Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C
Authors: Shen Qi , Liu Sichu , Chen Yu , Yang Lijian , Chen Shaohua , Wu Xiuli , Li Bo , Li Yangqiu 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML.

Disclosures:
Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.