| Breast cancer is the most common malignancy in women.The incidence rate of breast cancer ranks first among the female malignant tumors in Europe and the United States and other developed countries. In our country, breast cancer is also located in the female cancer incidence of the first place, and the incidence rate increases year by year. According to statistics released by the 2016 year’s report of National Cancer Registry, our country breast cancer incidence rate increased significantly in recent years, and which gradually tends to be younger and become threat to women’s physical and mental health, and to be life-threatening number one killer.There are 273 thousand new cases every year. In recent years with the continuous improvement of early diagnosis of breast cancer and health screenings, and individualized treatment design of breast cancer present has become the main treatment strategy, coupled with the comprehensive application of surgery, radiotherapy, chemotherapy, biological targeted therapy, hormonal therapy and other treatments, making the overall survival of breast cancer patients have been significantly improved, it has become one of the better prognosis of solid tumors. Cancer is a multi-factor, multi-step, multi-gene, multi-signaling pathways influence each other, the complex interaction of biological processes formed the joint action. Risk factors for breast cancer include genetic, lifestyle, hormone levels, mood etc. With the development of science and technology, such as molecular biology and genomics, many subjects have triggered a boom in the development of cancer from the perspective of molecular and gene.MicroRNAs (miRNAs) are a class of endogenous, short non-coding, single stranded RNA molecule, plays a key role in the post transcriptional regulatory factor in metazoans and plants gene expression. MiRNA can significantly affect cell proliferation, morphology, apoptosis and differentiation as well as in the occurrence and development of tumor. MiRNA regulates the biological behavior of tumor cells by regulating the mRNA of the target gene. MiRNA regulated gene expression has become a new hot spot in cancer research. At the present stage, tumor targeted therapy and individual therapy will be a breakthroughpoint, and have already obtained certain results. Therefore, to find new molecular targets for diagnosis and treatment of breast cancer and improve the prognosis of patients are of great significance. MiRNA-205 has been found for a long time, but its biological function has only just begun to be studied. Studies have shown that miRNA-205 expression is reduced in many tumors, miRNA-205 plays a role in tumor suppression and is involved in many physiological and pathological processes. The core of miRNA function research is the research of miRNA target gene, miRNA participates in the regulation of life activity through the regulation of target genes. Therefore, the key and difficult points in miRNA research work have been the discovery and clear miRNA control of target genes and the signal pathway involved.Angiogenesis is essential in the process of tumor growth, and it also plays a role in the stage of tumor invasion and metastasis. Angiomotin (AMOT) is a kind of angiostatin binding protein, is formed by the combination of a membrane associated protein and vascular myostatin, can advance cell invasion and migration, and the destruction of the connection between the endothelial cells. The expression of AMOT was significantly higher in invasive tumors than in non aggressive tumors, and in patients with metastatic breast cancer. However, the mechanism of AMOT is not clear.The expression of miRNA-205 in breast cancer was significantly decreased. These studies have shown a negative correlation between miRNA-205 and AMOT expression in breast cancer, so we choose miRNA-205 to analyze the role of AMOT in breast cancer. We hypothesized that miRNA-205 has a regulatory effect on the expression of AMOT. But there is no report on the regulatory effect of miRNA-205 on AMOT, and the mechanism of miRNA-205 in human breast cancer cell line AMOT. This study was divided into three parts content, respectively, described the possible role of miRNA-205 and AMOT in the development of breast cancer and their molecular mechanisms, diagnosis and treatment of breast cancer to bring new ideas and enhances our understanding of AMOT post transcriptional regulation.Part Ⅰ Expression of miRNA-205 and AMOT in breast cancerMethods(1) miRNA-205 mRNA expression was detected by qRT-PCR technology in 20 cases of breast cancer tissue and 20 paracancerous normal adjacent tissue specimens.(2) The expression of AMOT mRNA was detected by real-time quantitative PCR technology in 20 cases of breast cancer tissue and 20 paracancerous normal adjacent tissue specimens; AMOT protein was detected by Western Blot in 20 cases of breast cancer tissue and 20 paracancerous normal adjacent tissue specimens.(3) the expression of miRNA-205 in SKBR-3, MDA-MB-435S and MCF-7 was detected by Real time PCR assay.(4) Statistical analysis:Statistical analysis was performed by SPSS 18.0 software, all experiments were repeated three times, all data were expressed as x±s. Between the two groups was statistically significant difference using ANOVA or unpaired independent sample t-test, P value less than 0.05 was considered as statistical significance.Results(1) miRNA-205 mRNA expression was detected by qRT-PCR in 20 cases of breast cancer tissue and 20 paracancerous normal adjacent tissue specimens. The results showed that the expression of breast cancer tissue (0.130±0.003) was significantly lower than the paracanerous adjacent normal tissues (1.004±0.048), and the difference was statistically significant (P<0.05).(2) AMOT mRNA expression was detected by real-time quantitative PCR in tumor tissue (4.027±0.158) compared to the paracancerous normal adjacent tissue specimens (1.228±0.050) was significantly increased and the difference was statistically significant (P<0.0001). Western Blot at the protein level also shows the high expression of AMOT in breast cancer tissues.(3) Real time PCR to detect the expression levels of miRNA-205 in different breast cancer cell lines. The expression level of miRNA-205 in SKBR-3 cell line was 1, The expression of MDA-MB-435S are detected by RT-PCR method was 2.49± 0.21, MCF-7 expression was 0.25±0.01. (P<0.05) The expression of miR-205 in MCF-7 is the lowest, become subsequent experiments cell lines by screening.Part Ⅱ Effects of miRNA-205 on proliferation, apoptosis, invasion and migration of breast cancer cellsMethods(1) Transfected with miRNA-205 mimics,72h after transfection, to detect the expression of miRNA-205 by RT-PCR in breast cancer cell.(2) Transfected with miRNA-205 mimics, to detecte the growth of breast cancer cell by CCK-8 assay at 0,12,24,48,96h,and the draw growth curve.(3) Transfected with miRNA-205 mimics.to detect the apoptosis of breast cancer MCF-7 cell by flow cytometry.(4) Transfected with miRNA-205 mimics,to detect the migration of breast cancer cell MCF-7 cell by Transwell assay.(5) Transfected with miRNA-205 mimics,to detect the expression of AMOT in breast cancer cell MCF-7 cell by RT-PCR and Western Blot, to assure the influence of the overexpression of miRNA-205 over the expression of AMOT.(6) Statistical analysis:Statistical analysis was performed by GraphPad Prism6 software, all experiments were repeated three times, all data were expressed as x±s. Using ANOVA or unpaired independent sample t-test, P value less than 0.05 was considered as statistical significance.Results(1) In order to understand the expression level of miRNA-205 after transfecting miRNA-205 mimics. we made NC(negative control) as the control group. miRNA-205 was detected by RT-PCR after transfection 72h,U6 as refernce. The results showed that the transfection of miRNA-205 could significantly increase the expression of miRNA-205 (the relative expression of miRNA-205 was 82 times than that of NC group) (P<0.05).(2) we detected the growth of breast cancer cells before and after MCF-7 cell treated by miRNA-205mimics by CCK-8 method. The experimental results showed that with the prolongation of the observation time with 0,12,24,48,96h, the cell growth of MCF-7 cell decreased significantly, (P<0.05) and there was a time dependent effect.(3) After treatment with mimic/NC miRNA-205 for 24 hours, the Annexin V-FITC and PI staining were used to detect the effect of miR-205 on the apoptosis of MCF-7 cell line. P> 0.05, there was no influnce on the apoptosis of MCF-7 cell line.(4) Transwell test to detect the migration ability of tumor cells. By grouping cells, mimic group and (NC) with Transwell experiment to observe the ability of cell migration respectively. The experimental results show that miRNA-205 over expression group (175.1±9.16). NC group for (238.5±11.20) could significantly inhibit the migration ability of breast cancer cells. P<0.05(5) After treated by miRNA-205 mimics,the expression of AMOT in mRNA level was not obvious different P> 0.05.but at the protein level,the expression of AMOT became lower that of NC. P<0.05Part Ⅲ Target gene of miRNA-205 and its molecular mechanism of breast cancerMethods(1) In order to determine the miRNA-205 in the breast cancer can regulate the AMOT expression, we apply the prediction software targetscan (www.targetscan.org)、miRDB (http://mirdb. ORG/miRDB) and MICRORNA (www.microrna.org) on target miRNA-205 forecast database by the computer analysis.(2) Construction of plasmids and verification, gene mutation of AMOT 3 ’UTR, experimental grouping:NC+AMOT-3’UTR-WT; miR-205 mimics+AMOT-3’UTR-WT; NC+AMOT-3’UTR-MUT; miR-205 mimics+AMOT-3’UTR-MUT, luciferase assay to verify whether AMOT is the target gene of miRNA-205.(3) SiRNA or NC were transfected into breast cancer cell line MCF-7 cells, the proliferation, migration and apoptosis of breast cancer cells were detected after inerferring AMOT.(4) Transfection plasmid with overexpression of AMOT and detect the expression of AMOT by Western Blot. experimental grouping:miRNA-205+AMOT-3’UTR-WT, miRNA-205+Vector.(5) AMOT was overexpression in miRNA205-overexpression MCF-7 cell and detected by CCK-8 for proliferation,and then detected by flow cytometric analysis for apoptosis,and detected by Transwell assay for migration. To further test the effect of AMOT overexpression on the biological behavior of MCF-7 cells and to understand the effects on the overexpression of miRNA-205.Results(1) The use of bioinformatics analysis, according to the complementary pairing principle, we found that there was a sequence of fragments that may be combined with miRNA-205 in AMOT gene 3’-UTR 2938-2945.(2) By electrophoresis of PCR products, double enzyme digestion electrophoresis and plasmid DNA sequencing and Blast alignment to prove successful plasmid construction. Luciferase assay system to verify that AMOT is the target gene of miRNA-205.The relative activity values of each group were:NC+AMOT-3’UTR-WT (4.79±0.24); miR-205mimics+AMOT-3’UTR-WT (2.34±0.23); NC +AMOT-3’UTR-MUT (4.8±0.16); miR-205 mimics+AMOT -3’UTR-MUT (4.52±0.25), The results showed that the action sites of miRNA-205 and AMOT-3’ UTR-WT could be combined (P< 0.05).(3) The protein expression of AMOT was significantly decreased after AMOT was knocked down by siRNA which was detected by Western blot. The growth of breast cancer cells was detected by CCK-8 method before and after siRNA treatment.The experimental results showed that with the prolongation of the observation time, the cell growth decreased significantly of after cell were treated by siRNA with 0,12,24,48,96h, and there was a time dependent effect.That AMOT was knocked down by siRNA had no effect on apoptosis by flow cytometry. The migration of breast cancer cell line MCF-7 cells was detected by Transwell assay. The results indicated that the migration ability of MCF-7 cells was significantly decreased, and the NC group was (238.5±11.20); AMOT-siRNA group was (124.4±10.4), P<0.05.(4) AMOT overexpression experiments further confirmed that the AMOT is miRNA-205 target genes. After cotransfection miRNA-205 mimics and AMOT overexpression plasmid or empty plasmid,we detected the AMOT expression at the protein level by Western Blot.The result indicated that compared with the control group (empty plasmid) the AMOT expression was significantly enhanced and that AMOT overexpression can ameliorates the inhibitory effect of miRNA-205 overexpression on the expression of AMOT. P< 0.05.(5) Cotransfection of miRNA-205 mimics and AMOT overexpression plasmid could partly reverse the inhibitory effect of miRNA-205 on the proliferation and migration of breast cancer cell line MCF-7 cells. Results showed that MCF-7 cell growth inhibition is relieved, and the proliferation of cells was far more than the control group, with the time extended.the difference was more and more significant. P<0.05.The difference reached the most significant at 72h, but it was in plateau phase between the 72-96h. Apoptosis was detected again by flow cytometry, and the results showed that there was no significant difference. P>0.05. The cell migration ability was detected by transwell assay, the empty plasmid group was (136.1±13.8), and the AMOT overexpression plasmid group was (219.1±11.48). The two groups were significantly different, P< 0.05. After cotransfection of AMOT overexpression plasmid and miRNA-205 mimics, the inhibition by the overexpression of miRNA-205 was relieved,the migration ability was enhanced. P< 0.05.Conclusion(1) MiRNA-205 expression is down regulated in breast cancer;The expression of AMOT in breast cancer is significantly increased;The expression of miRNA-205 and AMOT was negatively correlated.(2) MiRNA-205 overexpression could inhibit the proliferation and migration of breast cancer cells, but had no significant effect on the apoptosis of breast cancer cells;The expression of miRNA-205 had no significant effect on the expression of mRNA in the AMOT level, but the expression of the protein level was inhibited.(3) AMOT which expression in protein level was inhibited through AMOT 3’-UTR specific point is a direct target gene of miRNA-205.The overexpression of AMOT could partially reverse the inhibitory effect of miRNA-205 overexpression on the proliferation and invasion of breast cancer cells. |
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