Role Of MicroRNA In Radiation Carcinogenesis And Radiation Biology

MicroRNA (miRNA) are noncoding RNA 18-25nt in length, first discovered in worms, that regulate a variety of biological processes by silencing specific target genes.Several studies have shown that miRNA play important roles in essential processes, such as differentiation, cell growth, and cell death. These miRNA can downregulate various gene products by translational repression when partially complementary sequences are present in the 3'untranslated regions (3'UTR) of the target mRNAs or by directing mRNA degradation. Using these posttranscriptional control mechanisms, mammalian miRNA appear to target a diversity of cellular functions, including cell proliferation and differentiation. Moreover, it has been shown that miRNA are aberrantly expressed or mutated in cancers, suggesting that they may play a role as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. In the last few years, it has become evident that miRNA expression is deregulated in human cancer, resulting in specific oncogenic events.Radiation therapy is now a routine treatment for certain types of cancer and over 20 percent of cancer patients will require radiation therapy during the treatment of their disease. However, Ionizing radiation is a well-known carcinogen for various human tissues and a complete carcinogen that is able to initiate and promote neoplastic progression. Studies show that radiation can cause many types of cancers, especially leukemia and lymphoma, and of the mechanism of this radiation carcinogenesis is still not very clear. It is true that there are differences between mice and humans, however the well established mouse model of radiation induced thymic lymphoma can help us understand more about this. Indeed, studies of radiation induced mouse thymic lymphomas, one of the classic models in radiation carcinogenesis, demonstrated that multi-steps and many factors, like oncogene ras and tumor suppressor gene pten or p53, were involved in radiation carcinogenesis. We previously found that expression changes of ERK1/2, STAT3 and SHP-2 in Bone Marrow Cells is highly associated with y-ray Induced Leukemia Mice. However, the mechanism of radiation carcinogenesis is still poorly understood. Specifically, the possible role of microRNA that participates in radiation carcinogenesis is still unknown.Here in this study, by using of radiation-induced mouse thymic lymphoma model, we explored the possibility that microRNA may be involved in adiation carcinogenesisPart 1:Identification of in radiation carcinogenesis associated miRNAs in BALB/c mice.The use of miRNA microarray technologies has been used as a powerful tool to recognize miRNA differentially expressed between normal and tumor samples and also to identify miRNA expression signatures associated with well-defined clinicopathologic features and disease outcome Several studies have also investigated the molecular mechanisms leading to an aberrant miRNA expression, identifying the presence of genomic abnormalities in miRNA genes. In this study, By miRNA microarray technology and cluster assay, we find that miRNA profiles are aberrantly expressed in split radiation induce thymic lymphoma tissues in comparison to normal thymic tissue. The overall miRNA expression could clearly separate normal versus cancer tissues. We next use q-PCR methods to validate that The most significantly overexpressed miRNA in thymic lymphoma were miR-762, miR-17-92 cluster, miR-106-363 cluster and miR-23a/b; whereas miR-200c, miR-486, miR-199a, miR-143, miR-145, miR-125b, miR-31, miR-26, miR-100 and let-7 were among the most down-modulated miRNA.Part 2:Role of selected microRNA in radiation carcinogenesis.1. miR-23, A novel miRNA signature assoiated with pathogenesis and diagnosis of radiation carcinogenesis.In this study, using bioinformatics methods and radiation induced thymic lymphoma model in balb/c mice, we identified the microRNA23 could directly targeting proapoptosis molecular FAS, which may also play an important role in radiation carcinogenesis.Our data also indicates that miR-23a could repress FAS much more potent than miR-23b and the additional region besides conserved seed pairing enables miR-23a's higher regulation.Besides, miR-23 can be detected in the serum of radiation leukemia mice. This results indicate that miRNA23 is a novel microRNA signature assoiated with pathogenesis and diagnosis of radiation carcinogenesis.2. miR-21 play an important role in radiation carcinogenesis by directly targeting the tumor suppressor gene Big-h3Deregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, only functions and targets of few mammalian miRNAs was known. Specifically, the miRNA that participates in radiation carcinogenesis and the miRNA that targets tumor suppressor gene Big-h3 are still unknown. Using a radiation induced thymic lymphoma model in balb/c mice, we identified that miR-21 could directly target tumor suppressor gene Big-h3, which may also play an important role in radiation carcinogenesis. We also found that miR-21 could be induced by TGF. and has both positive and negative effects on regulating TGF. signaling. In conclusion, this data strongly suggests that miR-21 also participates in radiation carcinogenesis and TGF. signaling regulation.3. Identification of microRNA targeting tumor suppressor gene P16.Then we focused on studying another tumor suppressor gene P16/INK4a and it's potential role in radiation carcinogenesis. We firstly found that significant downregulation of 16 protein expression was closely related to radiation induced thymic lymphoma. Next by using bioinformatics methods, we found that both miR-126-5p and miR-762could target murine p16 mRNA in vitro. Furthuemore, we found that miR-762 was enhanced significantly in radiation induced thymic lymphoma tissues when compared to normal thymus tissues. However we didn't find an obvious enhancement of miR-126-5p expression between thymic lymphoma tissues. Finally, there was an inverse correlation between P16 protein and miR-762 expression found in different radiation induced thymic lymphoma tissues and normal thymus tissues. Taken together, our data indicates that miR-762 may play an important role in radiation induced thymic lymphoma by directly targeting tumor suppressor gene P16. This work also demonstrates the difference between in vivo and in vitro.4. Citical role of DNTT and miR-125 in radiation induced thymic leukemia.Next we use FACS assay to Identify that the thymic leukemia/lymphoma cells are CD4+CD8+ and DNTT+. This is important because Dntt(Deoxynucleotidyl Transferase, Terminal)is also a marker of Pro and Pre-T. Studies also show that DNTT play critical role in TCR recomposition. Next we find that Dntt play critical role in radiation carcinogenesis and both silence and induction of DNTT are required for radiation carcinogenesis. Last, we find that miR-125 may target DNTT and play critical role in radiation carcinogenesis.Part 3:Role of selected microRNA in cancer threapy。Next we used overexpression experiment to find that miR-200c, miR-486, miR-199b, miR-193b, miR-143, miR-145, miR-125a/b, miR-31, miR-26a, miR-99a, miR-100, miR-29a and let-7e could enhance the apoptosis rate of U87MG cells, A549 cells and NIH3T3 cells. We also found that these miRNA could inhibit proliferation,induce cell cycle arrest and induce DSB foci of U87MG cells and other cell types. These data indicate that miR-200c, miR-486, miR-199b,miR-193b, miR-143, miR-145, miR-125a/b, miR-31, miR-26a, miR-99a, miR-100, miR-29a and let-7e could kill cancer cells in vitro. Then we selected 4 miRNAs, namely miR-200c, miR-486, miR-143 & miR-125b for in vivo usage for the prevention and therapy of radiation induce thymic T lymphoma. Now the in vivo work is still on going.Part 4:Role of selected microRNA in other aspects of radiation biology MiRNA34a have shown to exert potent antiproliferative effects and induced cell apoptosis, and can be induced by irradiation in vivo and in vitro. However, the relationship between miR-34a and radiosensitivity, and its potential diagnostic significance in radiation biology, remain unclear. This study found that differing responses to radiation of young and old mice were related to miR-34a. First, we found that miR-34a could be induced in many organs by radiation of both young and old mice. However, the level of miR-34a induced by young mice was much higher when compared to old mice. Next, we found that miR-34a played a critical role in radiosensitivity variations of different tissues by enhancing cell apoptosis and decreasing cell viability. We also found that the inhibition of miR-34a could protect cells from radiodamage, however. Finally, we concluded that miR-34a could be stable in serum after IR and serve as a novel biomarker of radiation injury. Taken together, this part strongly suggests that miR-34a may be a novel biomarker, mediator and target of radiodamage, radiosensitivity and radioprotection.In summary, our data strongly suggests that microRNAs play a critical role in radiation carcinogenesis.