Effect Of MiRNA-125B-1on Radiosensitivity And On Chemosensitivity Of SKBR-3Breast Cancer Cells

BackgroundBreast cancer, one of the most common malignant tumors in woman, is malignancy that happens at epithelial cells of mammary duct and accounts for about7%-10%of malignant cells in the whole body. There are about1.5million new cases of breast cancer around the world every year and570000cases of death. In western developed countries, North America and Europe for example, the morbidity rate is about4times higher than that in Asia, Africa and Latin America. Although China is one of the low incidences of breast cancer, yet the morbidity rate has been increasing over the past20years. Especially in economically developed areas, such as Beijing, Shanghai and Guangzhou, breast cancer has become a serious health hazard. Its incidence may be higher than that of cervical cancer and holds the first place. The generating process of breast cancer is a complex biologic process that involves polygenic mutations, which includes oncogene activation, anti-oncogene inactivation and the mutation of relevant regulatory genes. Then the cell genes lace the stability and the tumor occurs at last. And because lymphatic pipe network and reflow vein anastomosis are plentiful in female breast, breast cancer metastasis happened early to the lungs, bones, liver, brain, etc, making treatment become more difficult and the mortality rate increase greatly. The major breast cancer treatment is the traditional surgical, after which the local or systemic radiation therapy, chemotherapy and hormone therapy can be helpful. In recent years, with the rapid development of molecular biology, molecular immunology study, and cancerous tumors gene research theory, arly diagnosis and treatment in breast cancer become reality. The gene therapy research in breast cancer treatment including inhibit protocarcinogenic gene function, restore tumor-suppressor genes function, gene immune therapy, suicide gene therapy, many drug resistance gene therapy, antiangiogenesis gene therapy, formed apoptosis gene therapy, using soluble tumor virus, etc.In the C. elegans research, one of the exciting findings is identified a kind of the developmental regulation tiny RNA (microRNA, miRNA). In1993, miRNA was found in worm caenorhabditis elegans for the first time. It is now known that miRNA exists widely in eukaryotic cells and is one of the biggest gene families, accounting for about1%of the entire genomes. Each miRNA can adjust hundreds of the expression of genes, and multiple miRNAs may act on a target to cut a variety of protein expressions in the same way at the same time. miRNA in fine regulation of gene expression and biological growth process plays an important role. miRNA is one kind of long about22nt of the non-coding functions small RNA which exist in many biologicals. miRNA original transcription (Pri-miRNA), after a series of processing and modification in the nuclear and cytoplasmic, become a mature miRNA form. First of all, miRNA gene by the action of RNA polymerasell, transcription generate pri-miRNA; Then, under the action of Drosha cutting enzyme, pri-miRNA release60-70nt precursor (Pre-miRNA), the latter is not perfect hairpin structure; Finally, exportin5acts on Pre-miRNA into cytoplasm, which during DICER cutting becomes the mature form miRNA. Mature miRNA form not completely matching with target genes3’the translation section (3’UTR area), thus inhibiting target genes translation, or form close to complete matching, leading to target mRNA degradation. Recently people discover that in the human genome, there is about533miRNA gene seat, which is about1%of the protein-coding genes, but can control about one-third of the human genes, explaining miRNA is a regulation network. miRNA plays an important role in physiological and pathological process in many of the organisms, and influence cell proliferation and growth, apoptosis, and related with disease. More and more evidence show that many cancer occurrence and development have relation with miRNA abnormal expression, such as breast cancer, brain cancer, chronic myeloid leukemia, colon cancer, liver cancer and lung cancer. So some miRNAs may play an important role in cell proliferation and differentiation and apoptosis and other biological processes of evolution. Esquela-Kerscher, A report, the use of miRNAs gene therapy could be an effective method in inhibiting tumor cell proliferation. Now it has been confirmed that let-7has now been confirmed can inhibit RAS cancer gene, and miR-15and miR-16can inhibit the gene activity of BCL2. Most human miRNAs were located in mRNA coding area for protein or the non-coding introns area. Other miRNAs may be far from chromosome transcription area, locating in the mRNA non-coding explicit or in the mRNA3’UTRs non-coding areas, or together with other miRNAs (such as in19chromosome gathered54new discovery miRNAs).miRNA-125b-1is one of the miRNAs, and expresses lowly in most breast cancer cell lines, which play tumor-suppressor role presumably.In breast cancer, Scott, G K, found miRNA-125b-1can inhibit tumor cell growth by target to HER2and HER3in mRNA or protein level. Hofmann, M H, by the cell of MDA-MA-435found that, miRNA-125b-1can target to mRNA3’UTR area of the c-raf-1,cause the levels of C-Raf protein and downstream targets cell cycle protein in D1loss. Li, W, etc report, in the breast cancer cell lines, miRNA-125b-1expression can inhibit the ERK1/2phosphorylation, and reduce the transfer and aggressive of cells. Besides miRNA-125b-1target:ras related proteins Rab-13(RAB13), silk threonine protein kinase13(AURKC), protein tyrosine kinases receptor Tie-1precursor (TIE1), phosphoric acid creatine3kinase adjustment subunit5(PIK3R5) and so on, the results show that miR-125-b inhibiting cell proliferates by the protein kinase pathway. In other respects, Komagata S, etc, miR-125-b precursor in MCF-7cells can cause the content of vitamin D3hydroxyl enzyme (CYP24) down, presumably in cancer tissue CYP24high expression is relation to miR-125-b content decreases. Smirnov, D A research shows, ATM gene makes transcription factor CDX2content increase by adjusting, which role in miRNA-125b-1content down, and miRNA-125b-1targets to TNFSF4. ATM-CDX2-(miRNA-125b-1)-TNFSF4signaling pathways may be adjusting system of increasing risk of breast cancer. In addition, Iorio, M V, find the target cancer genes of miR-125b-1, including YES ETS1, TEL, AKT3, growth factor receptor FGFR2, silk crack grain activation signal transfer way branch:VTS58653, MAP3K10, MAPK14, etc.In prostate cancer, miRNA-125b-1in prostate cancer cells can be induced express increased by androgen, and the latter can inhibit Bakl (apoptosis induced factor) gene expression, finally stimulating the growth of cancer cells. miRNA-125b-1plays a cancer sexual function part in the process of the prostate cancer occurrence. And the content of miRNA-125b-1in the prostate cancer cells changes in different reports.In the nervous system, in the glioma lines, miRNA-125b-1can cut the cell cycle protein CDK6, CDC25A, plays the role of inhibiting cell proliferation, also can target to DGAT1and SGPL1, and for some apoptosis gene has negative adjustment, for example, can make the content of BMF (cell apoptosis related proteins Bcl-2repair factor) down and cause Bcl-2express increase and induce cells apoptosis. In addition, many scholars investigate the function of miRNAs in alzheimer’s disease and neurons process function, which include miRNA-125b-1participation. Such as Wu, L, etc in inducing rat P19embryonic cells into nerve cells process, finding miR-125a and miRNA-125b-1can cause Lin-28cancer gene expression inhibition by the two conservative reaction district of the Lin-28cancer gene3’UTR area.The blood system, miRNA-125b-1plays an important role in macrophages matured process, for example, Androulidaki, A etc, find LPS action to macrophages by LPS-(PI3K/AKT)-miRNAs-TNF to action, miRNA-125b-1also participating. Thus, we can also research this signal pathways in breast cancer cell lines. In addition, scholars discuss the role of miRNAs in the course of illness in children acute myeloid leukemia (AML), children acute lymphocytic leukemia, myelodysplastic syndrome (MDS) and acute myelogenous leukemia etc.There are miRNAs(including miRNA-125b-1) research in the head and neck squamous cell carcinoma, undifferentiated thyroid cancer, liver cancer, lung cancer, and bladder cancer and urinary tract epithelial cancer, ovarian cancer, cndometriosis, psoriasis, Down Syndrome etc.The research for miRNA-125b-1in breast cancer is little domestic. ShenShuRong etc, using Realtime RT-polymerase chain reaction (PCR) to test miRNA-125b-1expression in42cases infiltrating ductal carcinoma of breast cancer and the non-tumor tissue, found that the expression of miRNA-125b-1abnormal in infiltrating ductal carcinoma, which may be concerned with breast cancer malignant biological behaviors,such as invasion and metastasis.C-erbB-2protocarcinogenic gene, also known as HER2/neu gene, located in human chromosome17q21, code a active protein p185of tyrosine kinases (TPK) substances, known as her2/neu protein. Studies found that P185protein and the epidermal growth factor receptor (EGFR) has a high degree of homology, which may combine with unknown ligand, promoting cells divide, growth and transformation by increasing the protein kinase activity. c-erbB2/HER-2gene amplification and p185erbB2protein excessive expression appear in malignant tumor, including breast cancer, ovarian cancer, colon cancer, endometrial cancer, cancer of the stomach, prostate cancer and lung adenocarcinoma. As known in the body of most patients with breast cancer, HER2/neu has expression high. Therefore, taking HER-2/neu as target for cancer treatment has been widespread concerned. In HER-2/neu protein level, Genentech company developed herceptin according to the antibody erbB2has been approval in clinical application by American FDA, which have a very good treatment effect on the tumors expressed erbB2, the effectiveness of single herceptin for metastatic breast cancer about15%, and herceptin can induce SKBR3breast cancer cells erbB2high expressed stop at GO/G1phase, inhibiting SKBR3cell by about63%. Herceptin has been confirmed with growth resistance signal system, chemotaxis immune cells to attack the tumor cells and enhance the induction chemotherapy cytotoxic effects, and therefore, erbB2receptor has become a new antitumor target. In the HER-2/neu gene level, someone blocks gene by the RNA interference (RNA interference, RNAi) technology, someone by cancer gene that antisense deoxidizing nucleotide technology, can induce breast cancer cells SKBR-3apoptosis, reduced the cell vitality, make p185protein expression reduce But the research about miRNA-125b-1target to HER-2/neu cancer genes is very small Traditional resistance ErbB2monoclonal antibody herceptin combining specific with ErbB2receptor exocellular region, restrain ErbB2homologous dimerizate, achieving the purpose of blocking downstream signal. But this method is invalid for lack of extracellular ligand domain of receptors (P95ErbB2) and ErbB2heterologous dimers (ErbB2/ErbB3, ErB1/ErbB2, ErBI/ErbB3). But miRNA-125b-1can combine with mRNA3’UTR area specificity of c-erbB-2cancer gene, inhibiting ERBB2at the transcription and translation level.This study is to investigate the effect of miRNA-125b-1on gene expression of Her2and on radiosensitivity and chemosensitivity of SKBR-3cells(Her2positive).The new mechanism of miRNA-125b-1can block ErbB2receptor signals at the level of mRNA level, to suppress tumor cells growth. This provides a new approach to ErbB2positive breast cancer treatment for us. This study intends to synthetic miRNA-125b-1, using liposome guide miRNA-125b-1into SKBR-3breast cancer cells, inhibit or closed ErbB2gene expression, thus achieves the aim in suppressing the growth of tumors, value-added, differentiation and inducing apoptosis. At the same time, we detect miRNA-125b-1whether we can improve breast cancer the sensitivity of the radiotherapy. In addition, miRNA-125b-1can strengthen the effect of chemical function, applicating miRNA-125b-1towards the Her2, at the same time, such as application of taxol chemotherapy drugs, testing whether can improve breast cancer chemotherapy sensitivity in drugs paclitaxel etc. This kind method of cancer treatment combines the little interference RNA with the union of radiation or chemotherapy perfectly, has great clinical research value. We hope that the study can identify the ultimate effect of miRNA-125b-1on Her2positive breast cancer, which provide theoretic base for application of miRNA-125b-1to Her2positive breast cancer and reference for application of miRNA-125b-1to other solid tumors too.PurposeExplore the impact sensitivity of radiation and chemotherapy of miRNA-125b-1 on SKBR-3breast cancer cells, and the influence of target genes her2protein expression.MethodsSynthetic miRNA-125-b-1which was transfected into SKBR-3cells by the lipofectamineTM2000, and retrovirus-polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) and Western Blot methods were used to detect the change of HER-2mRNA and protein expression level; Flow cytometric testing the cell cycle; Method methyl thiazolyl tetrazolium (MTT) detect the inhibition rate of the cell proliferation; By different dose of X-ray irradiation, cell survival rate can be calculated by flat cloning experiments, using the click more mathematical models to curve cooperation figure, evaluating curvilinear parameter DO, Dq and N value, by which cells radiation sensitive changes can be compared. The effect of miRNA-125b-1-1on paclitaxel action on proliferation inhibitory rate of SKBR-3breast cancer cells was measured by MTT assay. The SPSS13.0statistical software were used for the statistical analysis. Measurement data show with χ±s, using Shapiro Wilk method for the normal distribution inspection, P>0.05to meet the normal distribution; Use Leven ’test for variance of all inspection, P>0.05to meet Homogenity of variances; In the variance not neat, choose approximate F inspection Welch with French poor analysis. To the cell cycle experimental results do rank and inspection after the inspection standards do adjust two more; Cell proliferation experimental results using Mauchly’s test of sepericity for spherical inspection, spherical test statistics P>0.05don’t refuse to spherical hypothesis, and the test of repetitive measure anova.Quantitative results of RT-PCR and Western blot between groups use the single factor analysis of variance, and the cell cycle and cell proliferation experimental results between groups use the of repetitive measure anova.ResultsIn the experiment group, both the mRNA and protein expression levels of HER-2gene in the transfected SKBR-3cells were reduced significantly(all P<0.05). Most of the transfected SKBR-3cells were blocked in G2M phase of cell cycle. The cell proliferation inhibitory rate was obviously higher than that in the negative control group (P<0.05). The values of DO, Dq and N were obtained through the cell survival curves. The values of transfected SKBR-3cells (D0=1.866, Dq=2.681, N=4.207) were significantly lower than those in the control group (D0=2.513, Dq=4.097, N=5.107; SER=1.347), which demonstrated that the radiosensitivity of these cells was enhanced. IC50of miRNA-125b-1-1treatment group of cells fell2.69times compared with untreated group (P<0.05), which showed that miR-125-b-1can increase the chemosensitivity of the paclitaxel on SKBR-3breast cancer cellsConclusionmiRNA-125b-1can markedly increase the radiosensitivity of SKBR-3breast cancer cells; miRNA-125b-1can sensibly increase the chemosensitivity of the paclitaxel on SKBR-3breast cancer cells, which inspires a new treatment for HER-2positive breast cancer.