| Research background:Chronic myeloid leukemia is an acquired malignant disease of hematopoietic stem cells or myeloid origin with the annual incidence rate of 1-2/10000 adults, accounting for about 15% of newly diagnosed adult leukemia. In China, CML ranks the third in leukemia, higher than in Europe and the United States. Most CML patients are in their fifties to seventies. The CML is thought to be a clonal myeloproliferative disorder that is derived from a single hematopoietic stem cell malignant transformation. The pathogenesis of CML is complex, and currently, the exact pathogenic factors are still unclear, even though two major factors, i.e., both environmental and genetic factors contribute to the pathogenesis of CML. More than 95% CML patients have characteristics of the Ph chromosome or BCR-ABL fusion gene, which encodes protein P210 with constitutively active tyrosine kinase activity, which eventually led to the extreme proliferation of immature myeloid cells. Many other oncogenes and tumor suppressor genes are also implicated in the disease process; however the exact underlying mechanisms are still unclear.Micro RNA (microRNA, miRNA) is a highly conserved non-coding single-standard small molecule RNA, which approximately 19-25 nucleotides in length, and is widely expressed in prokaryotic and eukaryotic cells specifically. MiRNA exerts biological effects by regulating expression of downstream target genes. So far,1600 miRNAs are identified in the human genome, and it is estimated that miRNA can regulate one-third of human genes. The study confirmed that miRNA plays important roles in the development of leukemia, acting as either the oncogenes or tumor suppressor. Currently, miRNA research is focused more on acute leukemia, therefore the roles of miRNA in CML is still unclear.MiR-362-5p was first discovered by Bentwich in mammals. It was reported that miR-362-5p was highly expressed in hepatocellular carcinoma, and suppressed the expression of cylindromatosis (CYLD) and contributed to the growth and metastasis of hepatocellular carcinoma cells. In the previous study, we found that in early stages of hematopoietic cells the expression of miR-362-5p was low, while in the leukemia cell lines, miR-362-5p was highly expressed. The knowledge of biological effects of miR-362-5p in CML is still sketchy.GADD45a plays the oncogenic role in many malignant tumors, which can inhibit the proliferation of pancreatic cancer cell and induces cell cycle arrest, and GADD45a is closely linked to the progression of CML. But it is still unclear whether the high expression of miR-362-5p also plays an oncogenic role in this context, or whether miR-362-5p works through or in part through the GADD45a to regulate the downstream targets?This paper will study the expression of miR-362-5p both in chronic myeloid leukemia cell lines and clinical cases. We then manipulated the level of miR-362-5p in vitro and in vivo to observe the proliferation, cycle, apoptosis and tumor changes. By the bioinformatics prediction and experimental verification, we identified GADD45a is a target of miR-362-5p. Investigating the roles of miR-362-5p in the pathogenesis of CML provides new clues for clinical diagnosis and treatment of CML.Research objective:Study the expression of miR-362-5p in CML cells and patients, and explore its role in pathogenesis in CML.Research contents:Part I. The high expression of miR-362-5p and its function in chronic myeloid leukemia1. Measuring the level of miR-362-5p expression in K562, BV173 CML cell lines,45 CML patients’samples and 26 healthy controls by qRT-PCR, samples from eight CML patients prior to treatment (at diagnosis time) and after acquisition of complete remission.2. K562 and BV173 transfected with miR-362-5p inhibitor& negative control by Cationic liposomes, reduced miR-362-5p expression. Followed by:(1) CCK-8 assay detected K562 and BV173 cell proliferation and draw the growth curve.(2) Flow cytometry for detection of cell cycle change K562 and BV173 cell(3) Annexin V/PI assay for detection of apoptosis in K562 and BV173 cell(4) Transwell/Matrigel observed K562 and BV173 cell migration and invasion abilities.(5) Soft agar semisolid colony forming assay is to observe the capacity of anchor-independent growth.3. Established the stable expression of miR-362-5p inhibitor and inhibitor control in K562 cell using lentivirus vector, and inoculated it to female mice subcutaneously, studied the two groups of nude mice and measured the tumor growth rate, tumor weight and immunohistochemical expression of GADD45a.Part II. Verification of GADD45a as a target of miR-362-5p1.Targetscan, miRanda and Dual luciferase reporter gene method confirmed that the miR-362-5p complementary to 3’non-coding regions of GADD45 a mRNA.2. Western blot detected the change of target gene after inhibition or overexpression of miR-362-5p3. analyzed high or low expression of miR-362-5p CML cell lines and patient GADD45a protein expression.4. The K562 and BV173was first transfected with miR-362-5p inhibitor, and then cotreated with miR-362-5p inhibitor and GADD45a interfering RNA. We observed that the knockdown of GADD45a neutralized the effect of miR-362-5p inhibition on cell proliferation and apoptosis.5. Investigated the roles of miR-362-5p and GADD45a on cell signaling pathways.Research results:PartⅠ1. miR-362-5p expression was significantly higher especially in the K562 and BV173 CML cell line, compared with the hematopoietic progenitor cells CD34+of normal healthy population, (p<0.01); the expression of miR-362-5p in 45 cases of patients with CML was significantly higher than the corresponding 26 healthy controls (p<0.01).2. The expression of miR-362-5p in K562 and BV173 was effectively inhibited after transfection miR-362-5p inhibitor, which inhibited the proliferation, migration, invasion and colony formation in soft agar of the CML cell lines. In addition, it promoted apoptosis and G1 phase arrest obviously.3. Nude mice was injected with K562 cells transfected with miR-362-5p inhibitor, and we found that the tumor volume and growth significantly slower than the control group (p<0.01) and the expression of GADD45 a was higher than the control group.Part II.1. Bioinformatics analysis showed that at 92-98 location of mRNA3’UTR in GADD45a, has 7 base seed sequence complementary with the miR-362-5p. Through the construction and transfection of GADD45a@ psi-CHECK2--Report-Luc plasmid and miR-362-5p mimic/inhibitor into the cell, which caused the fluorescence changes, compared with the control group.2. Overexpression or inhibition the level of miR-362-5p caused the protein expression level of GADD45 a decline or increase, respectively.3. High expression of miR-362-5p in CML cell lines correlated with low GADD45a protein, and the reverse is also true.4. Transfected of GADD45 a shRNA can partly neutralize the changes of cell proliferation and apoptosis when in cells with low level of miR-362-5p.5. Inhibiting the expression of miR-362-5p activated the P38/JNK signaling pathway of K562 and BV173 effectively, while transfection of si GADD45a offset the effect on the P38 and JNK phosphorylation.Conclusion:1. MiR-362-5p plays the oncogenic role in CML cells.2. miR-362-5p dampens the P38/JNK signaling pathway through target inhibiting the expression of GADD45α. This study provides new clues for molecular target of CML. |
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