| Background:colon cancer and colon cancer stem cellsColon cancer is a common malignancy. With the improvement of people’s living standards, changes of living habits, the incidence of colon cancer in our country showed a clear upward trend. With the development of adjuvant chemotherapy drugs and multidisciplinary treatment, the colon cancer survival rates in recent years have significantly improved. However, distant metastasis and recurrence of colon cancer is still main cause of death. Therefore, to fundamentally improve the diagnosis and treatment of colon cancer, we must study the mechanism.In recent years, more and more scholars believe that cancer is a disease caused by cancer stem cells. Research of cancer stem cell further deep the people knowledge of cancer development. Cancer stem cells presenting in the tumor tissue, with the nature of the cell populations, with the degree of self-renewal and differentiation potential uncertainty, are the root of tumor formation and the continuous growth of proliferation. Cancer stem cells have been isolated and screened in a variety of solid tumors and hematological tumors (such as colon cancer, breast cancer, prostate cancer, lung cancer, skin cancer, pancreatic cancer, leukemia, glioblastoma, etc.), fully described a common phenomenon.Tumorigenesis is a multi-factor, multi-step process. The cell signal transduction pathway abnormalities and disorders play a crucial role. The human body cell signal transduction pathways, related to ligands, receptors, intracellular signaling proteins and nuclear signaling proteins, form a set of very complex network, and have a very fine control mechanism. The occurrence of cancer stem cells controlled by Notch, Wnt /β-catenin, Hedgehog, Bmi-1, strictly control the receptor tyrosine kinase (receptor tyrosine kinases, RTKs) and other signaling pathways. Abnormal activation of these signaling pathways and disorders will promote the formation of cancer stem cells and tumorigenesis.Background:CCND1 and E2F3 researchCyclin is cyclin-dependent kinases (cyclin-dependent kinases, CDKs), with which cyclin-dependent kinase inhibitor (cyclin-dependent kinases inhibitors, CKIs) play together on the cell cycle regulation. The level of cyclin contents with cell cycle changes, different level of cyclin corresponding period when it reached its peak, followed by a rapid degradation of inactivation, and thus play a role in the regulation of the cell cycle. Thus, cyclin Dl as one factor regulating the cell cycle, and its overexpression is characterized by a variety of human primary tumors. Tumor diagnosis and prognosis is important. Cyclin D1 protein expression was mainly seen in some of the primary malignant tumors, such as parathyroid tumors, head and neck squamous cell carcinoma, breast cancer, esophageal cancer, and hepatocellular carcinoma.E2F transcription factor is cell cycle G1 to S phase of the important regulatory factors. Meanwhile, E2F factor has a close relationship with the occurrence and apoptosis of tumor cell. E2F target genes mediate agents include limiting DNA synthesis and cell cycle progression, proto-oncogenes and tumor suppressor genes. Studies have shown that, E2F genes play a role in cancer in most tissues, such as E2F overexpression can induce p53-def icient mouse skin tumor formation. In prostate cancer, breast cancer and other organizations also have abnormal expression of E2F.Background:microRNA and cancer stem cellsMicroRNA is a class of small non-coding single-stranded molecule RNA, by complete/incomplete target gene mRNA 3’-untranslated region of the complementary binding, cutting, degradation of target gene mRNA, inhibiting target gene mRNA translation, regulating a variety of pathophysiological processes, such as stem cell differentiation, cell proliferation and tumor formation. Certain microRNAs exist close contact with the function of self-renewal and differentiation in cancer stem cells. Researchers from mouse lung tissue successfully isolated stem cells, suggesting that microRNA regulation of tumor stem cells differentiation, invasion, apoptosis and other features. In addition, microRNA still by gene regulatory role in regulating the biological function of cancer stem cells. Thus, microRNA has become a new regulating way of cancer stem cell research in signal transduction pathways. PurposeThe topics proposed by Realtime-PCR, gene transfection, luciferase system validation, gene chips and other molecular biology, explore miR-449b expression differences and changes in colon cancer stem cells. And we will confirm by mining and bioinformatics their corresponding target genes, the establishment and development of colon cancer stem cell biology-specific signal-regulated pathways. Through in-depth study of these aspects, will help to reveal the microRNA regulation of signal transduction mechanisms for the development of colon cancer stem cells to learn, provide new therapeutic targets and strategies for the prevention and treatment of colorectal cancer.Methods(1) First, sorting and identification of cancer stem cells. According to the currently accepted cancer stem cell surface marker CD133 and CD44 by flow cytometry screening, colon cancer cell lines CD133+ CD44+ double-positive cancer stem cells and CD133-CD44-double negative cells are separated. Flow cytometry, Western-blot methods, the characteristics of colon cancer stem cells will be identified. (2) Differences in microRNA expression in colon cancer stem cells confirmed. Isolated colon cancer stem cells for amplification:serum-free culture amplified get colon cancer stem cells. Total RNA was extracted for two CD133+ CD44+ double-positive cancer stem cells and CD133-CD44-negative cells. And using microRNA chip comparison CD133+ CD44+ double-positive cancer stem cells and CD133-CD44- double negative cells microRNA expression profiling. Pick out a meaningful difference in microRNA expression. (3) A comprehensive database of mRNA, microRNA databases, bioinformatics analysis predicted target genes of miR-449b mRNA candidate. Use microarray to compare CD133+ CD44+ double-positive cancer stem cells and CD133-CD44-double negative cells mRNA expression profiles. Data on differential gene Gene Ontology (Gene Ontology, GO), signal pathway (Pathway), build Path-Net, building dynamic gene networks (Dynamic-Gene-Net). Predict difference microRNA target gene. Target gene prediction database:PicTar, TarBase, TargetScan, miRanda, DIANAmicroT. (4) The use of pMIR-REPORTTM microRNA expression of luciferase (luciferase) reporting system to verify the differential expression of microRNA:target mRNAs interaction target sites. (5) The use of microRNA inhibitors and pre-microRNA expression vector transfection, block microRNA molecular function or reproduction of colon cancer stem cells expressing, observing the difference microRNA (miR-449b)-CCND1, E2F3 pathway molecules phenotypic changes and differentiation of colon cancer stem cell functionResults(1) We use proven colon cancer stem cell surface marker CD133 and CD44 sorting CD133+ CD44+ double-positive cancer stem cells and CD133-CD44-double negative cells. Tumor stem cells were confirmed. (2) By comparing CD133+ CD44+ double-positive cancer stem cells and CD133-CD44-double negative cells microRNA expression profiles with microRNA chip, pick out differences in microRNA expression:miR449b. Confirmed the differential expression of microRNAs and mRNAs. (3) Using mRNA database and microRNA databases, bio informatics analysis of microRNA constructed differences. Establish GO mRNA expression analysis, Pathway analysis, Path-Net Fig. (4) The luciferase reporter system is used to verify the interaction with miR449b relationship between CCND1 and E2F3. (5) Verify that the miR-449b by acting on CCND1 and E2F3 targets, regulation of colon cancer stem cell self-renewal function by inhibiting the expression of CCND1 and E2F3 genes on colon cancer, plays a role in the development of tumor suppressor genes.Conclusions(1) miR-449b expression lower level in colon cancer stem cell lines, rarely reported prior as the experimental results. (2) Differences in colon cancer stem cells suggest that the functional change of cancer stem cells, including a variety differences in cell cycle, energy metabolism, cell connections. (3) miR-449b play an inhibitory effect on colon cancer stem cell function of self-renewal, suggesting that miR-449b plays a tumor suppressor gene in the molecular pathogenesis of colon role. (4) By directly acting on E2F3 and CCND1, miR-449b downstream their expression in colon cancer stem cells. (4) miR-449 targeting its role CCND1 and E2F3 and their downstream molecules, regulates cell proliferation pathway. miR-449b may inhibit cancer stem cell self-renewal and the tumor suppressor gene role. |
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