B-cell acute lymphoblastic leukemia (B-ALL) is the most commonly diagnosed childhood malignancy. Worldwide, B-ALL accounts for 85% of ALL cases [1
]. The five-year survival rate is >90%, and the cure rate is ~80% in developed countries [2
]. However, the cure rates are very low in developing countries [3
]. Several factors are associated with leukemia, including risks associated with clinical classification, cytogenetic alterations, and delayed diagnosis. Genetic alterations are also associated with ALL; however, the association between ALL and molecular alterations such as differential gene expression, transcriptional regulation, and epigenetic modifications is unknown. Therefore, identifying molecules that drive malignancy is a current challenge to improve the diagnosis, prognosis, treatment and understanding of leukemia biology.
Transcriptional regulation in eukaryotes is orchestrated by a wide variety of molecules, including DNA, RNA and proteins. Many of these proteins are involved in DNA binding and promote the regulation of gene expression. The zinc finger (ZNF) protein family is the largest family of DNA-binding proteins in mammals. The ZNF proteins have a large number of motifs that include Cys2-His2, GATA, RanBP, A20, LIM, MYND, RING, PHD, and TAZ. Of these, the most common motifs are the Cys2
domain-containing ZNF proteins [4
]. On the other hand, another conserved domain present in one-third of all ZNF proteins [6
] is Krüppel-associated box (KRAB) [7
]. The coding sequences of approximately 50% of ZNF proteins with a KRAB domain (ZNF-KRAB) are located on chromosome 19q13 [8
]. However, this cytogenetic location is not exclusive to ZNF-KRAB proteins. ZNF-KRAB proteins play several roles, including regulating gene expression mediated by RNA polymerases (Pol I, II, and III) [9
], binding to transcriptional repressors [10
], and regulating splicing [17
]. The KRAB domain is transcribed by independent exons, an advantage for its nascent transcript, which can undergo alternative splicing (AS) [11
], thus increasing the diversity of transcripts and the resulting proteins. However, the diversity of mRNAs encoding ZNF-KRAB proteins and the expression of these mRNAs in health or disease status are unknown.
In cell biology, many forms of processing occur by AS, including alternative 5′ splicing, alternative 3′ splicing, exon skipping, intron retention, mutually exclusive exon selection, and exon scrambling [29
]. AS is regulated by specific sequences, including sites delimited by specific sequences called intronic definition (ID) and exonic definition (ED) elements [30
] and a regulatory system comprising serine/arginine (SR)-rich proteins [30
] and heterogeneous nuclear ribonucleoprotein particle (hnRNP) A/B proteins [31
]. Specific pre-mRNA sequences play an important role as enhancers and silencers and can be classified as exonic splicing enhancers/silencers (ESEs/ESSs) or intronic splicing enhancers/silencers (ISEs/ISSs) according to their locations [32
]. Diverse reports have shown that alterations in ID elements [30
] as well as mutations in ESEs, ESSs, ISEs, and ISSs can promote intron/exon retention, skipping [34
], or both.
Aberrant AS contributing to structural protein variations results in functional and nonfunctional end products [38
], and aberrant AS has been associated with diverse cancer-associated processes, including cell death resistance, angiogenesis induction, genomic instability, tumor development promotion [38
], and cancer progression [39
]. The contribution of aberrant protein expression to the diversity of the cancer proteome and the functional impact of these proteins is unclear, and the diversity of alternative transcripts expressed is unknown. However, 98% of the human transcriptome is noncoding RNA. Noncoding RNA is divided into short noncoding RNA and long noncoding RNA (lncRNA). lncRNAs are transcripts with a full length of greater than >200 nucleotides [41
]. These transcripts are classified based on their location and include intergenic, intronic, intronic antisense, overlapping, and overlapping antisense transcripts [42
]. The expression of lncRNAs is highly spatially and temporally restricted [43
]; some cellular compartments can be enriched in specific lncRNAs. Moreover, lncRNAs can exhibit very diverse activities, acting in the cytoplasm and the nucleus as chromatin regulators, RNA-binding proteins, promoters, or enhancers [44
]. However, the diversity and functions of lncRNA expression are unclear.
Using high-density microarrays, we previously employed ovarian normal and tumor tissues to identify AS of ZNF695. Our findings showed two new alternative mRNA splice variants associated with ovarian cancer [46
]. However, the full-length sequence was not evaluated. In this work, we employed HeLa, MCF7, RS4 and SUP-B15 cells to evaluate the full-length transcript variants of ZNF695. Our findings showed the coexpression of six alternative transcripts of the ZNF695 gene. Additionally, we identified the prevalence of ZNF695 transcript variant 3 (a lncRNA) in B-ALL patients and the ability of this variant to predict overall survival.
2. Materials and Methods
2.1. Cell Lines and Growth Conditions
Cells were cultured at 37 °C and 5% CO2 in their preferred medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The following culture media were used: HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies, Carlsbad, CA, USA); MCF-7 and RS4 cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA); and SUP-B15 cells were cultured in Iscove’s modified Dulbecco’s medium.
2.2. Patients and Ethics Statements
Bone marrow samples were obtained from patients with a diagnosis of ALL who had previously provided signed informed consent, and the protocol was approved by the Institutional Ethics Committee (INP protocol 060/2016) and was in accordance with the Declaration of Helsinki. Leukemia cells were isolated using Lymphoprep density gradient medium (STEMCELL Technologies, St. Kent, WA, USA). According to the protocol, PBS was added to bone marrow (1:1, v/v), and the mixture was transferred to 3 mL of Lymphoprep and centrifuged at 1500 rpm for 30 min. Leukemia cells retained in the interface were transferred to a new tube and diluted with PBS (1:1, v/v). The cell suspension was gently homogenized by inversion and centrifuged at 3000 rpm for 5 min. Recovered cells were stored at −70 °C until they were used for nucleic acid purification.
2.3. RNA Purification and Reverse Transcription
Total RNA was purified from cultured cell lines and leukemia cells. Briefly, for cell lines, 1 mL of TRIzol reagent (Ambion, Life Technologies, Carlsbad, CA, USA) was added to the culture dishes, and the cells were scraped and collected in a 1.5 mL tube. Cells were disrupted with a TissueLyser at a frequency of 25/s for 30 s, and RNA purification was performed following the TRIzol manufacturer’s recommendations. Finally, RNA was quantified using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cDNA synthesis, 1 µg of total RNA was digested with DNase and incubated at 37 °C for 30 min, and 1 µL of 5 mM EDTA was then added to stop reaction the at 65 °C for 10 min. The cDNA synthesis reaction contained the following components: 1X RT buffer, 10 U Transcriptor Reverse Transcriptase (Sigma-Aldrich), 0.4 µM random primers, 1 mM dNTPs, and 20 U RNaseOut (Thermo Fisher Scientific). The reaction mixture was incubated for 10 min at 25 °C, for 30 min at 55 °C, and for 5 min at 85 °C.
2.4. Rapid Amplification of cDNA 3′ Ends (3′ RACE)
The 3′ RACE procedure was performed using a 3′ RACE System for Rapid Amplification of cDNA Ends Kit (Thermo Fisher Scientific) according to the protocol. cDNA synthesis was performed using 5 µg of total RNA. RNA was subjected to DNase treatment as previously described. After adding 10 µM adapter primer (AP), the mixture was placed in a thermal cycler for 10 min at 70 °C and was then transferred to ice for one minute. Finally, this mixture was added to a mixture containing 1X PCR buffer, 25 mM MgCl2, 50 mM dNTP mix, and a final concentration of 0.5 M DTT, and the mixture was placed in a thermal cycler for 5 min at 42 °C. Then, 200 U of SuperScript™ II Reverse Transcriptase was added, and the mixture was incubated for 50 min at 42 °C and 15 min at 70 °C and then placed on ice for one minute. Finally, 2 U of RNase H was added, and the mixture was placed in the thermal cycler for 20 min at 37 °C (Thermo Fisher Scientific).
2.5. PCR and Sequencing
PCR was performed using 25 ng of synthesized cDNA, and the reaction mixture contained 0.14 U Fast HotStart DNA Polymerase (KAPA2G, Kapa Biosystems, Wilmington, DE, USA), 0.2 mM dNTP mix, 1.5 mM MgCl2
, 5 µM forward primer, 5 µM reverse primer, and nuclease-free water up to 10 µL. The reaction mixture was incubated for 1 min at 95 °C, 15 s at 95 °C, 15 s at Tm
(the melting temperature of each primer is shown in Table 1
), 15 s at 72 °C, and 7 min at 72 °C for the final extension.
PCR products were purified using a Zymoclean™ Gel DNA Recovery Kit (ZYMO Research, Irvine, CA, USA) according to established protocols. After that, PCR products were sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA), according to established protocols. The master mix was placed in a Proflex thermal cycler for 25 cycles: 30 s at 95 °C, 15 s at 50 °C and 4 min at 60 °C. The samples were sequenced using an Applied Biosystems ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Finally, the sequences were analyzed using UGENE v1.23.1. The resulting sequences were aligned using Clustal Omega (Clustal Omega, EMBL-EBI, Cambridge, UK). The following reference sequences were used: ZNF695 transcript variant one (ZNF695_TV1, NM_020394.4), ZNF695 transcript variant two (ZNF695_TV2, NM_001204221.1), and ZNF695 transcript variant 3 (ZNF695_TV3, NR_037892.1, lncRNA).
2.6. Statistical Analysis
The clinical characteristics are summarized as absolute values and relative frequencies. Kaplan-Meier curves were generated to determine the survival and relapse rates. The differences in the survival rates among the ZNF695 transcript variants were determined by the Wilcoxon test. We considered p values of < 0.05 to indicate significant differences. Statistical analysis was performed using the commercial statistical package JMP11 from SAS Institute, Inc.
AS plays an important role in cell biology. However, little is known about the diversity of the transcripts expressed in several tissues and human diseases, including cancer. Several AS transcripts contribute to protein diversity [47
], but the diversity and function of RNA splice variants are unknown. In cancer, many AS transcripts encode proteins that contribute to the pathogenesis of the disease, conferring a gain, loss or change of function to the encoded protein. Tumor cells exhibit extensive dysregulation of normal biological processes. In these cells, AS is known as aberrant splicing, and this process is apparently a consequence of malignant transformation. Aberrant splicing occurs frequently in several types of cancers [48
]. However, identifying AS events is very difficult because AS patterns vary widely and include events such as alternative 5′ and 3′ splicing, exon skipping, and intron retention.
The molecular diagnosis of cancer is a serious challenge to modern medicine. Identifying the molecular markers that can predict prognosis, in addition to assigning specific treatments to oncology patients such as ER/PR-positive and ER/PR-negative breast cancer patients, is difficult [49
]. The genomic tools applied in cancer have revealed a comprehensive approach for detecting deregulation of the human transcriptome. We used high-density microarrays to identify new patterns of AS in ZNF695 transcripts [46
] and performed a simple procedure to evaluate the expression of the ZNF695 transcript variants. We designed specific primers to evaluate the new isoforms identified in a previous report [46
]. Surprisingly, we observed the co-expression of six alternative transcript variants in cancer cell lines and leukemia patients.
The complete sequence of the human transcriptome is unknown, but a gene:transcript ratio of 1:7 has been suggested [50
]. However, the diversity of the human transcriptome in healthy and disease states is unclear. ZNF695 transcript variants showed very low expression in healthy ovarian tissue [46
] or healthy lymphoid cells (Figure 5
C). We believe that the expression of ZNF695 increases during carcinogenesis and the subsequent generation of aberrant alternative splice variants. Moreover, we found nine patterns of AS in association with the ZNF695 transcript variants expressed in leukemia patients (Figure 7
We think that transcript variants one and three are regulated differentially because in some samples, the expression of these transcript variants is mutually exclusive. Thus, our results suggest two regulatory mechanisms for ZNF695 gene expression and AS. In contrast, ZNF695_TV4, ZNF695_TV5, ZNF695_TV6, and ZNF695_TV7 were not expressed alone (Figure 7
). To date, no previous studies have identified and quantified the expression of novel ZNF695 transcript variants in childhood leukemia. Some hypotheses suggest that the diverse expression patterns result from the heterogeneity of tumor samples. We discovered four novel ZNF695 alternative transcripts that are co-expressed in cell lines and leukemia patients. The results of the sequence-based bioinformatic analysis for identify the initiation codons in the cDNA sequence using the ATGpr program available in http://atgpr.dbcls.jp
] website suggested that ZNF695_TV1, ZNF695_TV4, and ZNF695_TV5 are coding RNAs, while ZNF695_TV3, ZNF695_TV6, and ZNF695_TV7 are lncRNAs. The diversity of these alternative transcripts of ZNF695 suggests differential functions, and changes in the resulting sequence are known to promote changes in the function of the resulting protein via topological changes, additionally recapitulating cancer-associated phenotypes such as angiogenesis promotion [52
], proliferation [53
], and apoptosis avoidance [54
Aberrant expression of alternative splice variants is a common event in cancer and is likely generated from somatic mutations [55
] or changes in the expression of the AS-associated proteins; however, this observation does not clarify the function of most of these resulting transcripts. The ZNF695 protein has been evaluated in breast cancer; interestingly, ZNF695 expression could classify the nonluminal A and luminal B subtypes [56
]. We evaluated ZNF695 expression in B-ALL and found some expression patterns. No specific antibodies against the ZNF695 proteins resulting from AS have been developed. Moreover, the function of ZNF695 is unclear. However, methylation-mediated silencing confers a complete therapeutic response in primary esophageal squamous cell carcinoma tumors [57
], suggesting that in normal cells, the ZNF695 gene is methylated and, consequently, unexpressed. Our results showed that some samples were positive (n = 26) and others were negative (n = 17) for ZNF695 expression; the negative samples were likely methylated. These variants are likely unmethylated in patients who express ZNF695 transcript variants, and the prognosis and survival of these patients are poor (Figure 8
B). Indeed, Li C et al. observed a strong correlation between the mRNA expression of ZNF695 and adverse prognosis in adult ALL [58
]. These results are very interesting and suggest that the expression of ZNF695 could be advantageous in malignances, probably via negative regulation of tumor suppressor genes.
The ZNF695 gene codes for three transcripts. In the full-length ZNF695 protein (ZNF695_TV1), which includes four exons, the terminal exon corresponds to the DNA-binding domain. However, the binding sites of the ZNF695_TV1 protein are unknown. The ZNF695_TV1 protein contains a KRAB-containing ZNF domain and belongs to a large family of proteins present in many species [12
]. ZNF proteins that contain a KRAB domain function as transcriptional repressors [19
]. Some KRAB proteins can bind to RNA and interact with RNA polymerase II [59
]. We believe that the ZNF695_TV1 protein plays a role as a transcriptional repressor; however, we do not know the mechanism by which this repressive activity occurs. Moreover, we detected partial loss of the KRAB domain in ZNF695_TV4 and ZNF695_TV5, suggesting a change in the repressive function. However, the protein products of ZNF695 AS cannot be detected because there are no specific antibodies against these proteins.
Approximately 90% of the human genome transcribes noncoding RNAs [60
], of which lncRNAs are a subcategory. Noncoding RNAs are classified based on the number of nucleotides and include short and long forms [62
]. lncRNAs can be classified as intergenic, overlapping, sense, and antisense according to their location. We observed that ZNF695_TV3 is an overlapping noncoding RNA, and based on its size, it can be classified as a lncRNA. However, the function of this transcript is completely unknown. A significant relationship between ZNF695_TV3 expression and patient survival was identified (Figure 8
A). The expression of the lncRNA ZNF695 transcript in cancer has not been reported. Moreover, several studies have suggested that lncRNAs regulate gene expression to mediate the interaction with chromatin and could play several roles, including oncogenic roles, as has been indicated for lncRNAs such as HOTAIR [64
], MALAT1 [65
], SPRY4IT1 [66
], and H19 [67
]. Our results are the first to show the expression of lncRNA ZNF695_TV3 and the role of this transcript as a predictor of survival in B-ALL patients. However, more extensive studies are necessary to identify the function of ZNF695_TV3.