| Breast cancer is not only the most common but also one of the major causes of cancer death in female malignant tumor. More than 90% of the death is attributed to the early invasion and distant metastasis. The tumors often metastasize to bone, lung, liver, brain, soft tissue etc.. The survival time for those patients who’s tumors metastasized to soft tissue and bone is 22 to 26 months, while for patients with lung and pleural metastasis is 10 to 12 months, and for patients with liver and brain metastasis is even shorter, which is only 4-6 months. Currently, the pathogenesis of breast cancer is not completely clear. Molecular biology study of the occurrence and development of breast cancer indicated that it is a complex long-term process which had multi genes, variation, multistage accumulation and interaction involved. Thus, targeted therapy for cancer-specific molecular structure, and appropriate pathways for blocking tumor cells proliferation, metastasis and apoptosis can effectively reduce the mortality, as well as improve the therapeutic effect dramaticlly.The miRNAs are a class of endogenous non coding single stranded RNA molecules, which are highly conserved and about 22-25 nucleotides in length (nt). They directly bind to the 3’untranslated region (3’-UTR) of the target gene to regulate the gene expression. Many studies show that abnormal miRNA expressions are related with tumorigenesis and development. About 50% of the known miRNAs are located in tumor-associated fragile sites (fragilesite) in the genome.More and more experiments also confirm the regulatory role of miRNAs on the biological behavior of cells, which is essential for tumorigenesis and development, such as cell proliferation, migration, invasion, apoptosis, ontogeny and so on. Furthermore, many studies even indicated that miRNAs may directly act as tumor suppressors or oncogenes in the process of tumorigenesis. For further understanding of the molecular mechanisms of miRNAs in disease, the researches had been expanded into the downstream target genes regulated by miRNAs. One miRNA molecule can regulate multiple target genes simultaneously, while one target gene can also be regulated by multiple miRNAs at the same time. Thus, to study the role of differentially expressed miRNA in tumorigenesis is essential for the research of molecular targeted therapy of tumor by the miRNA.The miR-32 locates in intron 14 of C9orf5 gene, and its biological study was focus in the replication in tumors and virus, regulation of androgen and so on. Lecellier et al. confirmed that miR-32, closely related with virus replication, could limit the accumulation of the type I reverse transcription primate foamy virus in human cells, restrain the replication of PFV-1. The over-expression of miR-32 can promote the growth of androgen-dependent LNCaP prostate cancer cell line. And miR-32 can also reduce apoptosis by directly regulating the expression of BTG2, while its up-regulation can inhibit the expression of apoptosis protein PIK3IP1 and regulate Ser/Thr protein kinase MAP2K. It acts as a common signal transduction element in different signal transduction pathways, plays an important role in the regulations of cell growth and differentiation, stress responses like inflammation and apoptosis, proliferations and angiogenesis of vascular endothelial cell. The miR-32 has tissue specificity in tumors, and it shows up-regulation in some tumors while abnormal low-expression in others. For example, it has significantly lower expression in lung cancer, and extremely higher expression in prostate cancer and renal cancer on the contrary, and the expression level is associated with the prognosis of patients. Its relationship with tumor metastasis, malignant evolution needs further study. It appears that the miR-32 may play different roles in different tumor types. The relationship between miR-32 and breast cancer has not been reported by now.FBXW7 is one of the F-box protein family members, which can specifically identify and degrade substrate proteins in ubiquitin-proteasome system (UPP), and important in cell cycle regulation, transcriptional regulation, apoptosis and cell signal transduction. Studies had shown that FBXW7 is a broad tumor suppressor gene. Its mutations and deletions can cause chromosome instability and accelerate the accumulation of cancer cell proliferation associated genes such as Myc, CyclinE and Aurora-A. Its abnormal expression is related with gastric cancer, breast cancer, cervical cancer, glioma and so on. The study of FBXW7 is very important for us to understand the mechanism of tumorigenesis, and to provide new targets for cancer diagnosis and treatment.Firstly, imported the miRNAs expression data of tumor and adjacent normal tissues from breast cancer patients (GSE45666:101 cases of breast cancer tissues,15 cases of normal tissues adjacent tumors) into Qlucore Omics Explorer software for analysis, and those data included 23cases of stage Ⅰ,44 of Ⅱ,29 of Ⅲ,3 of Ⅳ, and 2 of unknown stage, and 15 cases of normal tissues adjacent tumors(NA). The results showed that compared with breast cancer tissues, miR-32 had a lower expression in the 15 cases of normal tissues. Then in order to identificate the clinical breast cancer tissue samples and cells, we had collected 27 clinical cases of breast cancer tissues, paired normal adjacent tissues and MCF-10A, MCF-7, SK-BR-3, MDA-MB-231 cell lines. Total RNA was extracted from the cells and tumor tissues by Trizol reagent. The expression of miR-32 were detected by ABI 7500 real-time PCR Amplifyer through stem-loop RT-PCR, U6 was used as the reference gene. The results showed that in the detected 27 cases of fresh tissue specimens, the expression of miR-32 in breast cancer tissues was significantly higher than that of the adjacent normal tissues, which was in accord with the bioinformatics software. When the human breast cancer cell line MCF-7, MDA-MB-231, SK-BR-3 with normal mammary epithelial MCF-10A were compared, the miR-32 expression level in MCF-7 cells was the highest. We therefore selected MCF-7 as in vitro experimental cell line.Secondly, the studies of the function of the miR-32. MCF-7 breast cancer cell line was transfected with miR-32 mimic, miR-32 inhibitor and the NC control respectively, then the cell proliferation, apoptosis, cell period, and migration observed. The analysis conclusions were as follows:(1) The miR-32 mimic was successfully transfected into the MCF-7 cells and the miR-32 expression level was significantly increased. Meanwhile, in the MCF-7 cells transfected with miR-32 inhibitor, the expression level of miR-32 was significantly reduced compared with the control group.(2) MTT to detect cell proliferation. Compared with the negative control mimic NC, the independent samples T-test analysis of the cell proliferation transfected with miR-32 mimic were as follow:24h T=2.29, P=0.084; 48h T=5.098, P=0.007; 72h T=7.557, P=0.002; 96h T=11.226, P=0.000; and P<0.05 after 48h. Compared with the negative control inhibitor NC, the group transfected with miR-32 inhibitor inhibited the proliferation of MCF-7 cells, the independent samples T-test analysis were:24h T=-2.155, P=0.097; 48h T=-9.651, P=0.001; 72h T=-5.189, P=0.007; 96h T=-4.849, P=0.008; and P<0.05 after 48h. These results indicateded that up-regulation of miR-32 might promote the proliferation of MCF-7 cells, while down-regulation might cause inhibition.(3) Detection of cell apoptosis by flow cytometry. Compared with the negative control mimic NC, the independent samples T-test analysis of the apoptosis after transfected with miR-32 mimic, early apoptotic was T=-9.545, P=0.001; late apoptotic was T=-3.712, P=0.021; P<0.05. Compared with the negative control inhibitor NC, the independent samples T-test analysis of transfected miR-32 inhibitor of apoptosis, early apoptotic was T=9.307, P=0.001; late apoptotic was T=2.939, P= 0.042; P<0.05. The results indicated that the transfection of mimic would inhibit the apoptosis of MCF-7 by up-regulating the expression of miR-32; while the transfection of inhibitor would promote the apoptosis of MCF-7 by down-regulating the expression of miR-32.(4) Cell cycle analysis by flow cytometry. The miR-32 mimic/inhibitor were transfected into the breast cancer MCF-7 cells respectively, for the cells transfected with the miR-32 mimic, the cell ratio in G1 phase, G2 phase, S phase was 55.92±0.88,10.28±0.49,33.61±0.95; while mimic NC group was 53.49±0.28, 13.38±2.35,33.06±2.20. Another group transfected with miR-32 inhibitor was 62.04±0.64,10.4±2.47,27.51±1.86; while inhibitor NC group was 64.26±1.57, 4.65±1.03,31.09±2.56. According to the independent samples T-test, P>0.05, there was no significant difference in G1, G2, S phase.(5) The cycle was not affected by the expression of miR-32. Cell sratch tests showed that after 36h cultivated in serum-free culture, miR-32 mimic transfected group significantly reduced the scratches, but mimic NC group also had visible scratches. The scratches distance was measured then the independent samples T-test statistics analysis was performed. When compared the miR-32 mimic transfected group to mimic NC group, the result was Oh T=0.174, P=0.867; 36h T=-7.833, P =0.000, with statistical significance. miR-32 inhibitor transfected group compared to the control group with inhibitor NC, the result was Oh T=-0.354, P= 0.732; 36h T= 6.714, P=0.000, with statistical significance. It showed that up-regulation of miR-32 expression promoted migration of MCF-7 cells, while down-regulation inhibited.Furthermore, we carried on deeper studies on the target genes of miR-32 in breast cancer. After transfected with miR-32 mimic and the mimic NC group, the expression profile was screened by microarray. The results showed that in the mimic vs. NC,10 of the mRNAs who were down-regulated, participated in the ubiquitin protein degradation pathway, including E3 ubiquitin ligase FBXW7 who was down-regulated 3.1 times. It was clear that E3 ubiquitin ligase FBXW7 was involved in F-box from the analysis of KEGG pathway. We had predicted that E3 ubiquitin ligase related with miR-32 target genes were HECW1 and FBXW7, by multiple bioinformatics softwares, including TargetScan, miRanda, PicTar, miRBase, DIANA TarBase. Those results had intersections with the results of gene profile (microarray probes but not included HECW1 gene), indicated that E3 ubiquitin ligase HECW1 and FBXW7 might be target genes.This issue intended to take the miR-32, HECW1 and FBXW7 as a study object, to research the targeted regulation of miR-32 and its functions in breast cancer.1) Total RNAs of MCF-10A cells was used as a template, mRNA 3’UTR sequences of HECW1 and FBXW7 gene were amplified, the PCR products were then recombinated with psiCHECKTM-2 vector into the plasmid psiCHECK TM-2-HECWl-3’UTR, psiCHECK MT-2-FBXW7-3’UTR.2) The recombinant plasmid psiCHECKMT-2-HECWl-3’UTR, psiCHECK TM-2-FBXW7-3’UTR were used as templates for subcloning. SOE method were used to amplify the mutated sequences of HECW1-3’UTR miR-32 seed sequence binding sites, and to amplify the mutated sequences of FBXW7-3’UTR and recombinanted it to plasmid psiCHECKTM-2-HECW1-3’UTR-Mutant, psiCHECK TM-2-FBXW7-3’UTR-Mutant.3) The target gene of miR-32 was confirmed by dual luciferase reporter assay system. The miR-32 mimic group, mimic NC group, miR-32 mimic mutant group and mimic NC mutant group were established by transfecting the 293T cells respectively, the fluorescence were detected after 48h. The results were analyzed with One Way ANOVA analysis by statistical software SPSS 13.0.4) Statistical analysis showed that there were no significant differences (P>0.05) between miR-32 mimic+HECW1 and three control groups, which mean that miR-32 mimic hadn’t reduced the fluorescence value. And there was no significant difference between four control groups (P>0.05), indicated that HECW1 is not included in the target genes of miR-32; there were significant differences (P<0.05) between miR-32 mimic+FBXW7 and three control groups, which means that miR-32 mimic had reduced fluorescence value, and there was no significant difference among the other three control groups (P>0.05), indicated that FBXW7 is included in the target genes of miR-32. Western-blot was used to detect the expression of FBXW7 after miR-32 mimic transfected into MCF-7 cells. The FBXW7 protein level significantly reduced after the over-expression of miR-32 in breast cancer MCF-7 cells, which confirmed that miR-32 had a negative regulation on FBXW7.After FBXW7 had identified as target genes of miR-32, we moved forward for study the relationship between miR-32 and FBXW7. We detected the expression level of FBXW7 mRNA by real-time quantitative PCR in clinical tissue samples of breast cancer, and found that in 27 detected paired clinical samples,18 patients were with low expression level of FBXW7, compared with the matched adjacent normal tissue samples, which was negative correlation with the increased expression of miR-32 in these breast cancer tissue samples. Using siRNA method to inhibit the FBXW7 expression in MCF-7 cells, and detect by real-time quantitative PCR, the result showed FBXW7 expression significantly decreased and further experiments proved that FBXW7 down-expression could have impact on the cellular functions, such as cell viability, apoptosis, the situation of migration formation. The summary of the results was as following:we successfully inhibited the expression of FBXW7 in MCF-7 cells by siRNA method. Through MTT assay and independent sample t test, we found that after 48h that MCF-7 cells were transfected with FBXW7 siRNA, T=4.298, P=0.013, had statistically significant when compared with siRNA NC control group, indicating that FBXW7 down-regulation promote MCF-7 cells proliferation. Apoptosis experiments showed that after the transfection of FBXW7 siRNA, early apoptotic T=-13.253, P=0.000; late apoptotic T=-5.631, P-0.028; P <0.05, FBXW7 down-regulation inhibited MCF-7 cells apoptosis. Cell scratch tests showed that compared with the negative control group siRNA NC, Oh T= 0.25, P= 0.809; 36h T=-6.393, P=0.000, FBXW7 down-regulation will promote the migration of MCF-7 cells.In summary, this study had determined the expression of miR-32 in breast cancer and adjacent normal breast tissues by bioinformatic analysis, and confirmed the miR-32 expression level in clinical cases and in vitro cell lines. More over, the target genes of miR-32 were preliminary confirmed by expression profiling microarray and bioinformatics to prediction, and Dual-Luciferase Reporter Assay System. The study on the expression or over-expression in breast cancer MCF-7 cells, indicated the function of miR-32 in breast cancer, and proved the E3 ubiquitin ligase FBXW7 as one of miR-32 target genes. The down-regulation of FBXW7 in MCF-7 cells verified the biological experiments function in the reverse, which provided a potential target for the treatment of breast cancer. The results also showed a high expression of miR-32 in breast cancer that could accelerate tumors. Our study suggested that the high-expression of miR-32 was related with the mechanism of breast cancer, but it still needed further study. |
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