA可以有效抑制Skov-3细胞EGFR基因的表达；流式细胞分析显示，Skov-3细胞凋亡率明显增加，细胞周期出现明显的G0/G1期阻滞。结论 RNAi沉默EGFR表达可以诱导卵巢癌Skov-3细胞凋亡，并使卵巢癌Skov-3细胞停滞在G0/G1期，RNAi可作为研究卵巢癌发生发展机制和基因治疗的有效工具。
【关键词】 卵巢肿瘤；受体，表皮生长因子；RNA干扰；细胞凋亡
ABSTRACT］Objective To explore the effect of RNA interference （RNAi）-mediated inhibition on epidermal growth factor receptor （EGFR） gene expression in ovarian cancer cells. Methods The DNA sequence of EGFR gene was synthesized in vitro and cloned into pSilencer 2.1-U6neo plasmid vector to construct the shRNA expression vector. After the constructed vector was transfected into human ovarian cancer Skov-3 cells using lipofectamine 2000， semi-quantitative RT-PCR and immunocytochemistry assay were conducted to compare the changes in the expression levels of EGFR between before and after transfection. Moreover， cells were stained with Annexin V/PI and PI and then siRNA-induced apoptosis and cell cycle analyzed. Results SiRNA eukaryotic expression vector pSilencer-EGFR was successfully constructed; the siRNA targeted against EGFR could effectively inhibit EGFR gene expression in Skov-3 cells; the results of flow cytometric analysis indicated that the apoptosis rate of Skov-3 cells increased significantly and an obvious cell cycle arrest at G0/G1 phase was induced.Conclusion RNAi-mediated silencing of EGFR gene expression can induce cell apoptosis and cell cycle arrest at G0/G1 phase. Thus， RNAi can be used as an effective tool to explore the mechanism underlying the development and progression of ovarian cancer as well as conduct gene therapy of this disease.
［KEY WORDS］ Ovarian neoplams; Receptor， epidermal growth factor; RNA interference; Apoptosis
Ovarian carcinoma has a higher mortality rate among gynecological malignancies［1］. Over-expression of EGFR， as in most cases of ovarian carcinoma， is associated with the extent of malignancy and prognosis［2］. EGFR has recently received considerable attention in ovarian cancer research， since this 170 000 glycosylated membrane-spanning protein receptor is over-expressed up to 75% in primary cases［3］. Furthermore， activation of EGFR signaling pathway in can-cer cells is involved in increased cell proliferation， angiogenesis， metastasis and decreased apoptosis. EGFR is thus considered as a critical target in the treatment of this cancer［4］. RNA interference （RNAi）， also known as post-transcriptional gene silencing， is able to trigger some post-transcriptional control mechanisms via which mRNA is degraded［5-7］. For this reason， RNAi technology was used in this study. After eukaryotic expression vector of siRNA specific for EGFR was constructed and transfected into ovarian cancer Skov-3 cells， its inhibition on EGFR gene expression as well as its effect on the proliferation， cell cycle and apoptosis of ovarian cancer cells were observed. The results obtained lay a foundation for gene therapy of ovarian cancer using RNAi.
1 MATERIALS AND METHODS
1.1 Materials
Human ovarian cancer Skov-3 cell line was provided by West China University of Medical Sciences. E. coli DH5 alpha strain was obtained from the Department of Pathogenic Microbiology of Qingdao University. Eukaryotic expression plasmid pSilencer 2.1-U6neo was purchased from Ambion （USA）. Lipofectamine 2000 was purchased from Invitrogen （USA）. PureYield Plasmid Midiprep System was product of Promega. Propidium iodide （PI） and ribonuclease were products of Sigma. Annexin V-FITC/PI was product of Jingmei Bioengineering Company. Anti-EGFR antibody was product of Zhongshan Bio-tech Company.
1.2 Methods
1.2.1 Design and preparation of EGFR-siRNA The Ambion’s online software was used to design EGFR-siRNAusing the design strategy for small interfering RNAs according to the nucleotide sequence of EGFR retrieved from GenBank （Accession No. NM_005228）. The designed sequences of sense and antisense strands were 5′-GATCCGCACAGTG-GAGCGAATTCCTTTCAAGAGAAGGAATTCGCTCCAC-TGTGTTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAA-ACACAGTGGAGCGAATTCCTTCTCTTGAAAGGAATT-CGCTCCACTGTGCG-3′［8］. The sequences of these two DNA strands were composed of a BamH Ⅰ site， sense sequence， loop， antisense sequence， termination signal and a Hind Ⅲ site. After these two DNA strands were annealed and ligated into linear vector pSilencer 2.1-U6neo， the ligated products were transformed into competent E. coli DH5αstrain. The bacteria were then spread over a plate that contained ampicillin. After culture at 37 ℃ overnight， three single colonies were picked from each plate， inoculated into 3 mL of ampicillin-containing LB liquid medium and cultured at 37 ℃ overnight. Plasmids were subsequently isolatedusing plasmid DNA preparation kit， identified by restriction endonuclease digestion and sequenced.
1.2.2 Cell culture Skov-3 cells were routinely cultured in RPMI 1640 medium containing 10% fetal calf serum， 100 kU/L penicillin and 100 kg/L streptomycin at 37 ℃ with 5% CO2. The growth status of cells was observed regularly， and cells were subcultured by digestion with 2.5 g/L trypsin every two to three days.
1.2.3 Liposome-mediated transfection into Skov-3 cellsOne day before transfection， Skov-3 cells during logarithmic growth were seeded in six-well plates at 2×105 cells per well. When cells were grown to a confluence of 60% to 70%， transfection was conducted using Lipofectamine 2000 according to the manufacture’s instructions. A total of three groups， i.e. blank control group （untransfected Skov-3 cells）， non-specific transfection group （vector plasmid containing a gene segment which is non-complementary with any gene in vivo provided from the company.） and specific transfection group， were set.
1.3 Detection of EGFR mRNA expression
At 48 h after transfection， cells in the experimental group and control group were harvested and subjected to total RNA extraction using Trizol reagent according to theagent directions. After reverse transcription was performed using a specific downstream primer， two microliters ofcDNA were removed and subjected to PCR amplification in a25 μL reaction volume. PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 5 min， and followed by 25 cycles of 1 min at 94 ℃， 1 min at 55 ℃， 1 min at 72 ℃， and a final extension step at 72 ℃ for 10 min. Beta-actin gene was used as an internal control. After PCR cycling，3 μL of PCR products were analyzed by electrophoresis on a 12 g/L agarose gel， ultraviolet photography and scanning.
1.4 Immunofluorescent analysis of EGFR protein expression
A routine method was used to make cells adhere to slides. At 48 h after transfection of pshRNA-EGFR， slides were taken out and fixed with acetone at room temperature for 10 to 15 min. After blockage of endogenous peroxidase with 3% h3O2， the slides were incubated with the primary antibody （rabbit anti-human EGFR， 1∶60 dilution） at 37 ℃ for 1 h， followed by incubation with the secondary antibody （horseradish peroxidase-labeled goat anti-rabbit IgG） at 37 ℃ for 30 min. The slides were then subjected to DAB coloration， mounting in gum， observed microscopically and photographed. Immunohistochemial staining results were scored based on the extent of cell coloration to evaluate the intensity of EGFR gene expression. The scoring criteria were: 1， negative staining: no positively stained cells were seen， and zero was given; 2， mild staining: positively stained cells showed a yellow pigment， one point was given; 3， moderate staining: positively stained cells showed a buffy pigment， two points; 4， intense staining: positively stained cells showed a dark buffy pigment， three points. Five visual fields were randomly selected from each slide to count and score cells under high-power microscope. The expression intensity of EGFR was calculated by piding the sum of the score by the number of cells. Subsequently， the following formula was used to calculate the inhibition rate of EGFR expression: the inhibition rate of EGFR expression=（1- the expression intensity of EGFR in the experimental group/the expression intensity of EGFR in the control group）×100%.
1.5 Apoptosis detection and cell cycle analysis by flow cytometry
At 48 and 72 h after transfection of pshRNA-EGFR， cells in the experimental group and control group were digested with trypsin， harvested and resuspended in PBS at （1-2）×109/L. After being washed twice with precooled PBS（4 ℃）， these cells were sent for apoptosis analysis by Annexin V/PI staining. At 24 and 48 h after transfection of pshRNA-EGFR， cells in the both groups were assessed for their cell cycle by propidium iodide （PI） staining.
1.6 Statistical analysis
A statistical analysis was performed using SPSS 10.0. One-way analysis of variance was performed for group comparison.
2 RESULTS
2.1 Construction of plasmid expression vector of shRNA
The sequence of the DNA template that was inserted into the plasmid vector was composed of a BamH Ⅰ site， sense sequence， loop， antisense sequence， termination signal and a Hind Ⅲ site. Double digestion of pshRNA-EGFR with BamH Ⅰ and Hind Ⅲ would result in a 64 bp insert fragment and the 4.4 kb pshRNA linear plasmid. The culture of the transformed bacteria was sent to Shanghai Sangon Biological Engineering Technology && Services Co.， Ltd. for sequence analysis， and the results entirely met the requirements of the design.
2.2 Effect of siRNA on EGFR mRNA levels
Cells were harvested at 48 h after transfection and RT-PCRanalysisdoneforEGFRmRNAlevels.Abandof
bp （EGFR） was amplified in the former three groups while a band of 109 bp （β-actin） was visible in the latter three groups. The intensity of the band at 262 bp in the specific transfection group （lane 3） was significantly lower than that in both the non-specific transfection group （lane 2） and the blank-control group （lane 1） though no significant difference in the intensity of internal control band （β-actin） was observed among each group （see Figure 1）.
2.3 Analysis of EGFR protein expression by immunocytochemistry assay
The results of immunocytochemical staining showed that， after cell transfection for 48 h， the expression intensity of EGFR protein in blank-control， negative-control and positive-control groups was 2.91±0.057， 2.82±0.054， and0.97±0.048， respectively. The statistical analysis suggested that， after transfection of pshRNA-EGFR into Skov-3 cells， the expression of EGFR protein was significantly inhibited （F=1 702.63;q=69.767，73.058;P&<0.01）， with the inhibition rate of 66%. The difference between the blank-control and non-specific transfection was not statistically significant （q=3.290，P&>0.05）.
2.4 Flow cytometric
The results of flow cytometric analysis with AnnexinV/PI double staining showed that， at 48 and 72 h after transfection of pshRNA-EGFR into cells， the apoptosis rates were （19.13±2.68）％ and （23.25±9.33）％， respectively， of which， both differences were significant （F=12.08，q=5.642，7.217，P&<0.01） when compared with the blank-control （4.35±2.15）%. The apoptotic cells in the non-specific transfection group was not obvious， with apoptosis rate of （7.33±3.30）％， which was not significantly different compared with the blank-control （q=1.136，P&>0.05） （Figure 2）. The results of cell cycle analysis by PI staining showed that， at 24 and 48 h after transfection of pshRNA-EGFR into cells， the ratio of G0/G1 phase cells increased compared with the blank-control （F=158.70;q=19.450，24.047;P&<0.01）. However， compared with non-specific transfection group， there was no statistically significant difference in the blank-control （q=0.347，P&>0.05） （See Table 1）. Table 1 Comparison of cell cycle distribution in all groups of cells （略）
Ovarian cancer is one of the most severe malignancies in gynecological tumors. With the development of molecular biology in recent years， some studies have shown that 70%-100% of ovarian cancer are concomitant with high levels of EGFR expression［9-10］. The high-level expression of EGFR is associated with malignant transformation of cells， tumor proliferation and metastasis as well as tumor angiogenesis， which indicates a poor prognosis. In a prospective study of 71 cases of malignant ovarian tumor and 19 benign cases， TOMOV et al［11］ found that the positive expressions of EGFR in malignant and benign tumor were 62.9% and 38.6%， respectively. The positive expressions of EGFR in those with positive EGFR of peritoneal douche and advanced ovarian cancer were 77% and 75%， respectively. EGFR-positive tumor cells have a stronger invasiveness and metastasis， insensitive to radiotherapy and chemotherapy， with poor prognosis of higher recurrence and shorter survival time， which suggestive of EGFR is not only highly expressed in ovarian cancer but also closely related with the development and progression of this disease. Most literatures have documented that the increase of expression of EGFR mainly on transcriptional level. BAUKNECHT et al examined 47 ovarian cancer specimens and 21 normal ovarian tissue specimens by NorthernBlot analysis and found that the transcriptional level of EGFR gene in cancer tissue increased by 75% and no abnormal EGFR transcription was observed in normal ovarian tissue. Moreover， they found an abnormal gene structure in two cancer specimens though no increase in gene copy number was seen.
Using immunohistochemistry in combination with Southern Blot analysis， ITAKURA et al found that increased EGFR gene expression accounted for 71% in primary focus of esophageal cancer， and the gene copy number of EGFR increased 21%， all appearing as enhancement of the level of protein. Besides， it was noted that the abnormality in theamino acid structure of EGFR was also closely associated with tumors.
RNAi， also known as post-transcriptional gene silencing （PTGS）， is a gene silencing process mediated by endogenous or exogenous homologous double-stranded RNA （dsRNA）. When RNAi occurs， double-stranded RNA is cleaved into small interfering RNAs （siRNA） of 21 to 23 base pairs by ribonuclease Ⅲ enzyme （Dicer enzyme）. After these siRNAs are incorporated into an endonuclease-containing complex to form RNA-induced silencing complex （RISC） and pair with complementary sequences in target mRNA， they can induce the cleavage of target mRNA and thereby lead to gene silencing［12］.
RNAi is a recently developed short double-stranded RNA-mediated gene knockdown approach that is efficient and specific. Since this method can suppress the expression of target genes without inducing non-specific interference， its efficiency is significantly higher than that of the conventional method using antisense oligonucleotides. The expression-vector-based siRNA preparation method used in the present study possesses the advantages of not only cost-effective but also can be conducted for a long-term and steady study as compared with other methods commonly used in these days， such as chemical synthesis， in vitro transcription， “cocktail” preparation and expression cassettes. In the present study， the effect of RNA interference-mediated inhibition of EGFR gene expression on ovarian cancer cells was examined. After a eukaryotic plasmid expressing EGFR was constructed and transfected into human ovarian cancer Skov-3 cells， themRNA and protein expression levels of EGFR were determined. RT-PCR results showed that the brightness of the amplified band in the specific transfection group was significantly lower than that in the blank-control group and the non-specific-transfection group， indicating that RNAi was actually able to reduce the mRNA level of the target gene. Immunohistochemically， a comparison between the blank-control and the specific-interference showed the gene expression inhibition rate was 66%; compared with the EGFR-negative， the inhibition rate was 65%， indicating that EGFR gene expression was inhibited.
Flow cytometric results showed that specific interfe-rence with EGFR gene could not only induce cell cycle arrest at G0/G1 phase and inhibit cell growth， but also blockEGFR gene expression and effectively antagonize the anti-apoptosis mechanisms in Skov-3 cells， thereby inducing apoptosis.
Molecular-targeted therapy is the direction of anticancer therapy in the 21st century. RNAi-mediated silencing ofEGFR gene expression can induce cell apoptosis and cell cycle arrest at G0/G1 phase. Though the present study results offer a reliable base for gene therapy of ovarian cancer， a further study in this aspect is still needed.
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