The Correlation Between MiRNA-205Gene Rs3842530Polymorphism And Breast Cancer Pathological Classification

MiRNAs are endogenous small non-coding RNAs composed of19~26nucleotides, which mainly through their seed sequence recognize and bind to mRNA3’-untranslated region (3’-UTR) to downregulate target gene’s expression. There aremany evidences proved that deregulation of miRNAs correlated with carcinogenesisand development of breast cancer and genetic variants within certain miRNA codingregion attributed to this deregulation. For this part, through screening out breastcancer related miRNAs and effect analysis of single nucleotide polymorphism ontheir expression, this project will provide pre-clinical research foundation for miRNAcoding region variant as candidate biomarker for early diagnosis, disease progressionand prognostic prediction of breast cancer.Objective:To screen out and validate breast cancer related miRNAs and to analyze therelationship between miR-205coding region SNP and clinicopathological features ofbreast cancer.Methods:1. miRNA array was employed for screening out of differential expressedmiRNAs in12breast cell lines with different aggressiveness.2. miR-205expression detection with quantitative realtime PCR and in situhybridization in breast cell lines with different aggressiveness.3. miR-205expression determination with miRNA in situ hybridization on breastdisease tissue array.4.10cancer cell lines were included for SNP validation with miR-205codingregion amplification and sequencing.5. miR-205coding region were amplified and sequenced for tissue samples frombenign breast diseases and breast cancer. The correlation between rs3842530distribution in different breast diseases and clinicopathological features was analyzed.Results:1. miRNA array analysis showed that9miRNAs were differentially expressed in 12breast cell lines (p<0.01, Fold chang≥20or≤-20). Compared with low metastaticgroup cell lines(BT474,MCF7,MDA-MB-468,SK-BR-3,T47D,ZR-75-1,MCF10A,MCF12A), miR-100, miR-138and miR-146a were upregulated and miR-34a,miR-141, miR-200c, miR-203and miR-205were downregulated in high metastaticbreast cell lines(BT549,HS578T,MDA-MB-231,SUM159PT).2. Quantitative realtime PCR detection showed that miR-205expression washigher in immortal normal breast epithelial cell lines and low metastatic breast cancercell lines than high metastatic breast cancer cell lines. miRNA in situ hybridizationvalidated that miR-205expression were higher in BT549and MDA-MB-231thanMCF7and T47D cell lines.3. miRNA in situ hybridization on tissue array showed that, microRNA-205were positively expressed in33out of36(91.67%) normal tissues and benign tumorsand23out of36(63.89%) malignant breast cancer, respectively. microRNA-205expression negatively correlated with malignancy of breast diseases (p=0.011).4. miR-205coding region rs3842530SNP was extensively found in10cancercell lines(BT-549,MDA-MB-231,MCF7,T47D,95-D,A549,AGS,MGC-803,HEPG2,SMMC-7721). Cell line with homozygous13/13genotype has a lowest miR-205expression, while cell lines with heterozygous9/13or homozygous9/9genotypeshave a medium miR-205expression and heterozygous7/9genotype has a highest.5. miR-205coding region sequencing for195cases of benign breast disease and189cases of breast cancer indicated that homozygous9/9genotype of rs3842530polymorphism were more distributed in benign breast disease samples (61.03%) thanbreast cancer samples (56.08%), with no statistical difference (p=0.326).Homozygous9/9genotype were positively correlated with higher ER (p=0.014), PR(p=0.112) and ERBB2(p=0.391) expression in breast cancer samples.6.2new genetic variants of heterozygous C/T were discovered5bp and23bpdownstream of miR-205coding gene.Conclusions:1. miR-205was differentially expressed in breast cell lines. Compared with lowmetastatic group cell lines, miR-205was downregulated in high metastatic breast celllines;miR-205expression was lower in breast cancer samples than normal breast samples and benign breast disease samples.2. miR-205coding region rs3842530SNP was extensively found in cancer celllines. With the absence of increased repeating trinucleotides sequence of AGC, thereis a tendency for an increased miR-205expression.3. miR-205coding region rs3842530polymorphism associated with higher ERexpression in breast cancer samples.4.2new genetic variants were discovered5bp and23bp downstream of miR-205coding gene.