Cell-free Nucleic Acids As Biomarkers In Breast Cancer Patients Diagnosis

Objective Plasma free nucleic acids have great potential as tumor marker for early diagnosis of cancer disease. PIK3CA gene encoded a subunit of phosphatidylinositol 3-kinase (PI3K) and played an important role in cell signal transduction, proliferation and differentiation. Mutant PIK3CA is highly expressed in breast cancer. In this study, HRM and CAST-PCR were used to detect plasma PIK3CA gene mutations. We explore the possibility of digital PCR to quantitative detection the low concentration mutations in plasma samples. To identify the differential plasma microRNA related to breast cancer and elucidate the role of the microRNAs involed in the pathogenesis of breast cancer.Method E542K, E545K, H1047R mutations in PIK3CA gene were detected by HRM and CAST-PCR methods in both paraffin-embedded tissue and plasma samples from breast cancer. PIK3CA might be regulated by miR-19b, miR-302a, miR-520a and miR-525 based on the bioinformatics software prediction. MCF-7 cells were transfected with candidate microRNA mimics and negative control using HiPerFect Transfection Reagent. Dual luciferase report assay was used to validate the microRNA target genes. We determined the mRNA and protein expression levels of target genes by qPCR and Western blot. Apoptosis and cell cycle were analysed by flow cytometry. Cell viability was measured by CCK-8 assay and cell invasiveness was measured by transwell methods.Results(1) HRM and CAST-PCR were successfully used to screen for three "hot mutation" in PIK3CA gene. It was found that exon 9 mutations of PIK3CA gene were positively correlated to the breast cancer metastasis.(2) HRM and DNA sequencing showed no differential in detect PIK3CA gene mutations (p>0.05) and was suitable for mutation screening. CAST PCR allowed efficient amplification of both FFPE and plasma samples, probes were highly specific and all assays had a sensibility inferior to 1%. Combine CAST-PCR and HRM to detect PIK3CA gene mutations in breast cancer patients can be more effective.(3) The low abundance PIK3CA mutations in plasma can be sensitive detection and accurate quantification by digital PCR.(4) miR-19b, miR-302a, miR-520a and miR-525 were predicted to be possible regulate PIK3CA by bioinformatics software. Expression level of miR19b in breast cancer tissues was significantly lower than that in adjacent tissues (p<0.001). miR-302a, miR-520a and miR-525 in the cancerous tissue and benign tissue adjacent tissues were significantly increased the expression of the lowest in benign tissue (p<0.001).(5) Plasma miR-19b in breast cancer patients was significantly decreased than in normal control group (p<0.05). Therefore miR-19b may be a potential diagnostic biomaker. Combine the four microRNAs to diagnosis breast cancer, the sensitivity and specificity of ROC curve were 87% and 79%, respectively.(6) The results of dual luciferase report assay showed that the relative luciferase activity decreased when pmirGLO-PIK3CA 3'-UTR was co-transfected with miR-19b mimics, but not with mimics negative control (p<0.05).(7) The mRNA and protein expression level of PIK3CA were signicantly decreased in MCF-7 cells after transfection with miR-19b mimics compared with mimics negative control.(8) miR-19b promoted MCF-7 G1 cell cycle arrest, inhibited G1/S transition and cell proliferation, reduced cell invasiveness.Conclusion CAST-PCR is a simple-operation, fast method for mutation detection in plasma free-DNA and can be used for large scale screening. The sensitive detection and accurate quantification of low abundance mutations in plasma by digital PCR would be useful for early detection from breast cancer. miR-19b was considered as tumor suppressor by targeting to 3'-UTR of PIK3CA in process of development and cell progression of breast cancer.The expression of miR-19b was related to cell proliferation and invasiveness.