Mechanism Of UBE3A Affecting The Invasive Ability Of Breast Cancer Cells And Its Relationship With ANXA2

Part 1 Corelation of single nucleotide polymorphisms(SNPss)of ANXA2 with the pathogenesis of breast cancerObjective: To explore the relationship between ANXA2's SNPss and breast cancer's pathogenesis by PCR-SSCP and bidirectional sequencing analysis.Methods: 14 human breast cancer tissues and its adjacent normal tissues were collected from the Affiliated Hospital of North Sichuan Medical College from April 2015 to June 2015.Total RNAs of tissues were extracted,and then cDNAs were synthesized by reverse transcription timely.Five primers were constructed by NCBI and Primer3.0 Input,which covered major part of the coding region of ANXA2 gene.Semi-quantitative RT-PCR was performed to amplify the ANXA2 gene,and PCR-SSCP was used to examine mutation of ANXA2.Bidirectional Sequencing was done to detect the sequences of the ANXA2's bases,and sites of SNPss were located by BLAST analysis of the NCBI websites.Results: The significative band of SNPs was not observed by PCR-SSCP analysis in 14 human breast cancer tissues and its adjacent normal tissues,but The mutation of the ANXA2's bases were detected by Bidirectional Sequencing analysis,including 21 SNPss sites with 50 amino acides changed in cancer tissues,and 9 SNPss sites with 18 amino acids changed in normal group.Compared to adjacent normal tissues,there were statistically significant SNPss in 14 cancer tissues,including G/C mutation in 1071 site for 6 times(42.86%,and P=0.023),A/T and A/G mutation in 531 site for 7 times(A/T for 2 times,A/G for 5 times,the mutation rate of 50%,and P=0.006),GA/TG mutation in 260-261 sites for 5 times(35.71%,and P=0.041).However,the three mutations of ANXA2 gene were not reported up to now,so they may be new mutations and be closely associated with the pathogenesis of breast cancer.Moreover,G/T mutation in 380 site for respective 6 times(42.86%)and 2 times(14.29%)in adjacent normal tissues and cancer tissues,there was no statistically significant difference(P>0.05),as samples may be not enough.Conclusion: The stabile mutations of ANXA2 gene were observed in breast cancer tissues,including G/C mutation in 1071 site,A/T and A/G mutation in 531 sites,and GA/TG mutation in 260-261 sites.The results suggested that these SNPss may be closely associated with the genesis and developement of breast cancer.Part 2 Subcellular localizations of ANXA2 were changed in breast cancer cells after the knock-down of UBE3AObjective: To partly clarify the mechanism of UBE3 A affecting biological behaviors of breast cancer cells mediated by ANXA2 by observing the changes of subcellular localization of ANXA2 after knock-down of UBE3 A.Methods: Lentiviral vectors with UBE3A-shRNA were used to transfect breast cancer cells.RT-PCR and WB were performed to verificate the efficiency of the knock-down of UBE3 A in MDA-MB-231 cells.Laser Scanning Confocal Microscope(LSCM)was used to detect the subcellular distribution of ANXA2 in breast cancer cells.The data were analyzed by SPSS19.0 software,and there was statistically significant difference(P< 0.05).Results: MDA-MB-231 cells were transfected by lentiviral vectors with UBE3A-sh RNA,the efficiency of the negative control and experimental group were respectively 100% and above 95% after 96 hours.The differences of UBE3 A gene and protein between the blank group(0.59±0.029,0.35±0.017)and the negative control group(0.65±0.042,0.46±0.023)were not statistically significant(P>0.05).Compared to the blank group,the level of UBE3 A in experimental group(0.29±0.036,0.16±0.015)were lower with the inhibition rate 50.8% and 48.4% respectively,so there was statistically significant difference(P=0.000),and the results suggested that UBE3 A gene was effectively knocked-down.By LSCM analysis,ANXA2 were mainly distributed in cell membranes in the blank group and the negative control group,however,which were mainly distributed in the cytoplasm in the experimental group,and there was statistically significant difference(P=0.001).The results indicated that the subcellular localization of ANXA2 was changed after the knock-down of UBE3 A.Conclusion: After knock-down of UBE3 A,The function of the pathways(UBE3A/p53/ANXA2/EGFR)could be changed,and UBE3 A could impact the biological behaviors of breast cancer cells by affecting the subcellular localization of ANXA2.The results indicated that UBE3 A was tightly related with ANXA2,and both of them may play an important role in the pathogenesis of breast cancer.