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The Relationship between MicroRNA-182 and Glucocorticoids-resistance in Lymphoblastic Malignancies

Author: YangAPeng
Tutor: XieYanZuo
School: Fudan University
Course: Internal Medicine
Keywords: lymphoblastic malignancies glucocorticoid-resistance miRNA-182 FoxO3a apotosis
CLC: R392.4
Type: Master's thesis
Year: 2011
Downloads: 25
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Abstract

ObjectiveGlucocorticoids are the first-line chemotherapy drugs in many blood malignancies and their resistance is the main reason resulting in refractory and relapse. Currently the study about the mechanism of glucocorticoid resistance is a clinically urgent mission. FoxO3a, a key member of the FOXO family, plays a role in cell signaling pathway include the regulation of apoptosis, and works as an essential participant in apoptosis induced by glucocorticoids. However, the regulatory mechanism of FoxO3a is very complicated, but remains unclear. MicroRNAs (miRNAs) are a class of endogenous expression of small molecule non-coding RNA which could modulate posttranscriptional gene expression, has become a research hotspot. In our study, we intended to explore the regulation of FoxO3a by miRNA-182 and the relationship between miRNA-182 and glucocorticoid-resistance in lymphoblastic malignancies.Methodsa) Different level of miRNA-182 was detected by the method of qRT-PCR in lymphoblastic tumor cell lines.b) To screen and identificate the glucocorticoid-sensitive or glucocorticoid-resistant cell lines, we used flow cytometry to check the apoptosis induced by different level of concentration of dexamethasone. Used the method of CCK-8 to compare the coefficient of glucocorticoids in various lymphoblastic tumor cell lines. c) Used Western blot to detect the changes of total FoxO3a and phosph-FoxO3a in glucocorticoid-sensitive or glucocorticoid-resistant lymphoblastic tumor cell lines which were pretreated by different concentration of dexamethasone.d) The miRNA transfection efficiency on lymphoblastic tumor cell lines cells was detected through expression of green fluorescent by flow cytometry. miRNA-182 was overexpressed and measured by the method of RT-PCR in a higher transfection efficiency cell line. We used the Western blot to dectect the changes of total FoxO3a and phosph-FoxO3a after the over-expression of different dose of miRNA-182 and treatment with or without dexamethasone.e) Comparing with the NC control, we used the flow cytometry to detect the apotosis in the glucocorticoid-sensitive cell line CCRF-CEM which was over-expressed of miRNA-182-mimics, added with equivalent dexamethasone.f) Comparing with the NC control, we used the flow cytometry to detect the apotosis in the glucocorticoid-resistant cell line HUT-78 which was transfected with miRNA-182-ASO to knockdown the level of miRNA-182, added with equivalent dexamethasone.Results1. There were various expression levels of miRNA-182 in different lymphoblastic tumor cell lines. Glucocorticoid-resistant cell line Jurkat cells had the highest expression level. Molt-4 ranked second. While the expression in sensitive cell line SUP-B15 and wild-type splenocyte was very low.2.1n lymphoblastic tumor cell lines, Molt-4 and Jurkat were most resistant to glucocorticoid. Under dexamethasone up to 10μM their apoptosis rate was less than 5%; By contrast, splenocytes and SUP-B15 cell line were sensitive to dexamethasone. After treated with 100nM 8h or 36h respectively, their apoptosis rate up to 50%; Jurkat was more resistant than Molt-4, under different concentration of dexamethasone, the apotosis of Jurkat was lower than Molt-4. CCRF-CEM was more sensitive than HUT-78, its coefficient was significantly lower than the HUT-78.3. After dexamethasone treatment, there were increased FoxO3a expression and decreased phospho-FoxO3a expression in dexamethasone-sensitive lymphoblastic tumor cell lines treated by dexamethasone. While they did not change evidently in dexamethasone-resistant lymphoblastic tumor cell lines after receiving dexamethasone treatment.4. Select T leukemia cell line CCRF-CEM with higher transfection efficiency to overexpress miRNA-182.The level of total FoxO3a was inhibited by the increasement of miRNA-182.50nM could gain the best inhibitory effect, while the phospho-FoxO3a expression was not affected whether under the existence or absence of dexamethasone.5. Apoptosis on CCRF-CEM induced with equal dexamethasone was detected by flow cytometry. Compared to the control, the apoptosis of the sample over-expressed with miRNA-182-mimics was reducted and Bim protein appeared lower level.6. Apoptosis on HUT-78 induced with equal dexamethasone was detected by flow cytometry. Compared to the control, the apoptosis augmented in the sample whose miRNA-182 was knocked down and Bim protein achieved higher level.Conclusion1. In glucocorticoid-sensitive lymphoblastic tumor cell lines, the phospho-FoxO3a was significantly reduced and total FoxO3a was increased under the exposure of dexamethasone. While both of them did not change evidently in dexamethasone-resistant lymphocytes tumor cell lines after receiving dexamethasone treatment. We inferred that the level of total FoxO3a and phospho-FoxO3a changed in lymphoblastic tumors related to the sensitivity to glucocorticoids.2. miRNA-182 could reduce the protein level of FoxO3a but not phospho-FoxO3a in lymphoblastic tumor, the inhibitory effect was more obvious depend on the increasement of miRNA-182.3. The modulation of miRNA-182 in lymphoblastic tumor artificially could affect the sensitivity of glucocorticoids.4. Comparing the levels of miRNA-182 in different lymphoblastic malignancies, we found the level of miRNA-182 in glucocorticoid-sensitive lymphoblastic tumor cell lines were distinctly lower than that in glucocorticoid-resistant lymphoblastic tumor cell lines. We inferred that the resistance of glucocorticoids in lymphoblastic malignancies related to the aberrant miRNA-182 in a certain extent.

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