HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6

<div><p>All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia.</p></div

Fold resistance values for vincristine, arabinoside, doxorubicin, daunorubicin, and Cytoxan.

<p>Fold resistance values for vincristine, arabinoside, doxorubicin, daunorubicin, and Cytoxan.</p

Predicted HA117-RGS6 interactions.

<p>Predicted HA117-RGS6 interactions.</p

Chemoresistant cell lines showed increased DNMT1 expression.

<p>DNMT1 mRNA (a) and protein (b) expression was examined in all cell lines. HL60/ATRA, HA-con, and HA-si+117 cells showed higher protein but not RNA expression of DNMT1 than HL60, HA-si, or HA-si-con cells. Data are expressed as the mean ± SEM of five independent experiments (*p ≤ 0.05).</p

Sequences of the primers used for real-time PCR.

<p>Sequences of the primers used for real-time PCR.</p

Drug-resistant cells show increased expression of cancer stem cell markers.

<p>Total RNA and total protein were isolated from wild-type HL60, HL60/ATRA cell lines and subjected to real-time PCR (a) and SDS-PAGE gel electrophoresis/western blot analysis (b). Expression of Nanog, Oct-4, SOX-2, CD133 and CD123 mRNA and protein was then examined. HL60/ATRA cells showed higher expression of CD133, CD123, and Nanog than HL60 cells. Data are expressed as the mean ± SEM from five independent experiments (*p, 0.05).</p

RNAi-mediated inhibition or overexpression of HA117 up-regulates HA117 expression in HL60 cells.

<p>(a) HL60 cells transfected with HA117-specific shRNA or an empty lentivirus (images acquired under light and fluorescent microscopes). (b) HA-si cells transfected with HA117 overexpression lentivirus or empty lentivirus (images acquired under light and fluorescent microscopes). Real-time PCR analysis revealed that the expression of HA117 in HA-si and HA-con cells was considerably reduced after shRNA treatment compared with that in control cells. (d) Real-time PCR analysis revealed that expression of HA117 in HA-si+117 cells was significantly higher than that in HA-si-con cells after transfection with the HA117-overexpressing lentivirus.</p

Effects of down-regulating or up-regulating HA117 expression on the proliferation and migration of HL60 cells.

<p>(a) The CCK8 assays showed that up-regulating or down-regulating HA117 expression impacted cell growth in a time-dependent manner. Data are expressed as the mean ± SD of three independent experiments (*P < 0.05). HA117 expression or its down-regulation impacted cell growth in a time-dependent manner. (b) The number of migrating cancer cells was counted in three separate fields per experimental group. Data are expressed as the mean ± SD. (*P < 0.05; ANOVA). (c) The migration of cells showing low expression of HA117 was significantly lower than that of HA117-overexpressing cells.</p

Additional file 3: Figure S2. of Rapid evolutionary divergence of diploid and allotetraploid Gossypium mitochondrial genomes

Gene order comparison among mitogenomes of Gossypium. Colored blocks represent regions of conserved gene clusters in the Gossypium genomes and genes in bold are located in the repeat regions. Rpl2 and atp8 are shown in red bold to indicate that they are just close to or partially overlapped with the repeat sequences. (JPEG 1006 kb

DAVID KEGG pathway analysis.

<p>KEGG pathway analysis of the list of 50 miRNA targets. The vertical axis provides the names of the most significantly overrepresented pathways (P < 0.01), whereas the horizontal axis shows the -2log₁₀(P), where P was calculated based on Fisher’s exact test. The ratio (red) represents the numbers of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes in that pathway.</p