Transcriptome and Metabolome Analysis Reveals Salt-Tolerance Pathways in the Leaves and Roots of ZM-4 (Malus zumi) in the Early Stages of Salt Stress.

The breeding of salt-tolerant rootstock relies heavily on the availability of salt-tolerant Malus germplasm resources. The first step in developing salt-tolerant resources is to learn their molecular and metabolic underpinnings. Hydroponic seedlings of both ZM-4 (salt-tolerant resource) and M9T337 (salt-sensitive rootstock) were treated with a solution of 75 mM salinity. ZM-4's fresh weight increased, then decreased, and then increased again after being treated with NaCl, whereas M9T337's fresh weight continued to decrease. The results of transcriptome and metabolome after 0 h (CK) and 24 h of NaCl treatment showed that the leaves of ZM-4 had a higher content of flavonoids (phloretinm, naringenin-7-O-glucoside, kaempferol-3-O-galactoside, epiafzelechin, etc.) and the genes (CHI, CYP, FLS, LAR, and ANR) related to the flavonoid synthesis pathway showed up-regulation, suggesting a high antioxidant capacity. In addition to the high polyphenol content (L-phenylalanine, 5-O-p-coumaroyl quinic acid) and the high related gene expression (4CLL9 and SAT), the roots of ZM-4 exhibited a high osmotic adjustment ability. Under normal growing conditions, the roots of ZM-4 contained a higher content of some amino acids (L-proline, tran-4-hydroxy-L-prolin, L-glutamine, etc.) and sugars (D-fructose 6-phosphate, D-glucose 6-phosphate, etc.), and the genes (GLT1, BAM7, INV1, etc.) related to these two pathways were highly expressed. Furthermore, some amino acids (S-(methyl) glutathione, N-methyl-trans-4-hydroxy-L-proline, etc.) and sugars (D-sucrose, maltotriose, etc.) increased and genes (ALD1, BCAT1, AMY1.1, etc.) related to the pathways showed up-regulation under salt stress. This research provided theoretical support for the application of breeding salt-tolerant rootstocks by elucidating the molecular and metabolic mechanisms of salt tolerance during the early stages of salt treatment for ZM-4.

Transcriptome Analysis of the Effects of Grafting Interstocks on Apple Rootstocks and Scions.

Apples are a major horticultural crop worldwide. Grafting techniques are widely utilized in apple production to keep the varieties pure. Interstocks are frequently used in Northern China to achieve intensive apple dwarfing cultivation. High-throughput sequencing was used to investigate differentially expressed genes in the phloem tissues of two different xenograft systems, M ('Gala'/'Mac 9'/Malus baccata (L.) Borkh.) and B ('Gala'/Malus baccata (L.) Borkh.). The results showed that dwarfing interstocks could significantly reduce the height and diameters of apple trees while have few effects on the growth of annual branches. The interstocks were found to regulate the expression of genes related to hormone metabolism and tree body control (GH3.9, PIN1, CKI1, ARP1, GA2ox1 and GA20ox1), these effects may attribute the dwarf characters for apple trees with interstocks. Besides, the interstocks reduce photosynthesis-related genes (MADH-ME4 and GAPC), promote carbon (C) metabolism gene expression (AATP1, GDH and PFK3), promote the expression of nitrogen (N)-metabolism-related genes (NRT2.7, NADH and GDH) in rootstocks, and improve the expression of genes related to secondary metabolism in scions (DX5, FPS1, TPS21 and SRG1). We also concluded that the interstocks acquired early blooming traits due to promotion of the expression of flowering genes in the scion (MOF1, FTIP7, AGL12 and AGL24). This study is a valuable resource regarding the molecular mechanisms of dwarf interstocks' influence on various biological processes and transplantation systems in both scions and rootstocks.

Effects of Salt Stress on the Antioxidant Activity and Malondialdehyde, Solution Protein, Proline, and Chlorophyll Contents of Three Malus Species.

Understanding the different physiological responses of Malus species under salt stress in the seedling stages will be useful in breeding salt-tolerant dwarfing apple rootstocks. Seedlings of Malus Zumi (Mats.) Rehd. (M. zumi), Malus sieversii (Led.) Roem. (M. sieversii), and Malus baccata (L.) Borkh. (M. baccata) were treated with solution of 0, 0.20%, 0.40%, and 0.60% salinity. Physiological parameters of their leaves and roots were measured at 0 d, 4 d, 8 d and 12 d after salinity treatments. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA), solution protein (SP), and proline (PRO) initially increased and then decreased. The activities and contents of these parameters were higher in the 0.40% and 0.60% NaCl treatments than in the 0.20% treatment and in the 0% control. M. zumi was the most resistant to salt stress, showing the lowest content of MDA in the leaves and roots, which increased slightly under salt stress. M. baccata had the highest increase in both the content and proportion of MDA. High enzyme activity was shown to play an important role in the salt resistance of M. zumi. Moreover, it can be speculated that there are other substances that also play a major role. We found that osmotic regulation played a key role in response to salt stress for M. baccata even though it was sensitive to salt stress. For M. sieversii, both the osmotic regulation and enzymatic antioxidants were observed to play a major role in mitigating salt stress.

Expression Profiles and Characteristics of Apple lncRNAs in Roots, Phloem, Leaves, Flowers, and Fruit.

LncRNAs impart crucial effects on various biological processes, including biotic stress responses, abiotic stress responses, fertility and development. The apple tree is one of the four major fruit trees in the world. However, lncRNAs's roles in different tissues of apple are unknown. We identified the lncRNAs in five tissues of apples including the roots, phloem, leaves, flowers, and fruit, and predicted the intricate regulatory networks. A total of 9440 lncRNAs were obtained. LncRNA target prediction revealed 10,628 potential lncRNA-messenger RNA (mRNA) pairs, 9410 pairs functioning in a cis-acting fashion, and 1218 acting in a trans-acting fashion. Functional enrichment analysis showed that the targets were significantly enriched in molecular functions related to photosynthesis-antenna proteins, single-organism metabolic process and glutathione metabolism. Additionally, a total of 88 lncRNAs have various functions related to microRNAs (miRNAs) as miRNA precursors. Interactions between lncRNAs and miRNAs were predicted, 1341 possible interrelations between 187 mdm-miRNAs and 174 lncRNAs (1.84%) were identified. MSTRG.121644.5, MSTRG.121644.8, MSTRG.2929.2, MSTRG.3953.2, MSTRG.63448.2, MSTRG.9870.2, and MSTRG.9870.3 could participate in the functions in roots as competing endogenous RNAs (ceRNAs). MSTRG.11457.2, MSTRG.138614.2, and MSTRG.60895.2 could adopt special functions in the fruit by working with miRNAs. A further analysis showed that different tissues formed special lncRNA-miRNA-mRNA networks. MSTRG.60895.2-mdm-miR393-MD17G1009000 may participate in the anthocyanin metabolism in the fruit. These findings provide a comprehensive view of potential functions for lncRNAs, corresponding target genes, and related lncRNA-miRNA-mRNA networks, which will increase our knowledge of the underlying development mechanism in apple.

Expression Characteristics in Roots, Phloem, Leaves, Flowers and Fruits of Apple circRNA.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that play pivotal roles in various biological processes. However, circRNAs' roles in different tissues of apple are currently unknown. A total of 6495 unique circRNAs were identified from roots, phloem, leaves, flowers and fruits; 65.99% of them were intergenic circRNAs. Similar to other plants, tissue-specific expression was also observed for apple circRNAs; only 175 (2.69%) circRNAs were prevalently expressed in all five different tissues, while 1256, 1064, 912, 904 and 1080 circRNAs were expressed only in roots, phloem, leaves, flowers and fruit, respectively. The hosting-genes of circRNAs showed significant differences enriched in COG, GO terms or KEGG pathways in five tissues, suggesting the special functions of circRNAs in different tissues. Potential binding interactions between circRNAs and miRNAs were investigated using TargetFinder; 2989 interactions between 647 circRNAs and 192 miRNA were predicated in the present study. It also predicted that Chr00:18744403|18744580-mdm-miR160 might play an important role in the formation of flowers or in regulating the coloration of flowers, Chr10:6857496|6858910-mdm-miR168 might be involved in response to drought stress in roots, and Chr03:1226434|1277176 may absorb mdm-miR482a-3p and play a major role in disease resistance. Two circRNAs were experimentally analyzed by qRT-PCR with divergent primers, the expression levels were consistent with RNA-seq, which indicates that the RNA-seq datasets were reliable.

MiR-15b regulates cell differentiation and survival by targeting CCNE1 in APL cell lines.

MicroRNAs have been shown to be involved in various cell processes, including proliferation, apoptosis and differentiation. However, little is known about their function in granulopoiesis. In the present study, overexpression and knockdown experiments revealed that miR-15b was required to block the proliferation of NB4 and HL60 cells and induce them differentiated to granulocyte lineage. Moreover, we identified CCNE1 as a direct target of miR-15b, and demonstrated that CCNE1 was involved in cell differentiation and proliferation in acute promyelocytic leukemia cells. In addition, we demonstrated a novel pathway in which miR-15b regulated cells arrested in the G0/G1 phase and promoted terminal differentiation of cells by targeting CCNE1, which could modulate the cell cycle effort pRb in APL cells. These events blocked cell proliferation and promoted granulocyte differentiation. In conclusion, our data highlighted, for the first time, the important role of miR-15b in myeloid differentiation and suggested the potential role of miR-15b in cancer therapy.

RAS-Responsive Element-Binding Protein 1 Blocks the Granulocytic Differentiation of Myeloid Leukemia Cells.

RAS-responsive element-binding protein 1 (RREB1) is a transcription factor that is implicated in RAS signaling and multiple tumors. However, the role of RREB1 in acute myeloid leukemia has not been studied. We found that RREB1 is overexpressed in AML patients and myeloid leukemia cell lines (NB4 and HL-60), and RREB1 expression was significantly decreased during granulocytic differentiation of myeloid leukemia cells induced by all-trans retinoic acid (ATRA). Then we performed a RREB1 knockdown assay in NB4 and HL-60 cells; the results showed that knockdown of RREB1 upregulated expression of CD11b, CEBPß, and microRNA-145 (miR-145), which hinted that knockdown of RREB1 enhanced granulocytic differentiation of myeloid leukemia cells. In addition, inhibitor of miR-145 can offset the enhanced effect on granulocytic differentiation mediated by downregulation of RREB1. These collective findings demonstrated that RREB1 blocks granulocytic differentiation of myeloid leukemia cells by inhibiting the expression of miR-145 and downstream targets of the RAS signal pathway. These may provide a promising therapeutic target for AML patients.

ACTL6A interacts with p53 in acute promyelocytic leukemia cell lines to affect differentiation via the Sox2/Notch1 signaling pathway.

Actin-like 6A (ACTL6A), a component of BAF chromatin remodeling complexes, is important for cell differentiation. Nevertheless, its role and mechanism in acute promyelocytic leukemia (APL) has not been reported. To identify the genes that may participate in the development of APL, we analyzed data from an APL cDNA microarray (GSE12662) in the NCBI database, and found that ACTL6A was up-regulated in APL patients. Subsequently, we investigated the function and mechanisms of ACTL6A in myeloid cell development. The expression of ACTL6A was gradually decreased during granulocytic differentiation in all-trans retinoic acid-treated NB4 and HL-60 cells, and phorbol myristate acetate-treated HL-60 cells. We also found that knockdown of ACTL6A promoted differentiation in NB4 and HL-60 cells, and decreased the levels of Sox2 and Notch1. Mechanistically, ACTL6A interacted with and was co-localized with Sox2 and p53. Meanwhile, CBL0137, an activator of p53, decreased the expression of ACTL6A and promoted differentiation in NB4 and HL-60 cells. These findings suggest that the inhibition of ACTL6A promotes differentiation via the Sox2 and Notch1 signaling pathways. Furthermore, the differentiation promoted by inhibiting ACTL6A could be regulated by p53 via its physical interaction with ACTL6A.

miR-382-5p modulates the ATRA-induced differentiation of acute promyelocytic leukemia by targeting tumor suppressor PTEN.

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic differentiation and maturation. MicroRNAs play pivotal roles in formation of the leukemic phenotype. Previously, microRNA-382-5p (miR-382-5p) was upregulated in acute myeloid leukemia (AML) with t(15;17). In the present study, we found that miR-382-5p expression was elevated with ATRA-induced differentiation of APL. To investigate the potential functional role of miR-382-5p in APL differentiation, an APL cell line was transfected with miR-382-5p mimics, inhibitors, or negative control (NC). The results showed in APL cell line NB4 that miR-382-5p downregulation upon ATRA treatment was a key event in the drug response. Mechanistic investigations revealed that miR-382-5p targeted the ATRA-regulated tumor suppressor gene PTEN through direct binding to its 3' UTR. Enforced expression of miR-382-5p or specific PTEN inhibitors inhibited ATRA-induced granulocytic differentiation via regulation of the cell cycle regulator cyclinD1. Conversely, PTEN overexpression promoted differentiation and enhanced sensitivity of NB4 cell line to physiological levels of ATRA. Finally, we found that PTEN overexpression restored PML nuclear bodies (NBs). Taken together, these results demonstrated that up-regulated miR-382-5p in NB4 cell line inhibited granulocytic differentiation through the miR-382-5p/PTEN axis, uncovering PTEN as a critical element in the granulocytic differentiation program induced by ATRA in APL.

Salinomycin induces apoptosis and differentiation in human acute promyelocytic leukemia cells.

At present, acute promyelocytic leukemia (APL) is the most curable form of acute myeloid leukemia and can be treated using all-trans retinoic acid and arsenic trioxide. However, the current treatment of APL is associated with some issues such as drug toxicity, resistance and relapse. Therefore, other strategies are necessary for APL treatment. In the present study, we investigated the effects of salinomycin (SAL) on APL cell lines NB4 and HL-60 and determined its possible mechanisms. We observed that SAL inhibited cell proliferation, as determined by performing Cell Counting Kit-8 (CCK-8) assay, promoted cell apoptosis, as determined based on morphological changes, and increased Annexin V/propidium iodide (PI)-positive apoptotic cell percentage. Treatment with SAL increased Bax/Bcl-2 and cytochrome c expression and activated caspase-3 and -9, thus leading to poly(ADP-ribose) polymerase (PARP) cleavage and resulting in cell apoptosis. These results revealed that SAL induced cell apoptosis through activation of the intrinsic apoptosis pathway. The present study is the first to show that SAL induced the differentiation of APL cells, as determined based on mature morphological changes, increased NBT-positive cell and CD11b-positive cell percentages and increased CD11b and C/EBPß levels. Furthermore, SAL decreased the expression of ß-catenin and its targets cyclin D1 and C-myc. Results of immunofluorescence analysis revealed that SAL markedly decreased the ß-catenin level in both the nucleus and cytoplasm. Combination treatment with SAL and IWR-1, an inhibitor of Wnt signaling, synergistically triggered SAL-induced differentiation of APL cells. These findings demonstrated that SAL effectively inhibited cell proliferation accompanied by induction of apoptosis and promotion of cell differentiation by inhibiting Wnt/ß-catenin signaling. Collectively, these data revealed that SAL is a potential drug for treatment of APL.

Effects of lapatinib on cell proliferation and apoptosis in NB4 cells.

Acute promyelocytic leukemia (APL), characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid α receptor (RARα) fusion protein, responds to treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, drug resistance and side effects restrict the application of these reagents. Hence, the development of novel therapeutic drugs for APL treatment is critical. Lapatinib, a small-molecule tyrosine kinase inhibitor, has been used in the treatment of different tumors. However, it is unclear whether lapatinib exerts antitumor effects on APL. The present study investigated the antitumor effects and potential mechanisms of lapatinib on NB4 cells derived from APL. Cell Counting Kit-8 assay and colony forming analysis indicated that lapatinib inhibited NB4 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that lapatinib induced cell cycle arrest at the S phase and promoted cell apoptosis. Furthermore, Liu's staining and Hoechst 33258 staining revelaed that lapatinib treatment induced an apoptotic nuclear phenomenon. Furthermore, lapatinib induced apoptosis by decreasing Bcl-2 and PML-RARα levels, and by increasing the levels of Bax, cleaved PARP, cleaved caspase-3 and cleaved caspase-9. In addition, lapatinib increased the levels of phospho-p38 MAPK and phospho-JNK, and decreased the levels of phospho-Akt. The p38 inhibitor PD169316 partially blocked lapatinib-induced proliferation inhibition and apoptosis, whereas the JNK inhibitor SP600125 had no such effects. Therefore, treatment with lapatinib may be a promising strategy for APL therapy.

[Mechanism underlying inhibition of proliferation and promotion of apoptosis by lapatinib in HL60 cells].

Objective To investigate the effect of lapatinib on cell proliferation and apoptosis in acute myeloid leukemia HL60 cells in vitro and the related molecular mechanisms. Methods The HL60 cells were treated with 5, 10, 15 µmol/L lapatinib for 24 hours, and then morphological changes of the cells were observed under optical microscope. CCK-8 assay was used to assess the cell viability. Colony formation assay was performed to detect the cell proliferation ability. Cell apoptosis labeled by annexinV-FITC/PI were analyzed by flow cytometry. Wright modified LIU's staining and Hoechst33342 fluorescent staining were used to observe the morphology of the nucleus. Western blotting was utilized to detect the expressions of Bax, Bcl2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (cleaved PARP), cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A), c-MYC, AKT and p-AKT. Results Compared with the control group, lapatinib inhibited cell proliferation and promoted apoptosis, induced nuclear fragmentation, chromatin condensation of HL60 cells in a dose-dependent manner. Meanwhile, it down-regulated the expression of Bcl2, up-regulated the levels of Bax, cleaved caspase-3, cleaved caspase-9 and cleaved PARP, and decreased the levels of CIP2A, p-AKT and c-MYC. Conclusion Lapatinib could inhibit cell proliferation and promote apoptosis in HL60 cells by inhibiting the CIP2A/AKT/c-MYC signal pathway.

Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Human Leukemia NB4 Cells without Light Activation.

Background and Aims: Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has recently been proven a suppressor of yes-associated protein (YAP) and has shown potential in anticancer treatment. However, its anti-human leukemia effects in NB4 cells remain unclear. In this study, we investigated the effects of VP on proliferation and apoptosis in human leukemia NB4 cells. Methods: NB4 cells were treated with VP for 24 h. The effects of VP on cell proliferation were determined using a Cell-Counting Kit-8 assay (CCK-8) assay and colony forming assay. Apoptosis and cell cycle were evaluated by flow cytometry (FCM). The protein levels were detected by western blot. Results: We found that VP inhibited the proliferation of NB4 cells in a concentration and time-dependent manner. FCM analysis showed that VP induced apoptosis in a concentration dependent manner and that VP treatment led to cell cycle arrest at G0/G1 phase. Moreover, VP significantly decreased the protein expression of YAP, p-YAP, Survivin, c-Myc, cyclinD1, p-ERK, and p-AKT. In addition, VP increased the protein expression of cleaved caspase3, cleaved PARP, Bax, and p-p38 MAPK. Conclusions: VP inhibited the proliferation and induced apoptosis in NB4 cells.

Epigallocatechin-3-gallate promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells via PTEN.

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

Effect of YAP Inhibition on Human Leukemia HL-60 Cells.

Background: Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a candidate oncoprotein and participates in the progression of various malignancies. However, few reports have examined the effect of YAP inhibition in human leukemia HL-60 cells. Methods: We examined the effects of YAP knockdown or inhibition using short hairpin RNA (shRNA) or verteporfin (VP), respectively. Western blot assays were used to determine the expression levels of YAP, Survivin, cyclinD1, PARP, Bcl-2, and Bax. Cell proliferation was assessed using the cell counting kit (CCK-8) assay. Cell cycle progression and apoptosis were evaluated by flow cytometry, and apoptotic cell morphology was observed by Hoechst 33342 staining. Results: Knockdown or inhibition of YAP led to cell cycle arrest at the G0/G1 phase and increased apoptosis, inhibited cell proliferation, increased levels of Bax and cleaved PARP, and decreased levels of PARP, Bcl-2, Survivin, and cyclinD1. Moreover, Hoechst 33342 staining revealed increased cell nuclear fragmentation. Conclusion: Collectively, these results show that inhibition of YAP inhibits proliferation and induces apoptosis in HL-60 cells. Therefore, a novel treatment regime involving genetic or pharmacological inhibition of YAP could be established for acute promyelocytic leukemia.