DataSheet1_A circulating miR-19b-based model in diagnosis of human breast cancer.pdf

Abstract Objective: Breast cancer (BC) is becoming the leading cause of cancer-related death in women all over the word. Identification of diagnostic biomarkers for early detection of BC is one of the most effective ways to reduce the mortality.Methods: Plasma samples from BC patients (n = 120) and normal controls (n = 50) were collected to determine the differentially expressed circulating miRNAs in BC patients. Binary logistic regression was applied to develop miRNA diagnostic models. Receiver operating characteristic (ROC) curves were applied to calculate the area under the curve (AUC). MMTV-PYMT mammary tumor mice were used to validate the expression change of those circulating miRNAs. Plasma samples from patients with other tumor types were collected to determine the specificity of the model in diagnosis of BC.Results: In the screening phase, 5 circulating miRNAs (miR-16, miR-17, miR-19b, miR-27a, and miR-106a) were identified as the most significantly upregulated miRNAs in plasma of BC patients. In consistence, the 5 miRNAs showed upregulation in the circulation of additional 80 BC patients in a tumor stage-dependent manner. Application of a tumor-burden mice model further confirmed upregulation of the 5 miRNAs in circulation. Based on these data, five models with diagnostic potential of BC were developed. Among the 5 miRNAs, miR-19b ranked at the top position with the highest specificity and the biggest contribution. In combination with miR-16 and miR-106a, a miR-19b-based 3-circulating miRNA model was selected as the best for further validation. Taken the samples together, the model showed 92% of sensitivity and 90% of specificity in diagnosis of BC. In addition, three other tumor types including prostate cancer, thyroid cancer and colorectal cancer further verified the specificity of the BC diagnostic model. Conclusion: The current study developed a miR-19b-based 3-miRNA model holding potential for diagnosis of BC using blood samples.</p

Table1_A circulating miR-19b-based model in diagnosis of human breast cancer.XLSX

Abstract Objective: Breast cancer (BC) is becoming the leading cause of cancer-related death in women all over the word. Identification of diagnostic biomarkers for early detection of BC is one of the most effective ways to reduce the mortality.Methods: Plasma samples from BC patients (n = 120) and normal controls (n = 50) were collected to determine the differentially expressed circulating miRNAs in BC patients. Binary logistic regression was applied to develop miRNA diagnostic models. Receiver operating characteristic (ROC) curves were applied to calculate the area under the curve (AUC). MMTV-PYMT mammary tumor mice were used to validate the expression change of those circulating miRNAs. Plasma samples from patients with other tumor types were collected to determine the specificity of the model in diagnosis of BC.Results: In the screening phase, 5 circulating miRNAs (miR-16, miR-17, miR-19b, miR-27a, and miR-106a) were identified as the most significantly upregulated miRNAs in plasma of BC patients. In consistence, the 5 miRNAs showed upregulation in the circulation of additional 80 BC patients in a tumor stage-dependent manner. Application of a tumor-burden mice model further confirmed upregulation of the 5 miRNAs in circulation. Based on these data, five models with diagnostic potential of BC were developed. Among the 5 miRNAs, miR-19b ranked at the top position with the highest specificity and the biggest contribution. In combination with miR-16 and miR-106a, a miR-19b-based 3-circulating miRNA model was selected as the best for further validation. Taken the samples together, the model showed 92% of sensitivity and 90% of specificity in diagnosis of BC. In addition, three other tumor types including prostate cancer, thyroid cancer and colorectal cancer further verified the specificity of the BC diagnostic model. Conclusion: The current study developed a miR-19b-based 3-miRNA model holding potential for diagnosis of BC using blood samples.</p

(A) Western blot analysis of the , , , and expression in miR-17/20–transduced cells

served as loading control. (B) MCF-7 cells were transfected with cyclin D1 siRNA and control siRNA. Western blotting demonstrated the efficient knockdown of cyclin D1 after 72 h of siRNA treatment. (C) Cell cycle analysis indicated the increased population of cells at G/G phase and decreased S and G/M phase cells in miR-17/20–transduced MCF-7 cells under a cyclin D1 background. This difference was abolished by knockdown of cyclin D1 in cells. The analysis was performed in triplicates (data are equal to mean ± SEM). (D) Northern blot demonstrated the increased expression of miR-17/20 in the miR-17/20–transduced NAFA cells. tRNA served as a loading control. (E) The MTT assay showed the inhibited cell proliferation of NAFA cells by miR-17/20 transduction. The assay was performed in three independent experiments, and the data are presented as the mean ± SEM.<p><b>Copyright information:</b></p><p>Taken from "A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation"</p><p></p><p>The Journal of Cell Biology 2008;182(3):509-517.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500136.</p><p></p

(A) Quantitative real-time RT-PCR assay did not show significant difference in mRNA level of between miR-17/20–transduced cells and control cells

ABI1 was a positive control. 18S served as an internal control for normalization. This experiment was repeated three times in triplicate. Data are mean ± SEM. (B) BLASTN analysis of human and mouse mRNAs identified a miR-17-5p– and miR-20a–binding site at the conserved 3′ UTR region. (C) Sequence alignment of the miR-17-5p and miR-20a base-paring site in the 3′ UTR of mRNAs. The region complementary to the 2–10 nt of miR-17/20 is highly conserved among human, mouse, rat, and dog. The “seed” sequence of miR-17/20 that is complementary to cyclin D1 is shown in italics and boxed. The mutant sequence is identical to the wild-type sequence except the mutated nucleotides are shown in red. (D) Luciferase reporter assay constructs. 3′ UTR FL, the full-length 3′ UTR of inserted to the downstream of the luciferase coding region in the pGL3 vector; 3′ UTR-1, a fragment of 3′ UTR containing the miR-17/20–binding sequence; 3′ UTR-1-mu, the mutated construct identical to 3′UTR-1 but point mutated in the miR-17/20 binding site; 3′ UTR-2, another fragment of 3′ UTR without the miR-17/20–binding sequence. (E) Luciferase reporter assay showed the decreased luciferase activity in miR-17/20 overexpressed cells for both 3′ UTR FL and 3′ UTR-1 constructs, but not for the PGL-3 empty vector, 3′UTR-1-mu and 3′UTR-2 constructs. The luciferase activity was normalized to β-galactosidase. Data are derived from three independent experiments. Values are presented as the mean ± SEM ( = 3). *, P < 0.01<p><b>Copyright information:</b></p><p>Taken from "A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation"</p><p></p><p>The Journal of Cell Biology 2008;182(3):509-517.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2500136.</p><p></p

miR-10a does not influence hCMPC differentiation toward cardiomyocytes.

<p>A. Representative image of differentiating hCMPCs with manipulated expression of miR-10a or mock. Cells were stained with DAPI, α-actinin and Trop I (×200). B. Data collected from A. *P<0.05, n = 6. C. Relative expression of cardiomyocyte markers in differentiating hCMPCs transfected with miR-10a mimics. D. The same markers were tested in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 6.</p

miR-10a reduces proliferation of hCMPCs.

<p>A. miR-10a mimics decrease the BrdU incorporation rate of hCMPCs, whereas a miR-10a inhibitor does not significantly affect the process. *P<0.05, n = 6. B. miR-10a inhibits EdU incorporation of hCMPCs. Representative image of hCMPCs transfected with mock, miR-10a mimics or inhibitor stained with DAPI (blue) and EdU (red) (×200). *P<0.05, n = 5. C. Representative flow cytometry results of hCMPC manipulated with mock, miR-10a mimics or miR-10a inhibitor. hCMPCs overexpressing miR-10a show G1/S blocking, and the inhibition of miR-10a promotes G1/S transition compare with mock. D. Data collected from C. *P<0.05, n = 3. E. Relative expression of cell cycle regulatory genes in hCMPCs transfected with miR-10a mimics. F. The same set of genes was measured in hCMPCs with inhibited miR-10a. The expression level of GAPDH was used as the control. *P<0.05, n = 5.</p

miR-10a binds to GATA6 and suppresses GATA6 expression.

<p>A. Pmir- glo dual luciferase plasmid containing a wild type or mutant GATA6-3′UTR was cotransfected with miR-10a mimics or mock into 293 cells. The relative luciferase activity was shown. *P<0.05, n = 5. B. Western blot shows the expression of GATA6 in hCMPCs transfected with mock and miR-10a mimics. C. The same experiment with mock and inhibitor. Quantification of protein expression was normalized to GAPDH. *P<0.05, n = 3. D. Overexpression of GATA6 rescues the miR-10a inhibitory effect on hCMPC proliferation. Protein expression of GATA6 with different treatment was showed. BrdU absorbance of hCMPCs transfected with mock, miR-10a mimics and siRNA that downregulated GATA6 are shown. Similar to miR-10a, the downregulation GATA6 in hCMPCs reduces proliferation. miR-10a mimics and a plasmid that overexpresses GATA6 were cotransfected into hCMPCs, and a reduced BrdU absorbance was not observed. *P<0.05, n = 6.</p