Effect Of SiRNA Expressing Vector Targeting VEGF-C On Proliferation,Apoptosis And Chemosensitivity Of Human Breast Cancer Cell Line MCF-7

BackgroundBreast cancer is one of the malignant tumors threatening female health in the world.The incidence rate of breast cancer has recently been up in China.Nowdays, the popular treatment of breast cancer is still focus on surgery operation accompanied by radiotherapy,chemotherapy and hormone therapy.Although these approaches have made some considerable progress,questions such as metastasis and recurrence and drug resistance eventually make the therapeutic effect dissatisfactory.Therefore, the evaluation of novel targeted therapeutic agents is urgently needed.With the development of molecular biology,the mechanism of occurrence and development,especially about signal conduction that is pertinent to growth, proliferation and metastasis of breast cancer cells has been studied.Treatment targeting cancer genes related with occurrence and development and their expressed production,namely,molecule-targeted therapy,has currently been a new hotspot in the field of breast cancer therapy.Vascular endothelial growth factor-C(VEGF-C)is a new member of vascular endothelial growth factor(VEGF)family and stimulates the proliferation of vascular and lymphatic endothelial cells by working with tyrosine kinase receptors VEGF receptor-2(VEGFR-2)and VEGF receptor-3(VEGFR-3),suggesting that VEGF-C is associated with growth and metastasis of tumors.Recent studies have showed that the binding of VEGF-C and VEGFR-2/VEGFR-3 on tumor cells can stimulate proliferation of tumor cells,inhibit apoptosis and decrease sensitivity to chemotherapeutic drug by increasing the ratio Bcl-2/Bax.It is found that VEGF-C is over-expressed in breast cancer.Researches on that VEGF-C over-expression in breast cancer has close correlation with tumor growth and metastasis have been reported largely,whereas those on whether VEGF-C over-expression in breast cancer affects the sensitivity of chemotherapeutic drug have not been studied so far.VEGF-C has an important effect on growth,metastasis and drug resistance of tumor cells,therefore,inhibition of VEGF-C expression could be an effective therapeutic approach for breast cancer.At present,methods on gene therapy for targeting VEGF-C gene are as follows:1)utilization of antibody blocking VEGF-C or VEGFR-3.2)inhibition of activation of tyrosine kinase receptor.3)down-regulation of VEGF-C and VEGFR-3 expression by antisense oligonucleotide technology or ribozymes.However,these methods have their own limitations.The discovery of RNA interference(RNAi)phenomenon provides a new method for inhibiting tumor growth and metastasis and decreasing drug resistance by targeting VEGF-C gene.RNAi represents a phenomenon of double-stranded RNA(dsRNA)mediated post-transcriptional gene silencing(PTGS).During this process,siRNAs is the mediator,which is made up of 19～23bp small RNA fragments.By joining an effector complex termed RISC(RNA-induced silencing complex),siRNA binds to the cellular RNA with homologous sequences,and contributes to the degradation of the corresponding RNA.At present,RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing.In process of gene therapy,siRNA synthesized artificially or transcribed by siRNA expressing vector was transfected into tumor cells and specifically inhibited gene expression.Up to now,RNAi technology targeting VEGF has been applied in gene therapies for tumors,which have obtained great achievements.However,Such studies on VEGF-C gene lag behind,and are only limited to observing the effect of down-regulated VEGF-C on lymphatic vessels formation and lymph metastasis to distant organs.Research on whether down-regulated VEGF-C has an effect on tumor growth,apoptosis and chemotherapeutic resistance have hardly reported.Therefore,the present study is to inhibit VEGF-C expression in breast cancer cell line MCF-7 using RNAi technology and to find a new strategy of gene therapy based on inhibiting proliferation,promoting apoptosis and enhancing drug sensitivity of tumor cells.PARTⅠ:CONSTRUCTION AND IDENTIFICATION OF SIRNA EXPRESSING VECTOR TARGETING VEGF-CObjectiveTo construct a siRNA expressing vector targeting human VEGF-C gene and prepare for the further study.Methods1.Three complementary oligodeoxyribonucleotides encoding VEGF-C short hairpin RNA and one negative control were designed and synthesized.Then they were annealed and ligated into a linear pRNAT-U6.1/Neo containing U6 promoter to construct the recombined plasmid targeting VEGF-C.2.The ligation mixtures were transformed into competent E.coli DH5α.The recombinant plasmids were extracted from small-scale bacterial cultures by Alkaline Lysis and then identified by PCR and DNA sequence analysis.QIAGEN plasmid maxi-kit was employed for preparation at large quantity of recombinant plasmid.ResultsPCR and DNA sequencing has verified that the insert sequence in three siRNA expressing vector targeting VEGF-C and one negative control was successfully cloned into the vector.ConclusionThree expressing vector targeting human VEGF-C gene and one negative control have been successfully constructed by PCR and DNA sequencing.PARTⅡ:EFFECT OF SIRNA EXPRESSING VECTOR TARGETING VEGF-C ON VEGF-C EXPRESSION IN HUMAN BREAST CANCER CELL LINE MCF-7ObjectiveTo provide the experimental data for RNAi technology targeting VEGF-C applied for breast cancer gene therapy,siRNA expressing vectors targeting VEGF-C were transfected into MCF-7 cells via positive ion liposome Lipofectamine^TM2000, and VEGF-C mRNA and protein expression levels in MCF-7 cells were detected by RT-PCR,ELISA and immunocytochemistry,respectively.Methods1.Three siRNA expressing vector targeting VEGF-C,named pRNAT-VEGF-C-1 (P-1),pRNAT-VEGF-C-2(P-2),pRNAT-VEGF-C-3(P-3),were transfected into human breast carcinoma cells MCF-7 respectively via Lipofectamine^TM2000.At the same time,the untreated cells and the cells transfected with expressing vector targeting nonsense sequence,named pRNAT-Negative(P-N),were as the negative control.2.Transfection efficiency was quantified by determining the percentage of cells that were green fluorescence protein(GFP)-positive in the total cells under fluorescent microscope,and then transfection efficiency in every group was compared.3.VEGF-C mRNA and protein expression levels were detected by RT-PCR,ELISA and immunocytochemistry,respectively. Results1.GFP expression in MCF-7 cells was observed under the fluorescent microscope after transfection.Results showed that GFP in MCF-7 cells began to express at 24h and markedly expressed at 48h after transfection.The transfection efficiency in every group is above 70%.There was no significant difference in every transfection efficiency(P＞0.05).2.RT-PCR showed that VEGF-C mRNA andβ-actin as internal control were seen at 610bp and 197bp respectively through electrophoresis analysis.Three siRNA expressing vector targeting VEGF-C could effectively decrease VEGF-C mRNA expression in MCF-7 cells while P-1 more potently suppressed VEGF-C mRNA expression than P-2 and P-3.There is no significant difference in VEGF-C mRNA expression levels between P-N transfected cells and untreated cells.The relative expression rate of VEGF-C mRNA in P-1 transfected group,P-2 transfected group,P-3 transfected group,P-N transfected group and untreated group is 38.14%,63.42%,68.00%,98.00%and 98.63%,respectively.There was significant difference between controls and siRNA groups(P＜0.05).3.Results obtained from ELISA assay also demonstrated that compared with P-2 and P-3,P-1 could most significantly inhibit VEGF-C protein secretion.In contrast, P-N and untreated cells had no obvious silencing effect on secretion of VEGF-C protein(P＞0.05).The corresponding values in P-1 transfected group,P-2 transfected group,P-3 transfected group,P-N transfected group and untreated group is 117.81±24.22pg/ml,272.81±29.23pg/ml,289.60±31.00pg/ml, 385.40±31.42pg/ml and 367.12±34.90pg/ml,respectively.There were distinct differences between controls and siRNA groups(P＜0.05).4.The immunocytochemistry revealed that the positive expression of VEGF-C protein appears brown staining in the cytoplasm.P-N transfected group and untreated group displayed obvious brown immuno-reactivity.P-2 transfected group and P-3 transfected group showed light brown staining while P-1 transfected group hardly showed brown staining. Conclusion1.Lipofectamine^TM2000 transfected siRNA for VEGF-C gene into MCF-7 cells is convenient and has high transfection efficiency.2.RT-PCR,ELISA and immunocytochemistry have demonstrated at mRNA and protein level that three siRNA expressing vector targeting VEGF-C(P-1,P-2, P-3)could effectively inhibit VEGF-C mRNA and protein expression in MCF-7 cells.Compared with P-2 and P-3,P-1 most markedly suppressed the expression of VEGF-C gene.PARTⅢ:EFFECT OF SIRNA EXPRESSING VECTOR TARGETING VEGF-C ON PROLIFERATION, APOPTOSIS AND CHEMOSENSITIVITY OF HUMAN BREAST CANCER CELL LINE MCF-7ObjectiveTo assess the possibility of siRNA expressing vector targeting VEGF-C in combination with Epirubicin for breast cancer therapy,the cell proliferation, apoptosis and chemosensitivity was determined by MTT assay and flow cytometry, and the ratio Bcl-2/Bax was detected by Western blot.Methods1.The drug resistance concentration in vitro of MCF-7 cells was detected by MTT assay.2.Effect of siRNA expressing vector targeting VEGF-C in combination with Epirubicin on MCF-7 cells proliferation was analysed by MTT.3.Effect of siRNA expressing vector targeting VEGF-C in combination with Epirubicin on MCF-7 cells apoptosis at early stage was assessed by Annexin V/PI method.4.Effect of siRNA expressing vector targeting VEGF-C in combination with Epirubicin on the ratio of Bcl/Bax was determined by Western blot.Results1.MTT assay showed that low concentration of Epirubicin alone could not inhibit the proliferation of MCF-7 cells,while concentration of Epirubicin which is up to 8umol/L above could markedly inhibit the proliferation of MCF-7 cells and the suppression rate was a concentration dependent manner.2.MTT assay showed that with and without the addition of Epirubicin,the cell viability in P-1 transfected group was significant lower than that in P-N transfected group and untreated group and the difference is very significant (P＜0.05),while there was no significant difference between P-N transfected group and untreated group(P＞0.05).The cell viability in P-1 transfected group was markedly decreased,from 78.63%to 38.54%,when exposed to Epirubicin at 8umol/L for 24h.(8umol/L was determined corresponding to approximately less than 5%of growth inhibition rate of MCF-7 cells),and the difference was significant(P＜0.05).3.Flow cytometry showed that the rate of apoptosis in P-1 transfected group was only 13.10%without Epirubician treatment,but the rate of apoptosis in P-1 transfected group reached 38.91%after addition of Epirubicin for 24h.Moreover, the difference was more significant than that in P-N transfected group and untreated group(P＜0.05).There was no significant difference in apoptosis rate in P-N transfected group and untreated group in the absence or presence of Epirubicin(P＞0.05).4.Results from Western blot demonstrated that compared with controls,the expression levels of Bcl-2 in P-1 transfected groups was suppressed(P＜0.05) before addition of Epirubicin,and the expression levels of Bcl-2 was further suppressed by cotreatment with Epirubicin,while the expression levels of Bax had hardly changed with or without Epirubicin treatment,suggesting the ratio of Bcl-2/Bax was decreased towards proapoptotic(P＞0.05).ConclusionSiRNA expressing vector targeting VEGF-C combined with Epirubicin can significantly inhibit tumor cells proliferation,promote apoptosis and enhance the sensitivity to chemotherapeutic drug.This suggests that inhibition of VEGF-C expression in MCF-7 cells can accelerate Epirubicin-induced apoptosis by a down-regulation of Bcl-2 protein,resulting in the decrease of Bcl-2/Bax.Bring New Ideas1.SiRNA expressing vector targeting VEGF-C was first transfected into human breast cancer cell MCF-7 via Lipofectamine^TM2000,and successfully inhibited VEGF-C mRNA and protein expression in MCF-7 cells.2.SiRNA expressing vector targeting VEGF-C in combination with Epirubicin was first observed to effectively inhibit proliferation,promote apoptosis and enhance chemosensitivity of MCF-7 cells,which provides the data for RNAi targeting VEGF-C combined with chemotherapeutic drug in the treatment of breast cancer.3.The ratio of Bcl-2/Bax was first detected to study the mechanism that inhibition of VEGF-C expression in MCF-7 cells enhanced chemosensitivity.It is first demonstrated that VEGF-C expression inhibited by VEGF-CsiRNA expressing vector can enhance chemosensitivity by a down-regulation of Bcl-2 protein, resulting in the decrease of Bcl-2/Bax and inducing apoptosis.