The expression and functional characterization associated with cell apoptosis and proteomic analysis of the novel gene MLAA-34 in U937 cells
- Authors:
- Published online on: November 7, 2012 https://doi.org/10.3892/or.2012.2129
- Pages: 491-506
Abstract
Introduction
Leukemia is the leading cause of mortality worldwide in patients with malignant tumors under the age of 35 years. Patients with acute myeloid leukemia (AML) who have relapsed or are refractory to conventional chemotherapy have a poorer prognosis and response to chemotherapy than those with de novo AML, which remains a formidable therapeutic challenge even with the introduction of several new therapeutic strategies (1–3). M5 is largely incurable with high relapse rates, infiltration and a median remission duration of only six months, approximately (4). Moreover, M5 has been reported to have a worse prognosis than other subtypes of AML (5). Thus, a vaccine or a new drug against M5 is required as a strategic tool for the control of this disease, but none are currently available for practical use.
The MLAA-34 gene (GenBank no. AY288977.2) has been confirmed to be a novel splice variant of CAB39L (calcium binding protein 39-like). MLAA-34 was first discovered in M5 in an effort to identify monocytic leukemia-associated antigens by serologic analysis of a recombinant cDNA expression library (SEREX) that reacted exclusively with sera from allogeneic leukemia patients but not with normal donor sera (6,7). The 1671 kb gene is located on 13q14.2 and was initially cloned in our laboratory from U937 cells (7). CAB39L has three alternative transcripts and has been predicted to encode a 337 aa protein. The three alternative transcripts of CAB39L have been recognized to encode the same protein, differing only in their 5′ untranslated regions [GenBank nos. BC010993 (1482 bp), BX647518 (2371 bp) and AY288977.2].
In our previous study, MLAA-34 and CAB39L were identified with RNA interference (RNAi) in the U937 cell line as novel anti-apoptotic factors that are closely related to carcinogenesis or progression of M5 (7). Clinical research has shown that MLAA-34 mRNA expression is upregulated in refractory/relapsed M5 patients compared with newly diagnosed, healthy donors and AML patients in complete remission; high expression of MLAA-34 is more prominent in the M5 subtype than in other AML patients; MLAA-34 overexpression has been found to be associated with unfavorable clinical features at diagnosis and has been shown to be an independent prognostic factor (8). However, for MLAA-34, there are no exact reports regarding its cellular localization and expression in manifold cell lines; the anti-apoptotic mechanism of MLAA-34 remains unclear.
The purpose of this study was to conduct an in-depth search for the expression and anti-apoptotic mechanism of MLAA-34 through the lentivirus-mediated overexpression in the U937 cell line, and to then apply proteomics to identify its correlated proteins or pathways that might perform functions important for the apoptosis and proliferation of U937 cells.
Materials and methods
Cell culture
U937, HL60, K562, RPMI-8226, HepG2, Hep3B, MHCC97-H, RC-K8, SGC-7901, Eca109, BGC823, MKN45, GES-1, BxPC-3, A375, T24, HUVEC, BMSCs, LO2, HeLa, 293T, 293, RD, RT4, 5637, EJ, UM-UC-3, 2537, J82, Tsu-Prl, MAH, LiBr, Hut-78, HCT116+, FBL-3, C6, astrocyte, 3T3-L1, NIH3T3, Vero and MDCK cell lines were all maintained in our laboratory and cultured in RPMI-1640 or DMEM supplemented with 10% fetal calf serum. The medium for cell lines expressing the neomycin resistance gene was supplemented with 0.5 mg/ml G418. Human epithelial tissue, normal human peripheral blood mononuclear cells (PBMCs), M5 patient and non-M5 acute leukemia patient PBMCs were all obtained from over 30 cases of patients or healthy young individuals. Mouse splenocytes were obtained from 30 mice.
Antibodies and reagents
CAB39L and MLAA-34 share the same open reading frame (ORF), the CAB39L antibody was used in this report. Antibodies specific for CAB39L (sc-100390), β-catenin (sc-133240), Rab-3D (sc-26559), Rap-1B (sc-1481) and PGK1 (sc-130335) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A monoclonal mouse antibody against β-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA). The SAP kit and AP-Red kit were provided by Zhongshan Co. Beijing, China (SAP-9102, ZLI-9042). The lentivirus packaging system and enhanced infection solution (ENi.S) were purchased from GeneChem Limited Company (Shanghai, China). The SYBR Green PCR kit and SYBR Master Mixture were purchased from Takara Bio, Inc. (Dalian, China). The Endo-free Plasmid Mini kit was purchased from Qiagen, USA (12163). M-PER® Mammalian Protein Extraction Reagent was purchased from Pierce, Rockford, IL, USA (78503).
Western blot analysis
Cells were collected at a concentration of 2×107/ml. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene fluoride membranes, which were incubated with the primary antibody CAB39L (1:200). Western blot analyses were performed according to standard methods. The protein bands were visualized by applying SuperSignal West Pico Chemiluminescent Substrate (34079; Pierce). The exposed film was then analyzed using a densitometer.
Immunohistochemistry and immunofluorescence
For analysis of the subcellular localization of MLAA-34, U937 cells were washed with ice-cold PBS, blocked with 10% normal goat serum and incubated with a primary antibody against CAB39L at a dilution of 1:50 for 2 h at 37°C. Next, the cells were washed again and incubated with the appropriate biotinylated secondary antibody (goat anti-mouse IgG antibody) for 20 min at 37°C. Incubation with serum alkaline phosphatase (SAP; ALP) was then performed at 37°C for 20 min, and the immunolabeling was visualized with a mixture of AP-Red solution. Counterstaining with hematoxylin was performed. For immunofluorescence, the cell samples were incubated with the monoclonal antibody CAB39L (diluted 1:50) and fluorescein isothiocyanate (FITC)-labeled or rhodamine-labeled goat anti-mouse IgG as the primary and secondary antibodies, respectively. The mounted cells were visualized with a fluorescent microscope.
Construction and identification of the MLAA-34 lentivirus vector and upregulated MLAA-34 stably transfected cell line
The full-length MLAA-34 cDNA sequence was assembled by searching the NCBI database and amplified by RT-PCR from U937 cells. First-strand cDNA synthesis was performed using a commercial kit (Boehringer Mannheim, Milan, Italy). The restriction enzyme site for AgeI (ACCGGT) was introduced into the 5′ and 3′ PCR primers. To generate cDNA coding for full-length MLAA-34 by PCR, the following primers were designed using plasmid MLAA-34 as the template: MLAA-34-Age, I-F, GAGGATCCCCGGGTACCGGTCGCCACCATGAAAAAAATGCCTTTG and MLAA-34-Age, I-R, TCACCATGGTGGCGACCGGAGGGGCCGTTTTCTTCAAG. The PCR conditions consisted of 30 cycles, and the cycle parameters were: 94°C for 5 min, then 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 68°C for 1 min, followed by a final extension of 68°C for 10 min. The PCR product was purified using an Agarose Gel DNA Purification kit (Takara Bio, Inc.). The two recovered products were ligated using an In-Fusion kit (631774; Becton, Dickinson and Co., USA). To confirm that the ligation was correct, MLAA-34-SEQF, GACAGATAGGCACTCGGAG; Ubi-F, GGGTCAATATGTAATTTTCAGTG; and EGFP-N-R, CGTCGCCGTCCAGCTCGACCAG primers were designed. The cycle parameters were: 30 cycles of 94°C for 30 sec, 94°C for 30 sec, 60°C for 30 sec, 72°C for 50 sec, followed by a final extension of 72°C for 6 min. For detection of MLAA-34 expressed by recombinant lentivirus in vitro, purified pGC-FU-MLAA-34 vectors were transfected into 293T cells using Lipofectamine 2000 reagent (11668-019; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. This vector was termed MLAA-34-Lentivirus, and the vector without MLAA-34 cDNA was pGC-FU-GFP-LV. The titer of the recombinant lentivirus was determined by real-time qPCR on 293T cells. For identification of the recombinant MLAA-34 lentivirus vector, the virus was added to targeted U937 cells at multiplicity of infections (MOIs) of 10, 20, 50, 80, 100, 120 and 200 with ENi.S and 5 μg/ml polybrene. MLAA-34-Lentivirus and pGC-FU-GFP-LV transfected U937 cells were used as the test, and non-transfected cells were used as the control. The expression level of MLAA-34 was detected by western blot analysis and RT-PCR. The best MOI was chosen.
Cells were grown in selective media (containing G418) for two weeks, expanded and grown as independent clones for at least two weeks. Resistant colonies were counted, and the expression of GFP was confirmed by fluorescence microscopy, RT-PCR and western blot analysis.
Fluorescence microscopy, MTT, flow cytometry and DNA ladder
To determine the effect of upregulation of MLAA-34 by the MLAA-34-Lentivirus, non-transfected cells and cells transfected with pGC-FU-GFP-LV and MLAA-34-Lentivirus were examined. Cells were seeded in 96-well plates at a density of 1×104 cells/well. Cellular proliferation was measured once per day during a seven-day period. In brief, 20 μl of sterile MTT (Sigma) dye (5 mg/ml) was added to the cells, which were then incubated for another 4 h at 37°C. Then, 150 μl of dimethylsulfoxide was added to each well. The spectrophotometric absorbance was measured at a wavelength of 490 nm on an enzyme immunoassay analyzer.
Fixed cells were stained with 2.5 g/ml of DAPI (4′,6-diamidino-2-phenylindole) solution to detect apoptotic nuclei. Quantification of apoptosis was determined by counting the number of apoptotic cells. The cells were stained using an Annexin V-PE/7-AAD apoptosis detection kit (KGA1015; Nanjing KeyGen Biotech. Co., Ltd.) according to the manufacturer’s instructions and were analyzed by flow cytometry using a Beckman Coulter flow cytometer.
For cell cycle analysis, the cells were fixed in 70% ethanol and stained with propidium iodide (PI; Biosea Biotechnology Co., Beijing, China) at a final concentration of 20 μg/ml in Triton X-100 containing 10 mg/ml RNase. Following incubation, the samples were analyzed on a flow cytometer.
Fragmented DNA was isolated using a DNA extraction kit (C0008; Beyotime) according to the manufacturer’s instructions. The eluants containing DNA pellets were electrophoresed on a 1% agarose gel at 80 V for 1.5 h. The gel was examined and photographed using an ultraviolet gel documentation system.
Co-immunoprecipitation (Co-IP) and SDS-PAGE
Co-IP was performed using a Profound™ Mammalian Co-IP kit (23605; Pierce). Transfected U937 cells (2×107/ml) were washed, centrifuged and resuspended in lysis buffer for incubation. The cell lysates were centrifuged to remove the supernatant material, and the CAB39L antibody was cross-linked to the antibody coupling resin. The lysed cell sample was then applied to the antibody support to form immune complexes. Then, unbound proteins were washed away three times. The samples were then eluted, and coupling buffer was added to obtain the immunoprecipitated protein. Finally, the Co-IP protein concentrations were determined using a BCA Protein Assay kit (23225; Pierce). The proteins were analyzed by SDS-PAGE, and the gel was stained with Coomassie Blue.
Mass spectrometry analysis (MS, shotgun) and protein identification
After separation by SDS-PAGE, discrete bands were excised from and subjected to in-gel tryptic digestion. The extracted peptides were analyzed using shotgun HPLC-ESI-MS proteomics approach (LTQ; Thermo Finnigan, San Jose, CA, USA). High-performance liquid chromatography (HPLC) separation was performed with a capillary LC pump. The mobile phases used for the reverse phase were i) 0.1% formic acid in water, pH 3.0; ii) 0.1% formic acid in ACN. The collision energy was set automatically by the LTQ system. Following acquisition of full scan mass spectrum, three MS/MS scans were acquired for the next three most intense ions using dynamic exclusion. Peptides and proteins were identified using Bioworks Browser 3.1 software (Thermo Finnigan), which uses the MS and MS/MS spectra of peptide ions to search against the NCBI human protein database. The protein identification criteria that we used were based on Delta CN (≥0.1) and Xcorr (one charge ≥1.9, two charges ≥2.2, three charges ≥3.75). The protein identification results were extracted from the SEQUEST out file with in-house software (BuildSummary). The cellular localization, molecular function and biologic process were determined using the gene ontology annotation DAVID (http://david.abcc.ncifcrf.gov/). For pathway analysis, the KEGG database was searched. To identify the corresponding proteins in mixed protein obtained by Co-IP, western blot analysis was performed as previously described.
Statistical analysis
The RT-PCR results were analyzed by the self-contained software of iQ5 (Bio-Rad Co.). Statistical analyses were performed using an analysis of variance (ANOVA). All results are expressed as the means ± standard deviations from at least three experiments. P<0.05 was considered to indicate statistically significant differences.
Results
Expression of human MLAA-34 protein
With western blot analysis, a strong specific band of ~39 kDa was observed in U937 and MHCC97-H cells, and reduced expression was observed in other leukemia or lymphoma cell lines and PBMCs from leukemia patients. Much fainter bands were observed in solid tumor cell lines, and no expression was detected in normal human cell lines or primary animal cells (Fig. 1).
Identification and cellular localization of MLAA-34
Immunohistochemical staining confirmed the presence of MLAA-34 in U937 cells and the subcellular localization was detected primarily in the cytomembrane and cytoplasm (Fig. 2).
MLAA-34 is upregulated by the lentiviral vector
A human MLAA-34 lentivirus gene transfer vector encoding the green fluorescent protein (GFP) sequence was constructed. The pGC-FU-MLAA-34-GFP plasmid has an insert of ~771 bp, which is in accord with the MLAA-34 cDNA [identities, 1009/1012 (99%)]. The pilot experiments showed that 293T cells could be successfully infected by the packaged virus; the virus titer reached higher than 2×108 TU/ml, indicating that a high-titer lentiviral packaging platform was preliminarily established. The pGC-FU-MLAA-34-GFP plasmid was confirmed by western blot analysis. MLAA-34-Lentivirus and control pGC-FU-GFP-LV virus were produced. After obtaining ideal U937 cells, we transfected the cells with the MLAA-34-Lentivirus and pGC-FU-GFP-LV viruses at different MOIs. The transfection efficiency was ~95% or higher on Day 5 or later at the MOI of 50 (Fig. 3A). Five days after transfection, the recombinant MLAA-34-Lentivirus caused a pronounced increase in the expression of MLAA-34 compared with non-transfected U937 cells (Fig. 3B).
Establishment of U937 cell line stably overexpressing MLAA-34
In preliminary studies, 400 μg/ml of G418 were found to maintain adequate selection pressure. The expression of GFP and MLAA-34 were observed. After the cells had been frozen in liquid nitrogen for six months and revived monthly, the U937 cells expressed higher levels of MLAA-34 in ~400 μg/ml of G418, and ~95% of the lentivirus-transfected U937 cells overexpressed MLAA-34. These results suggested that the stably transfected U937 cell line was successfully established by lentivirus and that the expression of MLAA-34 can be long lasting even after passage.
Effect of upregulating MLAA-34 on apoptosis and growth of U937 cells
Observations of morphology revealed increasing cell shrinkage, nuclear condensation and fragmentation in non-transfected and pGC-FU-GFP-LV transfected cells. By contrast, cells transfected with MLAA-34-Lentivirus predominantly appeared uniformly stained without condensation (Fig. 3C). These results further support the findings that anti-apoptotic changes in the cell and nuclear morphology are induced by MLAA-34 overexpression. MTT assays suggested that the lentiviral overexpression of MLAA-34 induces anti-apoptotic effects that result in a promotion effect on U937 cells; these data suggest that MLAA-34 might accelerate cell proliferation (Fig. 3D). In agreement with the anti-apoptotic effects of MLAA-34, cells overexpressing MLAA-34 accumulated in the S-phase (~67.63% compared with ~49.6% of cells in the S-phase in the control) and showed a corresponding increase in cell numbers in the G2/M phase. The percentages of early (lower right) and late apoptotic (upper right) cells were markedly reduced in U937 cells after transfection with MLAA-34-Lentivirus (Fig. 3E). These results are in agreement with the DNA ladder assay and are even more evident at the MOI=50, in which the cells transfected with MLAA-34-Lentivirus showed a further increase. All of these results suggest that MLAA-34 inhibits apoptosis in U937 cells.
Co-IP, shotgun and western blot analysis
Protein extracts with Co-IP were separated by SDS-PAGE and the gel was cut into four pieces for shotgun ESI-MS analysis (Fig. 4A). A total of 256 proteins were identified by the LC ESI-MS analysis and BIOWORKS in the NCBI HUMAN protein databases, of which 225 (87.9%) proteins were annotated by DAVID and the remaining 31 (12.1%) proteins have no DAVID terms (Table I). The expression of Rap-1B, Rab-3D, β-catenin and PGK1 was verified by western blot analysis (Fig. 4B).
Classification of the 225 annotated proteins in terms of molecular function, biological process and cellular localization was performed according to the DAVID. Molecular function was clustered and the protein binding (140, 41.8%) and nucleotide binding (60, 17.9%) groups were the majority (Fig. 4C). For biological processes, annotated proteins are particularly involved in the cell process (167, 27.5%) and the multicellular organismal process (75, 12.4%) (Fig. 4D). Most (172, 18.6%) of the annotated proteins were localized in the intracellular (Fig. 4E). Distribution of molecular mass and isoelectric points (PI) of the annotated proteins was analyzed. Molecular mass ranged between 10.19 and 620.42 kDa in size, most of them were between 10 and 60 kDa (Fig. 4F). PI of the proteins ranged between 4.35 and 11.05 with the most PIs between four and ten (Fig. 4G). To uncover the signaling pathways of the 225 annotated proteins, the protein sequences were searched against the KEGG reference pathway database. The pathways were ascribed to metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems and human diseases (Fig. 5). Among them, the immune system, cancer and signal transduction were more than others. On the other hand, the specific expressed proteins related pathways displayed more differences and 71 proteins were involved in cell apoptosis or proliferation biological processes and KEGG pathways (Table I).
Figure 5Categories of related pathways in 225 annotated proteins according to KEGG pathway taxonomy. |
Discussion
In this study, to evaluate the function of MLAA-34 in M5 cells, we used the well-characterized cell line U937. In our previous research, we reported that the MLAA-34 protein is probably a cytoplasmic protein predicted by the amino acid sequence analysis of the encoded protein (7). Here, we verified that MLAA-34 is localized in the cytoplasm and cell membrane. Western blot analysis showed that the expression of MLAA-34 differed between different cell types and was observed to be stronger in U937. Although U937 cells are generally difficult to transfect, the U937 cells were transfected with MLAA-34-Lentivirus and pGC-FU-GFP-LV. A stably transfected U937 cell line was successfully established and expressed MLAA-34 at a high level, which aided in the study exploring the effect of MLAA-34 on M5 and will be critical for further research using U937 cells and animal models. In addition, an analysis of the cell morphology, apoptosis, proliferation and cell cycle revealed that the overexpression of MLAA-34 markedly inhibited apoptosis of U937 cells. These results suggested that MLAA-34 maybe a novel anti-apoptotic factor of M5, which is consistent with the RNAi in our previous study.
The proteins that interact with MLAA-34 or CAB39L remain unclear. To analyze complex mixtures of proteins, shotgun is considered the most powerful (9,10). Using the MLAA-34 protein as bait, 256 proteins were identified and 225 of them have DAVID terms. Among these proteins, 71 proteins correlated with cell apoptosis or proliferation biological processes and KEGG pathways. Twenty-eight proteins are involved in cell apoptosis or proliferation; nine proteins are associated with the calcium signaling pathway and seven proteins participate in the chemokine signaling pathway; 17 proteins are concerned with the Ras signaling transduction pathway and 8 proteins are concerned with Wnt signaling pathway. The Ras, Wnt, calcium and chemokine signaling pathways may be involved in anti-apoptosis with MLAA-34 in U937 cells. As is known, the Ras family plays an important role in the molecular pathogenesis of myeloid leukemia, and Ras mutations have been preferentially associated with monocytic subtypes in AML (11). The Ras and Wnt signaling pathways are known to be key anti-apoptosis pathways in AML-M5 (12). Understanding the molecular genetics of leukemia has led to an appreciation that particular molecular abnormalities give rise to specific subtypes of the disease. For example, in myeloid leukemogenesis, PML-RAR-α and BCR-ABL are defining features of acute promyelocytic leukemia and chronic myeloid leukemia, respectively (13). In this case, MLAA-34 may either play an important role in leukemogenesis or play a dual role in subsequent differentiation, as in the case of PML/RAR. The results suggest that MLAA-34 might be an important agent for subtype diagnosis in AML. However, an understanding of how these identified proteins or pathways interact with MLAA-34 requires further study.
In addition to the typical pathways such as pathways in cancer and apoptosis, there were several notable pathways such as the GPCR signaling, the insulin signaling pathway, the ErbB signaling pathway, the NOD-like receptor signaling pathway, the Ahr signal transduction pathway, the AKT signaling pathway, the Toll-like receptor signaling pathway, the RIG-I-like receptor signaling pathway, the ubiquitin mediated proteolysis, the hedgehog signaling pathway, the phosphatidylinositol signaling system, the PPAR signaling pathway, the VEGF signaling pathway and the TGF-β signaling pathway worthy of further validation (Table I). Otherwise, there are some proteins mainly involved in tumorigenesis concerned with MLAA-34 as discussed below. PGK1 is secreted by tumor cells and may play a role in inhibiting tumor angiogenesis (14). GAPDH has been shown to be upregulated in several types of cancer and downregulated by chemotherapeutic drugs, and could be considered a potential target to observe the effects of bisphosphonates on cancer cells (15). In addition, GAPDH was the best control gene in the apoptosis pattern on the myeloid cell lines (16). CRMP1 is a suppressor of tumor cell invasion of the local stroma and might be a functional modulator of the Wnt signaling pathway in vivo(17,18). As the trigger of TBK-1 pathway, TBK1 is important for tumor angiogenesis and tumor-associated microvascular inflammation and expressed at significant levels in many solid tumors (19,20). A recent study has demonstrated that SEPT7 could function in gliomagenesis and in the suppression of glioma cell proliferation (21).
Markedly, some p53 or caspase-related proteins were also identified, such as CLTC, PPP2CA, SOD2, PARK7, HSPA9, TXN, ESR1 and YWHAE. CLTC associates with p53 not only in nuclei but also in cytosol, and co-localizes with p53 at the plasma membrane in human cancer cells (22). CLTC expression enhances p53-dependent transactivation (23). As a downstream mediator of the antiproliferative effects of PPP2CA, p53 plays an important role in PPP2CA-directed cell cycle arrest and apoptosis (24). The SOD2 growth-retarding functions are at least partially due to triggering of a p53-dependent cellular senescence program (25). DJ-1 (PARK7) bound to p53 in vitro and in vivo and they were found colocalized. DJ-1 positively regulates p53 through Topors-mediated sumoylation (26). Previous studies indicated that HSPA9 could bind to p53 and sequesters it in the cytoplasm, thus providing a mechanism of inactivation of wild-type p53 and contributing to human carcinogenesis (27,28). Additional studies have shown that TXN induces p53 DNA binding activity in vitro and enhances p53-dependent expression of its target gene p21 and DNA repair genes (29). Additional studies also indicated that caspases could be activated by TXN due to its disulfide reducing properties (30). ESR1 might activate caspases-8, −9 and −3 and induce tumor cell apoptosis, it also showed the downregulation of β-catenin signaling implicating the suppression of proliferation and metastasis of tumor cells (31,32). The cleavage of YWHAE by caspase-3 during apoptosis might contribute to cell death by preventing the association of YWHAE with Bad (33). The key event during apoptosis that is common to all pathways is the activation of caspases. P53 is a well-known tumor suppressor gene, and mutational inactivation of p53 function or deletion of the gene increases susceptibility to cancer (34–37). On the basis of these findings, we will further study the interaction between MLAA-34 and caspases or p53 to investigate the anti-apoptotic mechanisms of MLAA-34 in U937 cells.
To our knowledge, this is the first report showing the cellular localization and expression of MLAA-34 in U937 cells. We have demonstrated for the first time that the overexpression of MLAA-34 by lentivirus can significantly suppress the apoptosis of U937 cells, and a cell line stably overexpressing MLAA-34 was successfully established. Another key finding of this study is the information from proteomics evidence that MLAA-34 may be a tumor-correlated gene, and this is the first time it is revealed that the preliminary framework of proteins and pathways interlink with MLAA-34 in U937. Furthermore, it will be essential to integrate data from many different sources to obtain an accurate understanding of MLAA-34 protein networks.
Gene therapy remains the most promising, if not the only, approach to treating genetic diseases. An example of this is the use of rituximab for the treatment of lymphoma and other types of cancer. Rituximab is a mouse/human chimeric IgG(1)-κ monoclonal antibody that targets the CD20 antigen found on the surface of malignant and normal B lymphocytes (38). Most cellular processes are performed by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (the proteome) is organized into functional units (39). Nevertheless, for a viable clinical approach, extensive research is needed in the future to regulate the expression of the target gene and improve its safety.
In conclusion, our current results provide new evidence that MLAA-34 may be a novel anti-apoptotic factor in vitro, and the data presented here show a strong correlation between anti-apoptosis with the upregulation of MLAA-34. In addition, preliminary proteomic analysis suggests that a number of genes belonging to different signaling pathways may be involved in apoptosis in U937 cells in association with MLAA-34, which would disclose a novel cross-link between MLAA-34 and the Ras, Wnt, calcium and chemokine signaling pathways. Findings of the present study will lead to a better understanding of the mechanisms involved in M5, and MLAA-34 may serve as a potential novel marker for the early diagnosis and gene therapy of M5.
Acknowledgements
This study was supported by the National Natural Science Foundation of China under award nos. 30971284, 81000219 and 18110021.
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