Study On Inhibitory Effects Of 2'-O-Methoxyethyl-Modified Antisense Oligonucleotides Targeting VEGF-A In SKOV3 Ovarian Cancer Cells

Ovarian cancers, one of the most common malignancies in gynecological cancers, have been the focus of gynecological research due to its high incidence rate, invasion and migration. Tumor angiogenesis is a process involving the growth of new blood vessels within or toward the tumor and is essential for tumor itself to grow and metastasize. Vascular endothelial growth factor (VEGF), also called vascular permeability factor (VPF), is a growth factor acting on the membrane of vascular endothelial cells. VEGF is widely expressed in human and animal tissues such as the brain, kidney, liver, spleen and bones. It was first purified from the bovine pituitary follicular stellate cells in vitro culture medium and was named by Ferrara and others in 1989. VEGF, a polypeptide cytokine of platelet-derived growth factor family, is one of the strongest vascular growth factor ever found. VEGF acts specifically on vascular endothelial cells and plays a key role in tumor genesis and growth by promoting endothelial cell proliferation and increasing the vascular permeability to induce tumor angiogenesis. Targeted tumor therapy by inhibiting VEGF expression has been widely investigated recently.As a new and fastly developing gene silencing technology, antisense oligonucleotides (ASODN) technology is regarded as a very effective tool in the field of genetic engineering. Antisense oligonucleotides technology can inhibit the expression of target genes in a specific, efficient and stable manner and can achieve relatively satisfactory gene silencing effect. Currently, ASODN has been widely and successfully applied to study the function of genes, the etiology and treatment of a variety of diseases.The purpose of this study is to:①determine the gene and protein expression of VEGF in ovarian cancers, in order to clarify the correlation between VEGF and the migration of ovarian cancers;②design and synthesize stable and efficient 2' -Methoxyethyl modified antisense oligonucleotides which can be transferred into ovarian cancer cells against VEGF gene;③study the silencing effects of the antisense oligonucleotides against VEGF-A expression in SKOV3 ovarian cancer cells, and further clarify the change of proliferation and apoptosis levels of SKOV3 cells before and after transfection, in order to investigate whether VEGF-A can be used as a potentially effective therapeutic target in the ovarian cancer gene therapy.④construct a nude mice model bearing human ovarian cancer cell line SKOV3 and observe the inhibitory effect of ASODN transfected using ultrasound irradiation treatment to endermic tumors cells of transferred ovarian cancers ascites in mice and evaluate tumor suppressive rate to evaluate the suppressive effects of ultrasound irradiation treatment and ASODN against ovarian cancers.Part I Expression of VEGF and its relationship with biological features in ovarian cancers ObejectveVascular endothelial growth factor (VEGF) is one of the strongest factors promoting the growth of blood vessels, its genetic expression is closely related to the increased invasion of tumor and decreased survival rates. The aim of this research is to determine the gene and protein expression of VEGF in ovarian cancers, clarifying the relationship between VEGF and the invasion and malignancy of ovarian cancers.Methods1 Samples preparationAll samples were obtained during 132 excision surgeries at Shandong Provicial Hospital affiliated to Shandong University during July 2008 and April 2010, including:①ovarian cancers (n=32), which include serous cystadenocarcinomas (n=31), endometrioid adenomas (n=23) and mucoid adenocarcinomams (n=14). The subjects' ages were ranged 29-77 years (mean age 55.3±6.3 years), they all received operations for the first time, no chemotherapy history, no tumor history, and all the diagnoses were confirmed by pathologicy.②benign ovarian tumors (n=34), including serous cystadenomas (n=22) and mucinous cystadenomas (n=12). The ages of the subjects were ranged 29-70 years old (mean age 39.7±7.5years).③normal control group (n=30), obtained from womb and ovary excison sugeries for hysteromyoma, dysfunctional uterine bleeding and hysteroptosis, and the ovary were confirmed as normal by histopathological studies. The ages of the subjects were ranged 42-70 years (mean age 54.1±7.1 years). All the diagnoses are confirmed by histopathological studies. 2 HistochemistryThe expression of VEGF protein in ovarian cancer, benign ovarian tumors and normal ovary tissues are determined by immunohistochemical staining with specific mouse anti-VEGF monoclonal antibody by streptavidin-peroxidase method (abbreviation:S-P).3. Analysis of immunohistochemical resultsVEGF immunoreactivity is evaluated semiquantitatively based on the intensity of staining.5 fields at a magnification of 400×at every slice are observed at random. The percent of positive staining cells in 200 tumor cells is calculated. The slides are analysed independently by four observers blinded for expression of VEGF.For the discrepancies a second evaluation course is running to reach agreement.4. Evaluation of B\VEGF protein expressionThe VEGF expression is scored as:strong (+++):if more than 50% of cells are positive or a strong diffuse reaction is seen; moderate (++):if less than 50% of cells are positive, or a moderate diffuse reaction is observed; slightly positive (+):if immunoreactions are found in less than 30% of tumour cells or the diffuse reaction is weak; and negative(-):if no VEGF is stained.5. Fluorescent quantitive PCR (FQ-PCR)Total RNA are extracted from frozen tissues obtained from ovary excision surgeries. cDNA is synthesized from total RNA by reverse transcription. mRNA expression of VEGF-A is determined by FQ-PCR.6. Western-blot to examine the protein expression of VEGF-AVEGF-A protein is extracted from 200mg of frozened ovarian cancer tissues according to standard experiment procedures, and then the difference of protein expression of VEGF-A is determined using western-blot.7. Statistical treatmentAll data are analyzed by statistical package for the social science 13 (SPSS 13.0). The data of histochemistry are treated by chi-square test, the semi-quantitive data of FQ-PCR and Wstern-blot were treated by one-way ANOVA, P<0.05 is considered significant value.Results1. Result of histochemistry In the ovarian tissues, a cytoplasmic VEGF reaction predominates and only scattered cells exhibited nuclear reaction. VEGF is expressed in 58(85.3%) ovarian cancers, in 12(35.3%) benign ovarian neoplasms and 4(13.3%) normal ovarian tissues. The chi-square test demonstrates a statistically difference in VEGF expression between vicious ovarian tumors and benign ovarian neoplasms (x2=22.310, P=0.000<0.05), between benign ovarian neoplasms and normal ovarian tissues(x 2=5.074, P=0.024<0.05), between vicious ovarian tumors and normal ovarian tissues(x 2=46.382, P=0.000<0.05), respectively. Among vicious ovarian tumors whose clinical stage are stageⅢand stageⅣVEGF staining is positive 41 (95.3%), while 23 (92%) specimens are positive stageⅠand stageⅡvicious ovarian tumors. No statistical deifferece is found between stageⅠ&Ⅱand stageⅢ&Ⅳvicious ovarian tumors (x 2=0.32, P=0.571). Furthermore, the strong and moderate positives (60.3%, 35/58) are most common in vicious ovarian tumors, while in benign ovarian neoplasms is slight (72.7%,8/11).2 FQ-PCR resultsThe onw-way analysis of variance(ANOVA) result showed the statistical difference of the expression of VEGF among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues:ovarian cancers vs benign ovarian neoplasms(P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05). No statistical difference of expression of VEGF was found between stageⅠ&Ⅱand stageⅢ&Ⅳovarian cancers (P=0.78).3.Western-blot analysisThe results of ANOVA showed that the differences of protein expression of VEGF-A among ovarian cancers, benign ovarian neoplasms and normal ovarian tissues were statistically significant:ovarian cancers vs benign ovarian neoplasms (P<0.05), benign ovarian neoplasms vs normal ovarian tissue (P<0.05), ovarian cancers vs normal ovarian tissue (P<0.05).Conclusions1. There is high expression of VEGF in ovarian cancers.2. There is a statistically difference in VEGF expression between vicious and benign ovarian neoplasms. VEGF is highly associated with malignant pituitary adenomas.3. VEGF can serve as a useful tool for confirmation of ovarian tumors and VEGF gene also may be an effective target for ovarian cancer gene therapy. PartⅡDesign and synthesis of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene and the assessment of the transfection efficiencyObjectiveDesign and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene, and test the inhibitory efficiency of the antisense oligonucleotides.Methods1. Design and synthesize 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A2'-Methoxyethyl modified antisense oligonucleotides were designed and synthesized by Oligos Etc Inc (Oregon),3 groups of ASODN targeting VEGF-A gene were synthesized and marked as 1#,2# and 3#, respectively.2. Test the efficiency of antisence oligonucleotides transfection in SKOV3 ovarian cancer cells)Firstly, SKOV3 ovarian cancer cells were cultured, and the cells in good growth condition were spread into a six-well cell culture plate one day before transfection. Secondly,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A was delivered into cultured cells according to previous groups design. Then we measured RFP expression under fluorescence microscope 48 hours after transfection to observe the transfectory potency of the recombined vectors.ResultsIn 3 test groups transfected with 2'-Methoxyethyl modified antisense oligonucleotides, the transfection potency values of 1#,2#,3# were 86.5±4.5%,91.2±5.7% and 92.1±2.8%, respectively.ConclusionsIn this part,3 groups of ASODN 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A were successfully designed and synthesized. PartⅢThe effect of 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cellsObjectiveStudy the influence and significance of 2'-Methoxyethyl modified antisense oligonucleotides 1#,2# and 3#, targeting VEGF-A gene on the proliferation and apoptosis level of SKOV3 ovarian cancer cells.Methods1. Transfect SKOV3 cells with ASODN 1#,2# and 3#Firstly, SKOV3 ovarian cancer cells were cultured in good growth condition, and then the cells were spread into the six-well culture plate one day before transfection. On the day of transfection, the experiment was performed with ASODN group 1#,2# and 3#, the negative control group and the blank control group according to the design of experiment.2. Measurement of the silencing effect on VEGF-A gene expressionReal-time PCR was performed to measure the mRNA expression of VEGF-A and Western-blot was performed to measure the protein expression of VEGF-A, testing the silencing effect of VEGF-A gene.3. Measurement of the proliferation level of cells with MTTSKOV3 cells which were in good growth condition and were seeded in 96-well culture plate before. On the second day after transfection, MTT was performed using standard methods to measure the changes of proliferation level before and after transfection.4. Investigation of pituitary tumor cell apoptosis level variation by PI dyeing FCMStable tansfecion SKOV3 are cultured in good condition. According to designed groups (ASODN 1#,2#,3# and controls), cells are collected. PI dyeing FCM method is employed to contrast the changes of cell apoptosis levels.Results1. Realtime-PCR results showed that, ASODN 1#,2#,3# had significant gene silencing effect against VEGF-A gene expression of SKOV3, and their silencing efficiencies were 68.4%,69.44%,55.7%, respectively. Western-blot showed a similar result. 2. MTT-detected cellular proliferation level test results showed that, the inhibition rate (IR) of experimental group 1#,2#,3# were 42.5%,38.7% and 44.5%, respectively, which had significant differences with the negative control group and the blank control group (P<0.05). The result indicated that when the VEGF-A gene were silenced, the proliferation of the ovarian cancer cells were significantly decreased, and the growth ability of the cancer cells were significantly inhibited.3. Analyzing the proportion of all period cells in cell colony by FCM, we found that, after VEGF-A was silenced, the proportion of apoptosis cell increased in the three experimental groups, comparing with control group.Conclusions1.2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly silence VEGF-A mRNA and protein expression in SKOV3 ovarian cancer cells. Besides,2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A gene can significantly inhibit the the proliferation and growth potency in SKOV3 ovarian cancer cells. It may indicate that VEGF-A gene can act as an effective target in the ovarian cancer gene therapy.2. The above results also indicate that 2'-Methoxyethyl modified antisense oligonucleotides targeting VEGF-A shows a promising potential in targeted gene therapy and research of ovarian cancers.Part IV Exploiting Inhibitory Effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer modle of nude mice by a novel combination:liposome microbubbles and site-specific ultrasound exposureObjectiveTo validate the inhibitory effects of the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene in Ovarian Cancer in vivo and to exploit the effectiveness of the transfection mediated by liposome microbubbles and ultrasound exposureMethodmethods:30 Balb/c nude mice were subcutaneously inoculated human ovarian cancer cell line SKOV3. When the endermic tumors were all larger than 300mm3 in size,the mice were then randomly divided into 3 groups (10 mice each group):①UM+ASODN (liposome microbubbles+ASODN+site-specific ultrasound exposure) group:400μL mixture of microbubble/ASODN/(with 2.5ug ASODN) /liposome was injected into the center of the tumor of every mice②; UM (liposome microbubbles+site-specific ultrasound exposure, no ASODN) group:300μL of microbubbles and 100μL of D-Hanks were injected into the center of the tumor of every mice③BC (blank control,no ASODN,no microbubbles,no ultrasound exposure) group: 400μL of D-Hanks was injected into the center of the endermic tumors.After all these have been done,site-specific ultrasound irradiation was carried out to each mouse with the parameter settting on a frequency 1 MHz and the intensity 1.0w /cm2.The exposure was given 30 seconds twice with an 30 seconds interval.The tumor volume was measured every 3 days from the irradiation on and the size of the endermic tumor was calculated according to the formula V=πab2/6 (a=length, b=width) 15 days after the irradiation, the nude mice were killed and the weight of the tumor was weighed.All data are analyzed by statistical package for the social science 16 (SPSS 16.0). The data of the size and the volume of the endermic tumors are treated by Anova analysis,and the comparison of the size between every two groups were carried out by LSD test. While the comparison of the weight of the endermic tumors between groups were carried out by DUNNETT T3 test, p<0.01 is considered to be of extremely significant value and p<0.05 is considered to be of significant value.Result(1)The mean volume of endermic tumor of UM+ASODN group, UM group and BC group was 108.2±26.42mm3,297.8±44.22mm3,339.7±41.32mm3 respectively. The ANOVA analysis demonstrates an extremely significant differnec between groups (F=104.67,p=0.00<0.01). LSD test told us that the volume of the ASODN+UM group was obviously statistically smaller than UM group and BC group (p=0.00<0.01) and the volume was statistically smaller in UM group than BC group (p=0.021<0.05).(2)the mean weight of nude mice tumor were 0.70±0.08g,1.45±0.14g,1.66±0.23g respectively. The weight of the ASODN+UM group was obviously statistically smaller than UM group and BC group (DUNETT T3 method,p=0.00<0.01).The analysis shows no significant difference between UM group and BC group(DUNETT T3 method,p=0.078>0.01). (3)The autopsy of the nude mice demonstrated that something like membrane surrounded the pale, smaller-sized tumors of ASODN+UM group. The boundary between tumors and normal tissues was obvious;While in UM group arid BC group, the tumor was red,huger in size, abundant in blood supply and mingled with the normal tissue that cannot be easily dissected.Conclusion(1) We successfully set up the Balb/c nude mice model bearing human ovarian cancer cell line SKOV3.(2) We successfully disposed of the nude mice model bearing human ovarian cancer cell line SKOV3 with Optison(?) ultrasound contrast agent microbubbles and ultrasound exposure.(3) We found that the 2'-O-Methoxyethyl-Modified Antisense oligonucleotides targeting VEGF-A gene greatly decrease the growth of the tumor of the SKOV3 ovarian cancer cells in vivo with site-specific ultrasound exposure and Optison(?) contrast agent microbubbles.