Expression And Clinical Significance Of Wnt5a Gene In Breast Cancer

ObjectiveHormone receptor positive breast cancer patients had better prognosis than negative cancers, hormone receptor positive was the indication of endocrine therapy. Therefore, regulation of hormone receptor expression has important clincical significance. Wnt signal pathway was closely correlated to the genesis of tumors, Wnt5a was significantly correclated with the status of hormone receptor. But the effect of Wnt5a on breast cancer biological characteristics and the mechanisms of regulation hormone receptor expression need to be further study. This thesis amis at investigating the effects of Wnt5a on biological characteristics of breast cancer cells, clinical significance of Wnt5a in the patients with breast cancer and the association between polymorphisms of Wnt5a and the occur and the hormone receptor status of breast cancer.MethodsPart I1. SiRNA for Wnt5a gene was designed and synthesized, then be transfeted into breast cancer cells line MCF-7. Western-blot was used to screen the most effective siRNA and Wnt5a protein changes. The effects of Wnt5a gene downregulation on proliferation and invasive ability were tested by CCK-8and transwell.2. The difference of miRNA-206expression between MCF-7and MDA-MB-231cell lines was determined by real-time quantitative PCR. Downregulation of MCF-7Wnt5a mRNAthen detected the miRNA-206changes by real-time PCR.3. Mimic-miRNA-206and inhibitor-miRNA-206were transfected into ER-positive breast cancer cell lines, transfect effiency was assessed by real-time PCR. Expression of Wnt5a and ER protein was detected by western-blot.4. Effects of zoledronic acid on the proliferation, apoptosis in ER-positive breast cancer cells were investigated by CCK-8and FCM. 5. CCK-8was used to observe the effect of Wnt5a downregulation on zoledronic acid sensitivity.6. The protein expression of Wnt5a, ER and LC3in different concentration zoledronic acid treated ER-positive breast cancer cells were detected by western-blot.7. Autophagy indued by zoledronic acid was investigated by transmission electron microscope.8. CCK-8was used to detect the effects of zoledronic acid combined with fulvestrant on growth of human breast cancer cell line MCF-7, western-blot was employed to detect the expression of Wnt5a and ER of human breast caner cells treated by the combination of zoledronic acid and fulvestrant.Part Ⅱ1. We used741breast cancer cases and293cancer free controls, controls were matched by age. Took5.0ml venous blood from every recruited patients after signing consent.2. To extract the DNA and to determine the association between SNPs in genes encoding Wnt5a and breast cancer risk. SNP genotyping was used MassARRAY system by means of matrix assited laser desorption ionization-time of flight mass spectrometry method (MALEI-TOF) according to the manufacturers instructions.3. We evaluated the odds ratios and their95%confident intervals of the associations between Wnt5a polymorphism and breast cancer risk and hormone receptor status among breast cancer patients.Part Ⅲ218patients of primary breast cancer resected between January2005to December2005in Fudan University Shanghai Cancer Center were included. Clinicopathologic data were obtained from patients" records. Immunohistochemical method was used to detect the expression of Wnt5a. The correlation among Wnt5a and clinicopathologic factors and survival were then statistically analyzed, using stata10.0. The statistical associations between clinical parameters and immunohistochemical staining were tested by y2test. The survival rate was calculated by the Kaplan-Meier method. Variables that seemed to be significantly associated with survival on univariate analysis were entered into multivariate analysis, which was performed with Cox proportional hazard model. A P value<0.05was considered significant.ResultsPart Ⅰ1. Downregulation MCF-7cell Wnt5a mRNA could inhibit the expression of ER protein and promote MCF-7cell proliferation, but had no effect on the invation of MCF-7cells.2. The expression level of miRNA-206in MCF-7cells was lower than MDA-MB-231cells. Downregulation of MCF-7cells Wnt5a mRAN could enhance the expression of miRNA-206.3. Upregulation miRNA-206of MCF-7cells could inhibit the protein expression of Wnt5a and ER, downregulation miRNA-206of MCF-7cells had no effect on the expression of Wnt5a and ER protein.4. Zoledronic acid could significantly decreased proliferation and induced apoptosis in breast cancer MCF-7cell line which depended on drug concentration.5. Human breast cancer MCF-7cells transfected by siRNA-Wnt5a could obviously decreased the sensitivity of MCF-7cells to zoledronic acid.6. Zoledronic acid could significanctly decreased the protein expression of Wnt5a and ER in MCF-7cells, and enhanced LC3protein expression.7. Autophagosomes in MCF-7cells induced by zoledronic acid were observed by transmission electron microscopy, and autophagosome formation enhanced with concentrations increased.8. Combination treatment of zoledronic acid and fulvestrant could significantly inhibit the growth of MCF-7cells, the protein expression of Wnt5a and ER decreased in MCF-7cells exposed to the combination of the two drugs.Part ⅡMultiple factor logistic regression analysis demonstrated that the polymorphism of Wnt5a was not associated with breast cancer risk after controlling the age, meno status and breast cancer family history.In the group of breast cancer, we found CG+CC genotype compared to GG genotype of SNP rsl1918967increased the positive proportion of PR and HR. Part ⅢHigh expression rates of Wnt5a was46.33%. A statistically significant association between the expression of Wnt5a correlated with tumor size, ER and PR. In the univariate survival analysis, we observed Wnt5a negative patients were more likey to recur than those Wnt5a positive patients. The Cox proportion hazards regression analysis showed that Wnt5a expression as well as lymph nodes was an independent prognostic factor for relapse-free survival. In subgroup of ER positive breast cancer patients, Wnt5a was also a independent prognostic factor.Conclusion1. Wnt5a gene is an important tumor suppressor gene in breast cancer, it might act together with miRNA-206to control the expression of ER. Zoledronic acid had a direct anti-tumor effect and enhanced the action of endocrine therapy, and this might be achieved through regulating Wnt5a-ER pathway.2. The polymorphism of Wnt5a gene have an effect on the ER and HR expression in breast cancer patients. It will lay the foundation of predicting endocrine therapy effect and prognosis model based on gene data.3. Expression of Wnt5a was significantly correlated with ER and PR. The survival rate of Wnt5a positive breast cancer patients was superior to negative patients, it was an independent prognostic factor.