Abstract
Severe hypoxia has been shown to favor the self-renewal of human hematopoietic stem cells. Recent studies demonstrate that hypoxia via hypoxia-inducible factor (Hif-1a) can modify the proliferation and differentiation of CML stem cells. The ubiquitin E3 ligase SIAH2 is an important regulator of the hypoxic response as it leads to the ubiquitin/proteasomal degradation of prolyl hydroxylases such as PHD3, which in turn increases the stability of Hif-1a. The Hif-1a has been linked to chemosensitivity while the underlying molecular mechanism remains elusive. Therefore, we comprehensively analysed SIAH2 and Hif-1a role in determining chemosensitivity via signal molecule vascular epithelial growth factor (VEGF) pathway.
We tested the level of Siah2, Hif-1a and VEGF in Imatinb-sensitive CML Patients (n=15) and insensitive CML patients(n=10) by real-time reverse transcription PCR and western blot analysis. K562-wild type (K562-W) and K562-imatinib-resistance type (K562-R) cell were cultured for 24h and 48h under the condition of normal and hypoxia concentration of oxygen (1%). Knockdown of SAIH2 by RNA interference ,Cell viability and IC50 under 1% concentration oxygen were tested by cck-8;detected cell cycle and apoptosis by flow cytometry (FCM) ; analyzed the expression levels of Siah2, Hif-1a and VEGF respectively by real-time PCR and western blot.
The level of mRNA and protein of SIAH2 ,Hif-1a and VEGF were significantly higher in IM- resistant CML patients compared to IM sensitive CML patients, respectively (P<0.05). The similar results were observed in K562-R and K562-W cells(P<0.01). Under 21%,1% oxygen concentration cultured for 24h ,the IC50 of K562-W and K562-R cells was no significant difference (P<0.05),but there was significant difference after cultured for 48h Cell cycle analysis showed that more G0/G1 cells in K562-R than K562-W after cultured for 48h under hypoxia condition(P<0.05). After being cultured in 1% oxygen concentration for 48 hours, we confirmed SIAH2, Hif-1a and VEGF were up-regulated in both cell lines, moreover, it was more obvious in K562-R cells. In SIAH2-sh K562-R cells, the apoptosis was higher than K562-R cells obviously under 1% oxygen concentration for 48 hours(P<0.05);the level of Hif-1a Hardly monitored, and the level of VEGF was also lower.
There were higher level of SIAH2, Hif-1a and VEGF in IM-resistant CML patients. Under hypoxia condition, K562 cells were likely to improve their resistance to Imatinib .After we Knockdown SAIH2, K562-R Cell apoptosis rate increased significantly, along with low level Hif-1a and VEGF. It indicated that in hypoxia micro-environment, Siah-2 might be one of the critical molecules that induce Imatinib-resistance in CML in the way of maintaining leukemic cell survival and stimulating them into quiescence phase.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
