Role Of MiR-15b Targeted Regulation Of EZH2Expression In Breast Cancer Cell

BACKGROUD AND PURPOSEBreast cancer is a complex disease that is caused by abnormal growth and uncontrolled division of cells within the terminal duct and lobular of the breast. It mainly occurs in women and less commonly in men. The majority of breast cancers are sporadic in origin and an appreciable fraction is caused by inherited predisposition. Breast cancer, with approximately one million new patients in each year, is the most common malignancy among women. Breast cancer is included about10percent of all cancers and23percent of women cancers in developed countries. Over15%of healthy women have at least one first-degree relative with breast cancer and experimental data showed that the risk of breast cancer in women has been doubled in recent years.At present the diagnosis and comprehensive treatment of breast cancer has been improved, but there still exists some problems. The diagnosis of breast cancer is mainly dependent on imaging techniques, such as Molybdenum target X-ray, ultrasonography and MRI, but it can’t detect the proliferation and micro-diffusion in blood circulation. The diagnosis of breast cancer in early stage is mainly dependent on carcinoembryonic antigen (CEA), CA153and other glycoprotein biomarkers, which are susceptible to non-tumor factors, such as hyperpyrexia, circadian rhythm and so on, are difficult to detect in the early stage of multiple kinds of cancer. It is normal to find that the abnormal expression of CEA is seen in a variety of tumors, which reduce the specificity and sensitivity. In terms of treatment including complete resection, radiation therapy and chemotherapy, can improve the prognosis of patients. But drug resistance and metastasis are still outstanding problems in treatment. As the conventional treatment is ineffective caused by chemoresistance or diffusion and recurrence because of viable cancer cells, it is urgent to find some new effective diagnostic and therapeutic strategies.It has shown that EZH2(Enhancer of zeste homolog2), highly expressed in invasive breast carcinoma, is an important biomarker. The gene of EZH2maps to chromosome7q35-6and consists of20exons, encoding746amino acids residues, of which there is a cysteine-rich domain known as pre2SET domain that is essential for the histone methyltransferase activity. EZH2is preferentially methylates Lysine27on Histone H3(H3K27) to influence basic cellular function and maintenance the self-renewal of adult and embryonic stem (ES) cells. EZH2turns out to be an oncogene and acts as a globally dual function transcription regulator by gathering the methyltransferase activity silencing tumor suppressor genes, which are implicated in neoplastic development and the transactivation property-activating genes involved in the late-stage process of cancer. The transcriptional repressor EZH2is also involved in other types of cancer progression, such as breast, prostate, bladder, colon, lung, pancreas cancer, sarcoma and lymphomas. Overexpression of EZH2is often associated with development and prognosis tumor in its advanced-stage. Recent findings implicate that EZH2deregulation could be an important driver of tumour development and progression. It reveals that EZH2can be can induce RAF1-ERK-β-catenin signaling, which in turn enhances the survival and proliferation of breast tumour-initiating cells (BTICs) and that inactivation of EZH2may be therapeutically effective in many cancers. As high EZH2expression is significantly associated with triple-negative breast cancer and decreased survival, it may represent a potential therapeutic target for this aggressive disease. Therefore it is considered that EZH2play a critical role for the diagnosis and therapy of breast cancer, but the mechanism of abnormal gene expression remains unclear, which warrants further investigation.MicroRNAs (miRNA) represent a diverse class of endogenous noncoding RNAs18-25nucleotides in length which post-transcriptionally regulate gene expression. Mature miRNAs mediates gene silencing via translational repression or target degradation, when it incorporates into the RNA-induced silencing complex (RISC). The human genome may encode over1000miRNAs, separation and sequencing has identified more than700miRNAs. Evidence has shown that each miRNA regulates hundreds of target genes and52.5%of miRNAs are located in cancer-associated genomic regions. Therefore miRNAs should be critical regulators at present. Most miRNA-mediated regulation occurs at the posttranslational level, primarily through the3’untranslated region (UTR) of target mRNA, leading to translational repression and/or degradation. Therefore, a large amount of miRNAs are involved in tumorigenesis and function as oncogenes or tumor suppressor genes depending on their targets.A growing body of evidence suggests that miRNAs, spatially/temporally and tissue-specific are critical regulators of widespread cellular functions, such as differentiation, proliferation, apoptosis, hematopoiesis, neurogenesis and oncogenesis. With the develop understanding of miRNAs, researchers have pay more attention to miRNAs in the pathogenesis of a variety of human diseases, including breast cancer diseases. It has been identified that dysregulation of several miRNAs in breast cancer, such as miR-21, miR-34a and miR-101, which contribute to the progression of malignancy. A recent study has suggested miRNAs involved in the regulation of EZH2, for instance, miR-26a, miR-101, miR-138å'ŒmiR-214, there still exists other miRNAs related to influence transcriptional EZH2.In this study we screen and quantitative analyze the novel miRNAs targeted to EZH23’untranslated region (UTR) which is recombined and over-expressed in MCF-7breast cancer cell with combination of controls MCF-7recombined with mock-vehicle, on the understanding of miRNAs regulate gene expression at the posttranscriptional level by binding to3’UTR of target mRNAs.Methods1. The reconstruction MCF-7breast cancer cells, which was constructed with EZH23’UTR, were infected with positive control virus (CD511VB-1) using a range of MOIs (1,5,10) to determine the MOI required to abtain optimal expression for our particular application with the help of fluorescence microscope. Efficiency of transduction of reconstruction MCF-7with the positive control virus (CD511VB-1) can be monitored by copGFP expression.2. After the reconstruction MCF-7breast cancer cells were infected with Lenti-miR Virus Library together with polybrene, target cells were screened with the help of cytotoxic drugs. MiRNA precursors were amplified by polymerase chain reaction from genome extracted from the target cells. The amplified fragments were cloned to the pMD-19T vector, and sequenced to determine the miRNAs type.3. Total RNA from carcinomatous tissues and the adjacent tissues as well as MCF-7and HBL-100cell lines was isolated using Trizol reagent according to the manufacturer’s instruction. The expression of miRNAs in breast cells and tissues was determined by Hairpin-it miRNAs real-time PCR analysis, choose one of them that was differentially expressed among them to do the target validation experiments4. To verify whether miR-15b can regulate the expression of EZH2, gain and loss of function was used. Firstly the expression of miR-15b was detected among breast cancer cell lines; the cell with higher expression of EZH2was transfected with miR-15b inhibitor, while the lower one was transfected with mimics. After that EZH2was measured with Western Blot.5. To perform luciferase activity assay, wild and mutant type EZH23’UTR was PCR-amplified from human genomes and cloned to the pMD-19T vector. After verified the sequence, wild or mutant type EZH23’UTR was digested from pMD-19T vector using restriction endonuclease, and two copies of the target sequence were cloned downstream of the Renilla luciferase in psi-CHECK2vector. Human embryonic kidney (HEK)293T cells were transfected with psi-CHECK2and miR-15b mimics. Cell lysate was collected and assayed30h after transfection and firefly and renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System and each transfected well was assayed in triplicate as described6. The ratio of gene expression level in breast cancer to that in the corresponding paracancerous tissue was calculated according to the standard curve. The relative amount of each miRNA was normalized to U6snRNA. Data are presented as the mean±standard deviation and analyzed by the Student’s t-test using the SPSS13.0Windows version software. P<0.05was considered statistically significant.Results1. Three days after the reconstruction MCF-7breast cancer cells, which was constructed with EZH23’UTR, were infected with CD511VB-1using a range of MOIs1,5,10, the expression of copGFP became lower with increasing positive control virus. Dilute an appropriate amount (MOI=1) of Lenti-miR Virus Library together with polybrene to infect the reconstruction MCF-7cells.2. After Lenti-miR Virus Library infected the reconstruction cells successfully, the Ctx drug was added to the cell culture medium. The cells died rapidly and were completely wiped out after10days unless a valid microRNA to3’UTR binding event occurs. Genomic DNA from selected cells was extracted by Phenol-chloroform extraction according to manufacturer’s recommendations. Seven effector miRNAs were indentified through PCR and sequencing methods, they were miR-217, miR-15b, miR-16-2, miR-329-1, miR-181b2, miR-224and miR-487b.3. Through Real-time PCR, we found that miR-217, miR-329-1and miR-487b were up-regulated expression in HBL-100, miR-15b, miR-16-2, miR-181b2and miR-224MCF-7were down-regulated expression in breast cancer cells MCF-7. The expression of miR-15b and miR-16-2was significantly increased in carcinomatous tissues compared with the corresponding paracancerous ones (P<0.05), other miRNAs didn’t have any no statistically significant difference.4. After measurement of miR-15b in HBL-100, MCF-7and MDA-MB-231cell lines, miR-15b was higher expressed in MCF-7, which was used to do loss of function, and lower in HBL-100that involved in gain of function. After MCF-7transfecting with80nM miR-15b inhibitor and HBL-100with lOnM miR-15b mimics, EZH2was down-regulated with the increase of miR-15b, on the contrary up-regulated compare with control.5. Wild or mutant type EZH23’UTR, the same with prediction, were cloned to dual luciferase vector. HEK293T was transfectied cells with psi-CHECK2and miR-15b mimics at the concentration of10nM. It showed that significant decrease in relative luciferase activity was noted when wild type EZH23’UTR was cotransfected with miR-15b mimics.ConclusionNovel targeted miRNAs are successful screened from MCF-7breast cancer cell which is restructured with EZH23’UTR together with Lenti-miR Virus Library and cytotoxic drugs. They are expressed differentially between normal breast cells and tumor. Gain and loss of fuction together with dual luciferase assay proves that miR-15b can regulate the expression of EZH2through interacting with3’UTR. These demonstrations confirmed the direct interaction of both miRNAs and EZH2. We can also use the selected system to screen miRNAs involved to ideal target genes. On the other hand, it needs further investigation to study the mechanism of EZH2abnormal expression and whether miR-15b can influence mammary epithelial function.