MicroRNA-106b/141/200c/205 And Its Targeted KLF12:Regulators Involved In Pathogenesis Of Basal-like Breast Carcinoma

BACKGROUND AND OBJECTIVE:MiRNAs are endogenous small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. They are thought that over one third of human genes appear to be conserved miRNA targets by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. MiRNAs have recently been identified as an important role in cellular processes including self-renewal, differentiation, growth and death, all of which are deregulated in carcinogenesis. More and more emerging evidence shows that miRNAs may be correlation to a large proportion of breast cancer heterogeneity. Because each miRNA can target up to one third of human genes, and mRNAs can have multiple miRNA target sites, so we think that much more miRNA regulators of these genes remain incompletely understood.Breast cancer presents as a heterogeneous disease, not only for the clinic and histology, but also in genetic expression profile. Recently, microarray-based gene expression profiling of breast cancer has been demonstrated that breast cancers can be classified into five main groups:basal-like, luminal A, luminal B, HER-2 over-expressing and normal breast-like breast carcinomas according to their profiles. The basal-like and HER2 groups are reported to have a more aggressive clinical behaviour when compared with carcinomas with luminal and normal breast-like phenotypes.Even though, recent studies shows some emerging miRNAs expression deregulation, however, to a large extent, the details of miRNAs expression deregulation and involved biological process including its target gene is still remained incompletely understood. Taking into account the potential role of miRNAs, more research is still required before final goal of completely understood its role can be achieved.MATERIALS AND METHODS:We obtained the breast cancer patient’s samples and the normal control from the School of Medicine Nanjing University/Nanjing Jinling Hospital under the procedures approved by the Ethnic Committee for Use of Human Samples of the Nanjing Jinling Hospital from Jan,2009 to Jan,2010. The fresh breast cancer tissues and the same patient’s normal control breast tissues were all collected in one hour after operation, and then stored at-80℃ refrigerator immediately in order to prevented RNA degradation.Identification of the basal-like breast carcinoma by using of immunohistochemistry (IHC) method and fluorescence in situ hybridization (FISH) assay. A panel of antibody was prepared for using in the study following Nielsen et al proposal in order to identify the basal-like breast carcinoma. IHC was done by using of EnVision methods. Tumor immunoreaction was scored "-"= negative, "+"= weak positive (0-25%), "++"= moderate (25-50%), and "+++"= strong (>50%) positive in combination with the percent of cells (per 100 cells) showing positive staining. FISH assay was also performed for detection of HER-2 gene amplification. Specimen with a HER2/CEH-17 ratio of> 2.2 was considered as HER2 gene amplification. Results at or near the cut-off (1.8-2.2) should be interpreted with caution. If the ratio was borderlines (1.8-2.2), counted an additional 30 nuclei and recalculated the ratio for the 60 nuclei. Specimens with a HER2/CEN-17 ratio of<1.8 was considered as none HER2 gene amplification.Total RNA from the basal-like breast carcinoma tissues (n=3) and the normal control (n=3) was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Purified RNA was labeled with a miRCURY Hy3/Hy5 labeling kit (Exiqon, Vedbaek, Denmark). The Hy3 TM-labeled samples and Hy5 TM-labeled reference pool RNA samples were then mixed pair-wise and hybridized to the miRCURY LNA array version 11.0 (Exiqon).Differentially expressed miRNAs of miR-106b, miR-141, miR-200c, miR-129-5p, miR-205, and miR-451 were selected at random for verification. These mature miRNAs were amplificated with SYBR Premix Ex TaqTM II Kit (TaKaRa) on a iQTM 5 real time PCR detection system (BIO-RAD) in order to assessed its expression by using of poly (A) miRNA-based real-time PCR approach.GO analysis was applied in order to organize genes into hierarchical categories and uncover the miR-Gene Regulatory Network on the basis of biological process and molecular function. In detail, the one-side Fisher’s exact test and x2 test were used to classify the GO category, and the false discovery rate (FDR). Afterwards, the biological process of positive/negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process related network of miRNA-mRNA interaction, representing the critical miRNAs and their targets, was established according to the miRNA degree.Western blot was used to study the KLF12 protein quantitative determination. Meanwhile, IHC was also used to detect the KLF12 protein localization in cell.Statistical analysis was done with the Student’s t-test for comparison of two groups, and ANOVA for multiple comparisons. A P-value of<0.05 was considered as statistically differences, a P-value of<0.01 was considered as significant statistically differences.RESULTS:18 samples were identified as basal-like breast carcinoma by using of IHC assay and prepared for LNA-based miRNA microarray analysis and QRT-PCR of miRNA verification. The up-regulated and down-regulated miRNAs in the basal-like breast carcinoma were 11 miRNAs and 18 miRNAs, respectively. The differential expression about up-regulation of miR-106b, miR-141, miR-200c, and down-regulation of miR-129-5p, miR-205, miR-451 was validated. The most significant differences GOs containing the positive/negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process-related targeted genes and miRNA-gene networks characterized by KLF12, which was targeted by the miR-106b/141/200c/205, uncovered the critical role of them in the positive/negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process. KLF12 protein expression level is obviously increased and diffused nucleus positive expression in the basal-like breast carcinoma.CONCLUSION:The expression deregulation about up-regulated miR-106b/141/200c and down-regulated miR-129-5p/205/451 is a frequent event in the basal-like breast carcinoma. Alteration of miR-106b/141/200c/205 may be essential for the biological process of "positive/negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process" by targeting KLF12. MiR-106b/141/200c and its targeted gene KLF12 may be a potential proto-oncogene in basal-like breast carcinoma. However, miR-205 may be a potential tumor suppressor gene in basal-like breast carcinoma. These miRNAs and its targeted gene KLF12, therefore, may be play an important role in the pathogenesis of the basal-like breast carcinoma through promote the proliferation and differentiation and are potential targets for intervention and biomarkers for diagnosis. Next works are required for characterization of these miRNAs and KLF12 as prognostic and/or diagnostic biomarkers in basal-like breast carcinoma by performing a number of specimens test as well as their in vivo role in the basal-like breast carcinoma pathogenesis by creating transgenic and/or knockout mice.