The Study Of Correlation Of Galectin-3 Gene Polymorphism And Breast Cancer And Preliminary Study Of Expression Of Poly(ADP-ribose) Polymers(PAR) In Triple-negative Breast

Objectivers4644 is a single nucleotide polymorphisms site locating at galectin-3 gene coding region. An allelic variation(C to A) in the DNA sequence of galectin-3 at position 191(rs4644), leading to the expression of either proline(P) or histidine(H) at position 64, which affects biological behavior of galectin-3. This study aims to analyze the distribution of rs4644 in breast cancer patients and explore the relations between rs4644 and breast cacer occurrence and development of chinese women. Poly(ADP-ribose) polymerase-1(PARP-1) is a DNA repair enzyme that can synthesize large branching chains of poly(ADP-ribose) polymers(PAR) involving in DNA single-strand break repair. PAR has been reported as a potentially powerful biomarker to indicate PARPs activity and predicted the response to PARP-1 inhibitors. A common PARP-1 single nucleotide polymorphism(SNP)rs1136410 affecting PARP-1 activity has been reported. Although PARP-1 inhibitors had entered Ⅲclinical trials in triple negative breast cancer treatment, however, a reliable biomarkers to assess of the sensitivity of PARP-1 inhibitors is lack.Therefore, the purpose of this report is to assess expression of PAR between triple-negative breast cancer(TNBC) and non- triple-negative breast cancer(non-TNBC). Correlation of PAR expression and PARP-1 active site SNP rs1136410 would be evaluated.MethodsGalectin-3 gene rs4644 polymorphisms was genotyped by polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP in 172 breast cancer patients and 17 controls. To verify the experimental results of PCR-RFLP, sample of 10 cases are sequenced randomly.Immunohistochemistry with a PAR antibody(MA1-90439 10 H Thermo) on the formalin-fixed paraffin-embedded tissue from 32 cases of TNBC and 39 cases of non-TNBC was used. The genomic DNA from same corresponding case of frozen tissue were extracted and used to detect differences between rs1136410 locus alleles.Results1. PCR products were digested after NCOI, band of 324 bp represented wild homozygotes(CC), heterozygous(CA) showed three bands of 324bp、171bp and 153 bp respectively, mutated homozygous(AA) were the 171 bp, 153 bp pairs of bands. Due to 2% agarose gel electrophoresis could not distinguish between 171 bp and 153 bp bands,only 160 bp fusion band were seen(figure 1). We selected 10 cases PCR products were sequenced randomly, the result is consistent with the electrophoresis(Fig. 2).2. The frequency of CC,CA and AA genotype in breast cancer group and normal control group was 72.7%,25.0%,2.3% and 52.9%,41.2%,5.9%,respectively. The frequency of C and A allele in breast cancer group and normal control group was 85.2%,14.8% and 73.5%,26.5%respectively.The risk of breast cancer in the individuals with rs4644 polymorphism was no significant difference.3. rs4644 C allele is more likely to occur in tumor T>2cm and ki-67<14% breast cancer population(P<0.05). There was no significant correlation with age, histological grade, lymph node metastasis, receptor status and p53 expression.4. Immunohistochemical staining with the PAR antibody revealed different cytoplasmic and nuclear PAR expression patterns, strong/intermediate nuclear reaction could be seen in the majority of breast carcinomas. In a small number of cancers both strong cytoplasmic and non/low nuclear expression was seen. This is similar to PARP-1expression pattern which we previous reported.5. The nuclear PAR expression in TNBC was low in 62.5 % and high in 37.5% of cases. Compared to that, the n PAR expression in non-TNBC was low in 28.2 % and high in 71.8 % of cases. Statistical analysis showed that n PAR expression was significantly higher in non-TNBC compared to TNBC(c2=8.404,P= 0.004).6. The cytoplasmic PAR expression in TNBC was positive in 50 % and negative in 50 % of cases. In contrast, c PAR in non-TNBC was positive in 20.5 % and negative in 79.5 % of cases. c PAR expression was significantly more often high in TNBC than in non-TNBC(c2=6.830,P= 0.009).7. We compares c PAR and n PAR expression in breast cancer, regardless of TNBC and non-TNBC origin. It shows that 46.5% of cases had high n PAR but negativec PAR expression and 23.9 % had positive c PAR but low n PAR expression. Statistical analysis showed that c PAR and n PAR expression was negatively correlated(r=-0.391,P= 0.001).8. Since we hypothesised that PAR would be dependent on the level of PARP-1 protein expression. We focused the analysis on the association between PAR and PARP-1expression which had been previously tested. It shows that 11.3% of cases had high n PAR but low PARP-1expression, 24.0% cases show high n PARP-1 but low n PAR expression.(P=0.054); 21% cases show positive c PAR but negative c PARP-1 expression, 12.7% cases show positive c PARP-1 but negative c PAR expression(P=0.448).There was no significant correlation between PAR and PARP-1expression regardless of cytoplasm or nuclear.9. The association between nuclear/cytoplasmic PAR expression and rs1136410 genotype and allele frequencies, respectively. There was no significant correlation between them.Conclusion1. There is no significant association between Galectin-3 gene 191 polymorphism rs4644 and susceptibility of breast cancer in Chinese female population, It is closely associated with the size of tumor and ki-67, suggesting that rs4644 play a biological role in the process of tumor growth and anti-cancer drug resistance.2. TNBC more often exhibits a high c PAR but low n PAR expression pattern,where as in non-TNBC, high n PAR expression is more common.c PAR and n PAR expression was negatively correlated. PAR levels and PARP-1 expression was not significantly correlated.PAR level and rs1136410 polymorphisms was not significantly correlated.