| Background and ObjectiveMiRNAs have good tissue specificity and specific expression pattern in different tumors, which have been confirmed in breast cancer, liver cancer, lung cancer, colorectal cancer, ovarian cancer and other malignant tumors. MiRNA expression in tumor tissue spectrum is related to tumorigenesis and prognosis, but the detection technology is complex, traumatic and can only obtained after the surgery, which is difficult to enter clinical application, In comparison, the easier to obtain and the easier detection of the peripheral blood serum, circulating miRNA is more convenient in clinical application. The discovery of circulting miRNA has aroused great interest of researchers. There are still many problems in research of serum miRNAs as tumor markers, such as the biological function of serum miRNAs has not been clear, the correlation of serum and tissue miRNA levels remains controversial, and so on. This study was to explore the correlation of miRNAs in the serum of patients with breast cancer and breast cancer tissues, and further clarify the feasibility and clinical value of serum miRNAs as a biological marker for breast cancer. To illustrate the above problems, this research will help to speed up the clinical application process of serum miRNAs as tumor biological marker for early diagnosis, judgment of biological behaviour and prognosis evaluation for breast cancer.MethodsMiRNA microarrays were used to analyze the miRNA expression profiles of breast tumors and the corresponding adjacent tissue. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to validate the results of the miRNA microarrays and to screen for miRNAs that show differential expression in breast cancer specimens and adjacent normal tissue. Real-time quantitative RT-PCR was applied to analyze the expression of target microRNAs in the serum of165untreated breast cancer patients and120healthy control subjects from March2011to October2011in Tianjin Cancer Hospital.Results1. MiRNA microarrays were used to analyze the expression of924miRNAs in breast cancer tissue and the adjacent normal tissue of8patients, and a total of23miRNAs showed differential expression. Of the23differentially expressed miRNAs, hsa-miR-155and hsa-miR-193b expression levels were up-regulated, and hsa-miR-145、hsa-miR-38、hsa-miR-132、hsa-miR-206、hsa-miR-199b-5p、 hsa-miR-100、hsa-miR-335、 hsa-miR-497、hsa-miR-99a、hsa-miR-10b、 hsa-miR-125b、hsa-let-7b、hsa-miR-10a、hsa-miR-126、hsa-miR-30a、hsa-miR-143、 hsa-miR-125a-5p、hsa-miR-19b、hsa-miR-26a、rno-miR-324-3p were down-regulated. miR-10b was up-regulated in breast tumors with lymph node metastasis but down-regulated in breast cancer tissue without lymph’node metastasis.2. MiRNA real-time fluorescence quantitative PCR was used to validate the screening results of the microarray analysis, and a total of10miRNAs showed differential expression. Of the10miRNAs that showed differential expression, hsa-miR-155and hsa-miR-193b were up-regulated, hsa-miR-100, hsa-miR-206, hsa-miR-381, hsa-miR-132, hsa-miR-199b-5p, hsa-miR-497, and hsa-miR-99a were down-regulated, and miR-10b was up-regulated in breast cancer tissue with lymph node metastasis and was down-regulated in breast cancer tissue without lymph node metastasis. Serum expression analysis was conducted using miR-10b, miR-100, miR-155, and miR-206because they were significantly differentially expressed in the microarray and the RT-PCR analyses (>2-fold and Ct value of RT-PCR was<30).3. Quantitative analysis was conducted on the expression of target miRNAs in the serum of165breast cancer patients and120healthy control subjects. The results showed that miRNAs were stably expressed in patient serum.4. Quantitative analysis was conducted on the expression of target miRNAs in the serum of165breast cancer patients and120healthy control subjects using U6as an internal reference. The results showed that miRNAs were stably expressed in patient serum and that U6was a stable and reliable internal reference. Compared to the normal control group, serum expression of miR-100and miR-206was significantly down-regulated in breast cancer patients, and this difference was statistically significant (P=0.012,0.010, respectively). miR-155expression was significantly up-regulated in breast cancer patients (P=0.001), and this difference was statistically significant. miR-10b expression was slightly down-regulated, and this difference was not statistically significant (P>0.05). Our study showed that the expression pattern of several miRNAs in the serum occurs in parallel with their expression in breast cancer tissue, suggesting that serum can be used as a sample source for breast cancer-related miRNA research, which could have higher clinical application value relative to tissue samples.5. The expression of miR-10b, miR-155, and miR-206correlated with some clinical and pathological profiles of breast cancer samples. miR-10b expression positively correlated with lymph node metastasis, as the expression level of miR-10b was lower in patients with no lymph node metastasis than in the normal control group (P=0.001). Likewise, the expression level of miR-10b was significantly higher in patients with lymph node metastasis than in the normal control group or breast cancer patients with no lymph node metastasis (P=0.001,0.001). The expression level of miR-206differed depending on the molecular subtype of the tumor (P=0.040). miR-206expression inversely correlated with the expression of ER and was significantly down-regulated in the ER-positive patients (P=0.035). miR-155expression positively correlated with the clinical stage of the tumor (P=0.001), Ki67expression (P=0.001), and p53mutation (P=0.027). miR-155was significantly up-regulated in patients with advanced stage cancer, high Ki67expression, and mutant p53status. We observed no significant correlation between the expression of miR-10b, miR-155and miR-206and any other clinical and pathological features of breast cancer. In addition, we observed no significant correlations between miRNA-100expression and any clinical and pathological profiles of the breast cancer samples (P>0.05).Conclusions1. MiRNA real-time fluorescence quantitative PCR was used to validate the screening results of the microarray analysis, and a total of10miRNAs showed differential expression, which initially establish miRNA expression profiling in breast cancer tissue.2. Our study showed that the expression pattern of target miRNAs in the serum occurs in parallel with their expression in breast cancer tissue, suggesting that serum can be used as a sample source for breast cancer-related miRNA research, which could have higher clinical application value relative to tissue samples. 3. This study also demonstrated that the expression levels of some miRNAs are associated with the clinical and pathological profiles of breast cancer patients. Thus, serum miR-10b and miR-206can be used for the early prediction of lymph node metastasis and the expression of ER in breast cancer patients, respectively, whereas miR-155can be used for the early judgment of the malignancy level of breast cancer cells, and for the prediction of the clinical stage and prognosis of breast cancer patients.4. Serum miRNA can become non-invasive and accurate new biomarkers for early diagnosis, predicting biological behavior and prognosis of breast cancer. |
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