Screening of Translocation Ets-Leukemia-Acute-Myeloid-Leukemia-1 Fusion Gene and Expression Pattern of Multidrug Resistance Protein Protein in Children with Acute Lymphoblastic Leukemia

The study aimed to investigate the positive rate of TEL-AML 1 fusion gene in acute lymphoblastic leukemia (ALL) children and the clinical characteristics of ALL patients with TEL-AML 1 fusion gene positively expressed, as well as the expression level of MRP-1. 40 ALL children were selected, with their medical records collected. The TEL-AML 1 fusion gene was screened by nested RTPCR. Bone marrow specimens were taken for G-banded karyotype analysis and flow cytometry immunophenotyping of the marrow chromosome. A semi-quantitative RT-PCR method was used to study the mRNA expression level of MRP 1. The results showed that the positive rate of TEL-AML 1 fusion gene in ALL patients was 22.5% (9/40). The positive group exhibited lower gene expression level, the hepatosplenomegaly degree, the total number of peripheral white blood cells, the absolute count of naive cells, and the Hb level at the first visit, indicating that the tumor burden of children in the positive group was lower. The complete remission rate of the positive group was higher (P < 0.05). The mRNA expression level of MRP 1 gene positive group was lower. In conclusion, patients with positive TEL-AML 1 fusion gene were more sensitive to chemotherapeutic drugs, and their treatment responses and prognosis were better.

Adoption of Biomedical Ceramic iRoot BP in the Treatment of Localized Pulpitis in Children

To explore the clinical efficacy of biomedical ceramic iRoot BP in the treatment of localized acute pulpitis in children, and the effect of iRoot BP on proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs), 72 localized acute pulpitis children admitted to our hospital from September 2018 to September 2019 were selected and divided into group A (treated with MTA pulp capping material) and group B (treated with iRoot BP material), and the clinical effect, pain degree, and adverse reactions (ADR) rate were compared. The effects of iRoot BP on hDPSCs proliferation and osteogenic differentiation were analyzed; the proliferative activity of cells in iRoot BP group, MTA group, and control group (C group) were measured by cholecystokinin-8 (CCK-8) assay, the ability of cell mineralized nodular formation was observed via alizarin red staining; and quantitative reverse transcription PCR (qRT-PCR) andWestern blot were adopted to determine the expression of osteogenic related genes of hDPSCs and key proteins of mitogen-activated protein kinase (MAPK) signaling pathway. After 1 week of treatment, the clinical efficacy of group B was more favorable in contrast with group A (P < 0.05); the pain of children in group B was notably better in contrast with group A, and incidence of ADR was notably lower in contrast with group A (P < 0.05). 5.0 mg/mL, 10.0 mg/mL, and 30 mg/mL iRoot BP or MTA could improve cell proliferation activity (P < 0.01); the effect of iRoot BP on proliferation of hDPSCs was greater in contrast with MTA (P < 0.05); and the integral optical density (IOD) value of iRoot BP group was notably higher in contrast with MTA group (P < 0.01). The mRNA expression levels of collagen-I (COL-I), bone sialoprotein (BSP), and osteocalcin (OC) in MTA group and iRoot BP group were notably higher in contrast with C group (P < 0.01); the COL-I mRNA expression level of iRoot BP group was notably higher in contrast with MTA group (P < 0.01); the mRNA expression level of BSP in MTA group was notably higher in contrast with iRoot BP group (P < 0.01); the relative protein expression levels of phosphorylated ERK (p-ERK) and phospho-Jun N-terminal kinase (p-JNK) in MTA group and iRoot BP group were notably higher in contrast with C group (P < 0.01); and the relative expression level of p-ERK protein in iRoot BP group was higher in contrast with MTA group (P < 0.05). These results indicated that the clinical efficacy of biomedical ceramic iRoot BP was better than MTA in the preservation of live pulpitis in children, and the patients treated with iRoot BP had better pain recovery effect and lower risk of ADR. The effect of iRoot BP on the proliferation and mineralization of hDPSCs was better than that of MTA, and it may promote the osteogenic differentiation of hDPSCs by activating MAPK signaling pathway and regulating gene expression of COL-I, BSP, and OC.

Association of SOCS1 mRNA expression with hepatitis B virus infection-related hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the predominant form of liver cancer worldwide, can be triggered by a variety of causes such as chronic hepatitis B virus (HBV) infection. The immune response to HBV activates the janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. Suppressor of cytokine singnaling 1 (SOCS1) is a negative feedback regulator of JAK/STAT pathway. Our study was carried out to evaluate SOCS1 mRNA expression and its correlation with paraclinical characteristics in patients with HBV-related HCC. The SOCS1 mRNA expression level in adjacent non-tumour tissues and tumour liver tissues were determined in 44 patients with HBV infection-associated HCC by real-time RT-PCR. Our results showed that SOCS1 mRNA expression level in adjacent non-tumour tissues were significantly higher than in HCC tissues (p=0.003). High expression of SOCS1 in adjacent non-tumour tissue was observed in patients with single tumour (p=0.024), tumour size ≤5 cm (p=0.011), vascular invasion (p=0.047), and no vascular invasion (p=0.007). Red blood cell counts were positively correlated with SOCS1 gene expression (Spearman’s rho=0.359, p=0.018). Our results suggested that SOCS1expression may be considered as a potential factor involved in the pathogenesis of HCC.

Kinetic characteristics of transcriptional bursting in a complex gene model with cyclic promoter structure

While transcription occurs often in a bursty manner, various possible regulations can lead to complex promoter patterns such as promoter cycles, giving rise to an important issue: How do promoter kinetics shape transcriptional bursting kinetics? Here we introduce and analyze a general model of the promoter cycle consisting of multi-OFF states and multi-ON states, focusing on the effects of multi-ON mechanisms on transcriptional bursting kinetics. The derived analytical results indicate that bust size follows a mixed geometric distribution rather than a single geometric distribution assumed in previous studies, and ON and OFF times obey their own mixed exponential distributions. In addition, we find that the multi-ON mechanism can lead to bimodal burst-size distribution, antagonistic timing of ON and OFF, and diverse burst frequencies, each further contributing to cell-to-cell variability in the mRNA expression level. These results not only reveal essential features of transcriptional bursting kinetics patterns shaped by multi-state mechanisms but also can be used to the inferences of transcriptional bursting kinetics and promoter structure based on experimental data.

Olfm4 Is Highly Expressed in HCC Patients and as a Biomarker and Therapeutic Target for HCC

Hepatocellular carcinoma (HCC) is one of the primary types of cancer that claims many lives worldwide, and its incidence continues to increase. Conventional therapies against liver cancer are inadequate, and the pathogenesis of HCC remains unclear. Thus, not only are more effective therapies to treat HCC required but also identification of the key genes involved in its pathogenesis is important for developing such therapies. This study found that olfactomedin 4 (OLFM4) level is higher in HCC patients than in healthy individuals. Furthermore, HCC patients also have higher messenger ribonucleic acid (mRNA) expression level in HCC tissues than in liver paracancerous tissues. OLFM4 has high predictive capacity as a biomarker for HCC and closely correlates to tumor size. It is confirmed that OLFM4 contributes to cancer cell proliferation, and HIF1α is involved in this process. Thus, the OLFM4/HIF-1α axis might be a target signaling pathway for developing novel drugs to treat HCC.

The Effect of Epigallocatechin gallate on Cross-talk Between Autophagy and Apoptosis in NALM-6 Cell Line

Background: Acute lymphoblastic leukemia is a prevalent hematological malignancy in 2-5-year-old children. Chemotherapy, as the most common treatment for ALL, is not usually responsive. Epigallocatechin gallate (EGCG), a small molecule extracted from green tea, has significant effects on tumor cells through different mechanisms, such as DNA damage, cell cycle arrest, oxidative stress, apoptosis, and autophagy. In this study, we investigated the impact of EGCG on autophagy and apoptosis in NALM-6 cell line. Methods and results: Cell viability and apoptosis were assessed by MTT and Trypan blue exclusion assay, and flow cytometry. It was shown that EGCG remarkably inhibited proliferation, reduced cell viability, and induced apoptosis in NALM-6 cell line (P<0.05). In addition, real-time PCR and western blot analysis were used to examine autophagy. It was observed that EGCG resulted in a 4-fold increase in LC3 protein level (P<0.05) while reducing the mRNA expression level of LC3B, P62/SQSTM1, and Atg2B genes (P<0.01). It also caused around 1.3-fold increase in DRAM1 mRNA expression level (P<0.05). Finally, it was indicated that the inhibition of autophagy affects apoptosis neither in untreated nor treated cells with EGCG.Conclusion: These results show that EGCG can induce apoptosis and autophagy in NALM-6 cell line while inhibition of autophagy cannot affect apoptosis in this cell line.

Role of FRG1 in predicting the overall survivability in cancers using multivariate based optimal model

FRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis.

Stratification of Intermediate-Risk Acute Myeloid Leukemia According to the Expression Level of WT1 mRNA at Diagnosis

Introduction Despite advances in the genetic analysis of acute myeloid leukemia (AML), Wilms' tumor oncogene 1 (WT1) mRNA remains an important pan-marker of AML. A log reduction in WT1 mRNA expression after chemotherapy is a predictor of prognosis, and WT1 mRNA can be used as a marker of minimal residual disease or relapse. In the 1990s, a high expression level of WT1 mRNA at diagnosis was considered a poor prognostic factor. However, recent analyses have found that WT1 mRNA expression varies with AML type. Since the prognosis is affected by cytogenetic abnormalities and therapeutic intensity, we re-evaluated the clinical significance of WT1 mRNA expression at diagnosis in the context of the European Leukemia Net (ELN) risk classification and treatment. Method We retrospectively analyzed 216 patients at five institutions between 2011 and 2020. The expression level of WT1 mRNA was measured for all patients at diagnosis using bone marrow (BM) samples from 191 patients and peripheral blood (PB) samples from 25. WT-1 mRNA expression was measured using real-time quantitative polymerase chain reaction and the measured values were converted in normal logarithm. The median age at diagnosis was 62 (range: 23-93) years. The cytogenetic risk of ELN was classified as favorable (n = 41), intermediate (n = 123), and adverse risk (n = 41). Selected therapeutics were standard chemotherapy (n = 182, 84.3%, including CAG regimen; cytarabine, aclarubicin and G-CSF), hypomethylating agents or low-dose cytarabine (n = 19, 8.8%), and best supportive care (n = 15, 6.9%). Also, 143 patients (66.2%) received one or more courses of standard consolidation chemotherapy and 61 (28.3%) underwent allogeneic hematopoietic stem cell transplantation (allo-HCT). The median observation period was 518 [1-3418] days (Table 1). The overall survival (OS) and event-free survival (EFS) rates were assessed. Relapse or death was defined as an event, and OS was evaluated on the date of death. Result The median expression level of WT1 mRNA was 4.68 (range: 1.0-5.72) [log copies/µg RNA, units are omitted thereafter] in BM and 3.66 (range: 1.34-5.20) in PB (Table 1). Favorable-risk AML had the highest WT1 mRNA expression level in BM (4.95, p = 0.0054), whereas there was no difference between the expression levels in intermediate- and adverse-risk AML in BM (4.63 and 4.47, p = 0.711, Fig. 1A). WT1 mRNA expression in PB were 4.04, 3.84, and 3.05 for favorable-, intermediate-, and adverse-risk AML, respectively, and were higher for those with a favorable-risk AML (vs. adverse risk, p = 0.048, Fig. 1B). When WT1 mRNA expression level in BM was compared between the two groups, i.e., &lt;4.0 (BM-WT1 low) vs. ≥4.0 (BM-WT1 high), 2-year EFS rates were 26.8% and 49.7% (p = 0.0035) and 2-year OS rates were 44.9% and 61.5% (p = 0.00053), respectively. When WT1 mRNA expression level in PB was compared between the two groups, i.e., &lt;3.0 (PB-WT1 low) vs. ≥3.0 (PB-WT1 high), 2-year EFS rates were 0% and 51.5% (p = 0.023) and 2-year OS rates were 0% and 73.1% (p = 0.01), respectively. PB- or BM-WT1 low showed a worse prognosis at diagnosis. Since AML prognosis is affected by ELN risk and selected therapeutics, we evaluated 109 intermediate-risk patients who had received at least one course of standard chemotherapy. Fifty of them (45.9%) underwent subsequent allo-HCT. The 2-year EFS rates of PB or BM-WT1 low (n = 25, PB = 2 and BM = 23) vs. PB or BM-WT1 high (n = 84, PB = 23 and BM = 61) were 23.9% and 50.4%, respectively (p = 0.023, Fig. 2A) and the 2-year OS rates were 51.2% and 64.6%, respectively (p = 0.03, Fig. 2B). PB or BM-WT1 low of intermediate-risk AML had a poor prognosis even with standard chemotherapy. Multivariate analysis adjusted for ages, the reduction rate of WT1 mRNA after induction chemotherapy, duration of receiving consolidation chemotherapy, allo-HCT, and PB or BM-WT1 low at diagnosis were independent poor prognosis factors for EFS in intermediate-risk AML (HR: 3.32, 95%CI: 1.29-8.53, p = 0.013). The OS rate of PB or BM-WT1 low also worsened (HR: 2.33, 95%CI: 0.88-6.18, p = 0.089) (Table 2). Whereas, the outcome of adverse-risk AML was not affected by PB or BM-WT1 low/high. Conclusion The expression level of WT1 mRNA at diagnosis was negatively correlated with prognosis. Favorable-risk AML had higher expressions of WT1 mRNA. PB or BM-WT1 low in intermediate-risk AML was associated with a poor prognosis. In standard-risk AML, WT1 mRNA may be a useful pan-marker to predict prognosis at diagnosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Pyruvate Kinase M2 Regulates Hif-1alpha Activity in Myeloid Dendritic Cells from Patients with Severe Aplastic Anemia

Hypoxia-inducible factor 1 (HIF-1) is a nuclear protein with transcriptional activity. HIF-1 can be activated by immune cells exposed to hypoxia and regulate glycolysis. In this regulation, pyruvate kinase M2 (PKM2), a key enzyme in the cell glycolytic pathway, is an important regulatory site. Our previous studies suggest that PKM expression is increased in myeloid dendritic cells (mDCs) in SAA patients. Therefore, we investigated the expression level of HIF-1α in mDCs and its interaction with PKM2 in SAA patients. The HIF-1α expression of mRNA and protein on mDCs in SAA untreated patients was significantly higher than that in SAA remission patients and normal controls. In the SAA patients, the HIF-1α expression on mDCs was positively correlated with the numbers of mDCs (p &lt; 0.01), CD80+mDC/mDC (r = 0.689, p &lt; 0.01), and CD86+mDC/mDC in PB (p &lt; 0.05). In SAA patients, the HIF-1α expression on mDCs was negatively correlated with the CD4+T/CD8+T ratio in peripheral blood (PB) (p &lt; 0.05), the percentage of granulocytoid and erythroid cells in bone marrow (p &lt; 0.05), the WBC count in PB (p &lt; 0.05), ANC in PB (p &lt; 0.05), and the percentage of Rets in the PB (p &lt; 0.05); was positively correlated with the percentage of lymphoid cells in bone marrow(p &lt; 0.05); and was not statistically correlated with the megakaryocyte number in bone marrow, absolute PBL count, HGB in PB, absolute Ret count in PB, or PLT count in PB. In the correlation analysis we observed that there was a positively correlation between HIF-1α and PKM2 expression on mDCs in SAA patients (p &lt; 0.001). To evaluate whether there is a mutual adjustment relationship between HIF-1α and PKM2, we successfully reduced PKM2 gene expression in this cell population via siRNA transfection. This process resulted in significantly lower levels of PKM2 protein expression relative to cells transfected with siControl which were evaluated by western blotting. We observed that the relative expression levels of HIF-1α mRNA in mDCs transfected with PKM2 siRNA was lower than siControl group（P&lt; 0.01）. Conclusions In this study, we found that untreated patients with SAA had higher HIF-1α expression on mDCs compared with recovering SAA patients and normal controls. The expression of HIF-1α was correlated with the number and function in mDCs and the severity of pancytopenia of SAA. The results indicated that the mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The mRNA expression level of HIF-1α in mDC of SAA patients at the onset was significantly higher than that of the remission group and the control group, and was significantly positively correlated with the expression of PKM2. The above experimental results confirmed that HIF-1α plays an important role in the abnormal immune response in patients with severe aplastic anemia, mainly by regulating the activity of PKM2 and thereby affecting the energy metabolism in immune cells. Disclosures No relevant conflicts of interest to declare.