MiRNAs In Bone Marrow Stromal Cells Mediated Drug Resistance Of AML Cells

Objective:Mesenchymal stem cells(mesenchymal stem cells, MSC) in the bone marrow microenvironment, can contribute to cancer progression, metastasis and resistance to chemotherapy by providing a favorable microenvironment. Leukemia stem cells(LSCs) once adhere to MSC, which can support tumor self-renewal and growth, foster therapeutic resistance, and provide a niche for dormant metastases to thrive. The interaction between AML cells and their microenvironment has become increasingly apparent when solving disease progression and drug resistant.mi RNA is a kind of short, non-coding small RNA. It plays an important role through post-transcriptional regulation of gene expression. Several studies showed that mi RNA have important functions of regulation in cell proliferation, differentiation, apoptosis, secretion, migration, malignant transformation and other cellular functions. Studies on the mi RNA expression and functions have important value in prevention and treatment of tumor diseases. Researches focus on the mechanism of AML in the degree of mi RNA just started in recently years, and mainly pay attention to the level of expression, few research refer to the functional study, and the study of mi RNAs in bone marrow stromal cells mediated drug resistance of AML cells has not been reported.In our previous work, we found that AML cell line resistant to chemotherapy and have a decreased apoptosis, when adhered to MSC. RT-PCR and Western Blot analyses showed an up-regulation of cell cycle related gene C-Myc. In this study, we separate human bone marrow MSC, establish an AML cell and BM-MSC co-culture system, and according to gene microarray chips to find out the mi RNAs,which may relative to regulate the C-Myc, then verifies the expression quantity of this mi RNAs, making a preparation for the follow-up functional verification. Methods:Methods:1. Recovery, culturing and subculturing KG1 a, which was the cell line of AML. Collection the bone marrows of 15 healthy donors`.2. Extraction, culturing and subculturing MSC. 3. Establishing an co-culture system of KG1 a and MSC, after co-culture for 48 hours, selecting CD33 positive KG1 a cells(sample number 4D,5E,6F), with AML cell line KG1 a training alone(sample number 1,2,3) as a control. 4. Adding these samples separated into six different tubes and dissolved with Trizol, and then sending to company for sample quality tests and gene microarray chips. According to the results of gene microarray chips to find out the mi RNAs, which may relative to regulate the C-Myc. 5. Repeating steps 1, 2, 3, RT-PCR was used to detect the changes of mi RNA expression quantity of two kinds of culture condition.Results:1. The gene chip samples testing was passed. 2. According to the results of gene microarray chips, let-7a, mi R-17-3p, mi R-34 c, mi R-135 a, mi R-494 were found out maybe relative to regulating C-Myc, U6 was used as a control. 3. RT-PCR shows an increased expression of let-7a, mi R-34 c, mi R-135 a, mi R-494 for 3.39, 621.67, 13.00, 17.51 times alone and a decreased expression of mir-17-3p for 3.70 times compared with control in co-culture condition. 4. mi R-17-3p may participate in regulating C-Myc.Conclusion:Once AML cells adhere to MSC, there was a series of mi RNA expression changes, which induce an up-regulation of C-Myc, and among them, mi R-17-3p may participate in the regulation of C-Myc.