Role And Regulation Of MiR-205 In Breast Cancer Chemoresistance Through Targeting VEGFA And FGF2

Background: Multidrug resistance(MDR) still remains one of major obstacles which lead to the failure of chemotherapy and tumor recurrence.While the majority of cancers initially respond to chemotherapeutic drugs, acquired cross resistance to a variety of structurally and mechanistically unrelated drugs develops during the treatment, leading to relapse and disease progression. MDR is Tumor appeared to a chemotherapy drug resistance, At the same time with different structure and function of other drugs it also produce cross resistance phenomenon. Almost inevitably chemotherapy drug resistance has been the bottleneck of effective control of breast cancer. So to study the breast cancer drug resistance mechanism, looking for a new resistance markers for effective reverse breast cancer drug resistance, to improve the level of breast cancer treatment is of very important theoretical significance and potential clinical application value. micro RNAs(mi RNAs) is composed of 18-24 nucleotides, endogenous highly conserved noncoding single-stranded RNA, Mature mi R molecules function as negative regulators of gene expression by triggering translation repression through partial complementation to 3'-untranslated region(UTR) of target m RNAs or by the degradation of m RNAs bearing fully complementary target sites. It becomes more and more important in gene expression regulation as one gene could be modulated by different mi RNAs and one mi RNA can modulate different genes depends on different contexts of tumor cells. The gene expression regulation can tell us a more comprehensive understanding of tumor occurrence and development, including the MDR of tumor cells,it has important significance in understanding the underlying molecular mechanisms of drug resistance of tumor. In this work, we mainly study the multidrug resistance of breast cancer related mi RNA: mechanism of chemoresistance mediated by mi R-205 in breast cancer,Clear and direct binding of mi R-205 and putative target genes, and whether mi R-205 through the target genes influence breast cancer drug resistance and related molecular mechanism is studied and discussed in this paper.Methods:1. We used micro RNAs Chip to compare the expressions of 1409 differents mi RNAs in sensitive breast cancer cell line MCF-7 and MDR breast cancer cell line MCF-7/A02. We select one of the most significant difference one in the two kinds of cell lines to do the follow-up study : mi R-205. 2. To verify the correlation between mi R-205 expression and drug resistance of tumor cells in different breast cancer cells and the corresponding resistance cell lines with Taqman real-time PCR, and further validation in other drug resistant cell line.The detection the expression of mi R-205 before neoadjuvant chemotherapy in breast cancer tissue, to investigate the relationship between the mi R-205 expression and the sensitivity to neoadjuvant chemotherapy in breast cancer. 3. We overexpressed mi R-205 in MCF-7/A02 cells and CALDOX cells by lenti-virus infection, MTT method was used to examine cell IC50 of doxorubicin, paclitaxel, etoposide, to detect the resistance changes.Clone formation experiment method was used to evaluae of the sensitivity of tumor cells to doxorubicin, paclitaxel.Annexin V/PI staining, caspase-3/7 activity detection and caspase-9 detection method was used to evalue the effect of apoptosis induced by paclitaxel on target cells. At the same time, apoptosis related genes' m RNA level were detected by using real-time quantitative PCR, western- blotting was used to detect the PI3K/AKT/ signal transduction pathway and apoptosis related protein changes. 4. Comprehensive bioinformatic softwares predict VEGFA and FGF2 is mi R-205' candidate genes. Differential expression of FGF2 and VEGFA were detected in MCF-7,MCF-7/A02,CAL51 and CALDOX cells by real-time quantitative PCR, while VEGFA and FGF2 of four groups were detected by ELISA in the supernatant.Further we detected the VEGFA and FGF2 gene expression in MCF-7/A02 and CALDOX transfected with mi R-205 by real-time quantitative PCR, and VEGFA and FGF2 factors in the supernatant of cells by ELISA. We analysised and comparied various cell lines the expression of mir-205,VEGFA and FGF2,to findout the correlation. We performed luciferase assays to verify whether VEGFA and FGF2 were the direct target gene that mi R-205 regulates inMCF-7/A02 and CALDOX cells. 5. We use real-time quantitative PCR to detect the of mi R-205, VEGFA and FGF2 before neoadjuvant chemotherapy in breast cancer tissue, to analys the correlation of them.MTT method was used to test the IC50 of cells to doxorubicin, paclitaxel, and etoposide after overexpression of VEGFA and FGF2 and silence both of them,inorder to evalue the changes of their drug sensitivity; and further to detect the effects of VEGFA and FGF2 on cell apoptosis. 6. The rescue experimental was used to test whether VEGFA and FGF2 expression can reverse mi R-205's inhibitory in breast cancer chemotherapy drug resistance, to confirm VEGFA and FGF2 were the direct targets of mi R-205.MTT method was used to test the IC50 of different cells with lenti-virus infectied mi R-205 to doxorubicin, paclitaxel, and etoposide after overexpression of VEGFA and FGF2.Clone formation experiment method was used to evaluae of the sensitivity of the above tumor cells to doxorubicin, paclitaxel.Annexin V/PI staining, caspase-3/7 activity detection and caspase-9 detection method was used to evalue the effect of apoptosis induced by paclitaxel on target cells. At the same time, apoptosis related genes' m RNA level were detected by using real-time quantitative PCR,western-blotting was used to detect the PI3K/AKT/ signal transduction pathway and apoptosis related protein changes. 7. Cells were treated with PI3 K inhibitor LY294002(10?M for MCF-7/A02 and 2 ?M for CALDOX) for 24 h and treated by taxol(0.25 ?M for MCF-7/A02 and 2.5 n M for CALDOX) for 48 h, We used the above methods to detect the effects on apoptosis after PI3 K inhibition, mi R-205 inhibits PI3 K / AKT pathway activity induced apoptosis in breast cancer cells and increases chemosensitivity. The establishment of subcutaneous tumor model in nude mice. BALB/c mice were randomly divided into four groups,MCF-7/A02-mi R-205, MCF-7/A02-mi R-NC, CALDOX-mi R-205 and CALDOX-mi R-NC cells cell suspension liquid were inoculated in nude mice subcutaneously in the right axilla, about 1 week after the start,we use vernier caliper to measure the transplanted tumor in mice the longest size L and the shortest path W every 3 days morning, and the tumor volume was calculated and we make a record. In D15 the last time the tumor volume was measured,we administrated DOX(1mg/kg) and Taxol(2.5mg/kg) 1 times every three days by intraperitoneal injection. Every 3 days measurement in transplanted tumor of mice to calculated tumor volume, On the forty-fifth day of the last time measuring tumor volume, all mice were killed by cervical dislocation?The tumor mass were weighed and stored in liquid nitrogen reserve. We used the real time quantitative PCR to detect the changes of mi R-205, VEGFA, FGF2, Bax and survivin in tumor tissue.Also we detected the changes of expression of VEGFA and FGF2 in the blood of the animal model by ELISA.Results: 1. We obtained 19 differentially expressed mi RNAs after the comparison of MCF-7 cells with MCF-7/A02 Cell using the mi RNA microarray expression profiling, combined with our group' early study, we mi R-205 for further study. 2. q PCR method was used to validate the chip results, we found that in breast cancer MCF-7 cells, Cal51 cells, MTMEC cells four cell and their corresponding resistant cell lines,mi R-205 is low expression in the resistant cells and also in leukemia K562 cells and HL60 cells, human lymphoma cell line BJAB and the corresponding resistant cells,the expression of mi R-205 amount and drug resistance were negatively correlated. The mi R-205 expression was positively correlated with response to neoadjuvant chemotherapy in breast cancer patients. 3. The sensitivity of tumor cells to adriamycin increased when mi R-205 was overexpression of in MCF-7/A02 cells and CALDOX cells, while sensitivity to paclitaxel,etoposide chemotherapy was improved.Colony formation assay found that in the same chemotherapy drug concentration, mi R-205 overexpression can inhibit the colony formation of MCF-7/A02 cells and CALDOX cells. For the paclitaxel-induced apoptosis detection experiments, the degree of apoptosis in breast cancer cells was significant increased when mi R-205 was overexpressed. The m RNA Expression levels of Bax was increased and survivin was decreased in transfected drug-resistant cells.At the protein level of detection, the expression of p AKT was decreased, pro-apoptotic protein Bax was increased, anti-apoptotic proteins survivin was decreased. 4. Bioinformatics forecast software shows VEGFA and FGF2 m RNA 3'UTR have two conserved sequences and can be binded to mi R-205. VEGF and FGF2 expression was higher in drug-resistant cell lines, than that in control cells the expression of both two factors in the cell supernatant were also significantly higher. And in mi R-205 over expressed breast cancer cell lines, it can inhibit VEGFA and FGF2 not only in the m RNA level but also at cytokine level. For the wild type VEGFA and FGF2 reporter, over-expression of mi R-205 significantly reduced luciferase activities compared to controls.These results strongly suggest that mi R-205 down-regulates the expression of VEGFA and FGF2 by directly targeting it's 3'-UTR. 5. By analyzing the mi R-205,VEGFA and FGF2 expression before and after neoadjuvant chemotherapy in breast cancer patients, we found a negative correlation between the them, and patients with high expression of VEGFA and FGF2 show poor response to neoadjuvant chemotherapy.After addition of VEGF and FGF2 factor in MCF-7 cells and CAL 51,it led directly the cell resistance to doxorubicin and reduce apoptosis. Bax m RNA and protein expression were decreased, while survivin m RNA and protein expression were increased,also Activated PI3K/AKT pathway,The the sensitivity of CALDOX to adriamycin was increased when transfected with VEGFA si RNA and FGF2 si RNA. 6. Exogenous VEGFA/FGF2 attenuates the effect of drug-sensitivity restoration of mi R-205,reverse mi R-205's function of promotes apoptosis induced by paclitaxel, activate PI3K/AKT signaling in breast cancer cells. Bax m RNA and protein expression were decreased, while survivin m RNA and protein expression were increased. 7. The application of PI3 K inhibitor LY294002, Bax m RNA and protein expression were decreased, while survivin m RNA and protein expression were increased. It also indirectly suggests that mi R-205 is to promote apoptosis through inhibition of PI3K/AKT pathway activity 8. mi R-205 impairs the growth of breast cancer MCF-7/A02 xnenografts and increases chemosensitivity in vivo, VEGFA, FGF2 and Survivin were decreased in tumor tissues, while Bax was increased. VEGFA and FGF2 were decreased in serum of Mice.While mi R-205 overexpressing CALDOX xenograft models show no obvious tumor.Conclusion: 1.The mi R-205 expression was positively correlated with response to neo- adjuvant chemotherapy in breast cancer patients. 2. mi R-205 is involved in the development of MDR in breast cancer cells via targeting VEGFA and FGF2 simultaneously. 3.mi R-205 can promote apoptosis through targeting VEGFA and FGF2 simultaneously to inhibite PI3K/AKT pathway activity. 4.Targeting mi R-205 may thus provide a potential strategy for reversing drug resistance in breast cancer.