A new tandem repeat-enriched lncRNA XLOC_008672 promotes gastric carcinogenesis by regulating G3BP1 expression.

Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.

PIK3CA mutations-mediated downregulation of circLHFPL2 inhibits colorectal cancer progression via upregulating PTEN.

BACKGROUND: PIK3CA mutation and PTEN suppression lead to tumorigenesis and drug resistance in colorectal cancer (CRC). There is no research on the role of circular RNAs (circRNAs) in regulating PIK3CA mutation and MEK inhibitor resistance in CRC. METHODS: The expression of circLHFPL2 in PIK3CA-mutant and wild-type cells and tissues was quantified by RNA-sequencing and qRT-PCR. CCK-8 assay and colony formation assay were used to evaluate cell viability. Annexin V/PI staining was implemented to assess cell apoptosis. Luciferase assay, biotin-coupled microRNA capture, and RIP assay were used to validate the interaction among potential targets. Western blotting and qRT-PCR assays were used to evaluate the expression of involved targets. Xenograft tumor in a nude mouse model was used to explore the role of circRNAs in vivo. RESULTS: RNA sequencing defined downregulated expression of circLHFPL2 in both PIK3CAH1047R (HCT116) and PIK3CAE545K (DLD1) cells. CircLHFPL2 was also downregulated in PIK3CA-mutant CRC primary cells and tissues, which was correlated with poor prognosis. CircLHFPL2 was mainly localized in the cytoplasm and its downregulation was attributed to the PI3K/AKT signaling pathway activated by phosphorylating Foxo3a. CircLHFPL2 inhibited PI3KCA-Mut CRC progression both in vitro and in vivo. Furthermore, our work indicated that circLHFPL2 acts as a ceRNA to sponge miR-556-5p and miR-1322 in CRC cells and in turn modulate the expression of PTEN. Importantly, circLHFPL2 was able to overcome PIK3CA-mediated MEK inhibitor resistance in CRC cells. CONCLUSIONS: Downregulation of circLHFPL2 sustains the activation of the PI3K/AKT signaling pathway via a positive feedback loop in PIK3CA-mutant CRC. In addition, downregulation of circLHFPL2 leads to MEK inhibitor resistance in CRC. Therefore, targeting circLHFPL2 could be an effective approach for the treatment of CRC patients harboring oncogenic PIK3CA mutations.

CD63 negatively regulates hepatocellular carcinoma development through suppression of inflammatory cytokine-induced STAT3 activation.

Tetraspanin CD63 has been widely implicated in tumour progression of human malignancies. However, its role in the tumorigenesis and metastasis of hepatocellular carcinoma (HCC) remains unclear yet. In the present study, we aimed to investigate the specific function and underlying mechanisms of CD63 in HCC progression. CD63 expression in HCC tissues was detected using immunohistochemistry and quantitative real-time PCR analyses; effects of CD63 on HCC cell proliferation and migration were investigated by CCK-8 assay, colony formation assay, transwell assay and a xenograft model of nude mice. RNA-sequencing, bioinformatics analysis, dual-luciferase reporter assay and Western blot analysis were performed to explore the underlying molecular mechanisms. Results of our experiments showed that CD63 expression was frequently reduced in HCC tissues compared with adjacent normal tissues, and decreased CD63 expression was significantly associated with larger tumour size, distant site metastasis and higher tumour stages of HCC. Overexpression of CD63 inhibited HCC cell proliferation and migration, whereas knockdown of CD63 promoted these phenotypes. IL-6, IL-27 and STAT3 activity was regulated by CD63, and blockade of STAT3 activation impaired the promotive effects of CD63 knockdown on HCC cell growth and migration. Our findings identified a novel CD63-IL-6/IL-27-STAT3 axis in the development of HCC and provided a potential target for the diagnosis and treatment of this disease.

FAPP2 promotes tumor cell growth in human colon cancer through activation of Wnt signaling.

Human phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2) is well-known to function as a cytoplasmic lipid transfer protein during vesicle maturation. However, the expression and role of FAPP2 in tumor remain elusive. In this study, data from immunohistochemical assays displayed that FAPP2 was remarkably upregulated (57.8%) in 90 cases of colon cancer samples in contrast to their corresponding adjacent tissues. Disruption of FAPP2 by CRISPR/Cas9 technique in colon cancer cells led to an attenuated effect on cell growth analyzed by CCK8 and colony formation assays. Meanwhile, the tumorigenicity of FAPP2 downregulated cells also decreased in nude mice model. Accordantly, CCK8 assays also indicated that FAPP2 overexpression could promote colon cancer cell growth. In addition, dual luciferase reporter assays and western blot analyses revealed that Wnt/ß-catenin signaling was involved in the FAPP2-regulated tumor cell growth. These findings suggest that FAPP2 could act as an oncogene in the regulation of tumor growth and may provide a new therapeutic target for human colon cancer.

SRPK1 facilitates tumor cell growth via modulating the small nucleolar RNA expression in gastric cancer.

Serine-arginine protein kinase 1 (SRPK1) is the main regulator in alternative splicing by phosphorylating splicing factors rich in serine/arginine repeats. Its overexpression has been found in multiple cancer types and contributes to cancer development. Here we report the role of SRPK1 and underlying mechanism in gastric cancer (GC) cell growth. We found that SRPK1 was frequently upregulated in GC samples compared with their adjacent corresponding normal tissues by immunohistochemistry and western blot analysis. Knockdown of SRPK1 in GC cells suppressed cell growth in cell viability assays, colony formation assays and nude mice xenograft model, whereas overexpression of SRPK1 promotes opposite phenotypes in these assays. By a complementary DNA microarray analysis, we found that SRPK1 knockdown had significant inhibitory effects on a majority of small nucleolar RNAs expression. Among them, snoRA42, snoRA74A, and snoRD10 were selected for further functional experiments. Cell growth curves on a plate and in soft agar indicated that the three snoRNAs play potential oncogenic function in GC. In addition, SRPK1 could co-immunoprecipitated with NCL, a nucleolar phosphoprotein involved in the synthesis and maturation of ribosomes. These results suggested that SRPK1 contributes to GC development by a new possible mechanism involving snoRNAs mediated signaling.

B-cell lymphoma-2-associated transcription factor 1 is overexpressed and contributes to sorafenib resistance in hepatocellular carcinoma.

AIM: B-cell lymphoma-2-associated transcription factor 1 (BCLAF1) is involved in various biological processes including tumorigenesis, but its function and expression in hepatocellular carcinoma (HCC) is little known, and its clinical value in HCC has not yet been defined. METHODS: The protein level of BCLAF1 in HCC specimens and paired adjacent normal tissues was examined by immunohistochemical staining. The effects of BCLAF1 on autophagy in HCC cells were detected by confocal microscopy, transmission electron microscopy, and western blot analysis. Cell proliferation and tumorigenicity assays were carried out in vitro and in vivo. Flow cytometry assay was used to determine the apoptosis level of HCC cells. The correlation of BCLAF1 and sorafenib resistance in HCC was analyzed by the Kaplan-Meier survival method. RESULTS: High expression of BCLAF1 was found in HCC tissues compared with adjacent normal tissues, and higher BCLAF1 expression was correlated with higher tumor-node-metastasis stage, worse differentiation, and worse prognosis of HCC patients. BCLAF1 could induce autophagy in HCC cells in response to starvation and BCLAF1-mediated autophagy could enhance cell proliferation and impede cell apoptosis under stress conditions. Animal experiments indicated that BCLAF1 promoted tumorigenicity of HCC cells in vivo. More importantly, high expression of BCLAF1 might contribute to sorafenib resistance in HCC patients. CONCLUSIONS: BCLAF1 is a potential oncogene in HCC by inducing autophagy to maintain tumor cell growth in response to stress conditions, and it could serve as a potential biomarker for predicting the prognosis of HCC patients and screening patients who are suitable for sorafenib therapy.

Downregulation of STRAP promotes tumor growth and metastasis in hepatocellular carcinoma via reducing PTEN level.

The serine-threonine kinase receptor-associated protein (STRAP) has been implicated in multiple human cancers. However, its expression and function are currently unclear and controversial in different tissue types. In the present study, we report that aberrant downregulation of STRAP in hepatocellular carcinoma (HCC) facilitated tumor cell growth and metastasis in a phosphatase and tensin homologue (PTEN)-dependent manner. Immunohistochemical analysis and quantitative real-time polymerase chain reaction results indicated that STRAP was frequently downregulated in HCC samples. Functionally, knockdown of STRAP by RNA inference in HCC cells promoted proliferation and migration in vitro and tumorigenicity and lung metastasis in vivo. Through detecting the expression of some tumor-related genes using western blot analysis, we found the tumor suppressor PTEN was decreased upon STRAP silencing. Further analyses demonstrated that silenced STRAP led to PTEN protein degradation. Immunohistochemical analysis revealed that STRAP expression was closely associated with PTEN expression in 30 cases of HCC samples. These findings strongly suggest that STRAP plays an inhibitory role in HCC via regulating PTEN expression and could be a potential therapeutic target for this disease. © 2017 IUBMB Life, 70(2):120-128, 2018.

SDCBP modulates tumor microenvironment, tumor progression and anti-PD1 efficacy in colorectal cancer.

Anti-programmed cell death 1 (aPD1) therapy has yielded limited success in patients with colorectal cancer (CRC). Syndecan binding protein (SDCBP), encodes a PDZ domain-containing protein that is essential for cellular processes, including cell adhesion, migration, and signal transduction. Here, we investigated the effect of SDCBP on tumor progression, immunotherapy, and the tumor microenvironment (TME) in CRC. High expression of SDCBP is associated with non-response to immunotherapy and correlated with poorer disease-free survival (DFS) in CRC patients. Inhibiting SDCBP by transfecting shRNA or using its inhibitor zinc pyrithione (ZnPT) hindered proliferation and metastasis while enhancing the efficacy of aPD1 treatment in a mouse xenograft model and liver metastasis model. The TME of CRC was significantly altered following ZnPT treatment characterized by a reduced amount of M2 macrophages and a heightened percentage of M1 macrophages. The co-culture system of CRC cells and macrophages provided evidence that SDCBP silencing promoted the repolarisation of M2 macrophages into M1. SDCBP promotes the proliferation, metastasis, and immunotherapy resistance of CRC. Thus, ZnPT represents an effective SDCBP inhibitor and exhibits considerable potential for combination with aPD1 to enhance immunotherapy efficacy.

SP1 Mediated PIK3CB Upregulation Promotes Gastric Carcinogenesis.

PIK3CB, one of catalytic subunits of PI3Ks kinase family, is implicated in several cellular processes such as cell growth, proliferation, mobility and neoplastic transformation. Its abnormal expression has been found in several human cancer types. However, the regulation pattern and function of PIK3CB in gastric cancer (GC) are still unclear. Here, we demonstrated that PIK3CB and SP1 (special protein 1) were both upregulated in GC samples compared to adjacent non-cancerous stomach tissues at mRNA and protein levels. The expression of the two genes also displayed a significant positive correlation in GC samples. Dual-luciferase assays and chromatin immunoprecipitation (ChIP) assays revealed that SP1 could bind to the -771~-605 region of the promoter of PIK3CB and enhance transcription. Furthermore, we discovered that SP1 induced AKT activation through PIK3CB and accelerated GC cell proliferation and migration in a PIK3CB/AKT signaling dependent manner. TGX-221, a PIK3CB-selective inhibitor, which can block this signaling transduction pathway, was found to inhibit the growth of GC cells and induce apoptosis in vitro, implying that it may act as a potential development agent for GC. These collective findings provide a new insight into PI3K/AKT signaling that SP1 may function as an upstream factor on PI3K, forming a new signaling axis to promote the progression of GC or other malignancies.

WNK1 Interaction with KEAP1 Promotes NRF2 Stabilization to Enhance the Oxidative Stress Response in Hepatocellular Carcinoma.

Cellular oxidative stress plays a key role in the development and progression of hepatocellular carcinoma (HCC). A better understanding of the processes that regulate reactive oxygen species (ROS) homeostasis could uncover improved strategies for treating HCC. Here, we identified WNK1 as an antioxidative factor and therapeutic target in HCC. In human HCC, WNK1 expression was increased and correlated with poor patient prognosis. WNK1 knockdown significantly inhibited cell proliferation and xenograft tumor growth. Mechanistically, WNK1 competed with NRF2 for binding to the partial Kelch domain of KEAP1, reducing NRF2 ubiquitination and promoting NRF2 accumulation and nuclear translocation to increase antioxidant response. WNK1 silencing increased H2O2-induced apoptosis and inhibited cell growth by elevating reactive oxygen species (ROS) levels, which could be rescued by treatment with the antioxidant N-acetylcysteine (NAC) and NRF2 activator tert-butylhydroquinone (tBHQ). Liver-specific WNK1 knockout mouse models of HCC substantiated that WNK1 promoted HCC development by regulating ROS levels. WNK463, an inhibitor of the WNK kinase family, suppressed HCC progression and altered the redox status. These findings suggest that WNK1 plays a critical role in HCC development and progression and that the WNK1-oxidative stress axis may be a promising therapeutic target for HCC.

Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

Evaluation on the Growth Performance, Nutrient Digestibility, Faecal Microbiota, Noxious Gas Emission, and Faecal Score on Weaning Pigs Supplement with and without Probiotics Complex Supplementation in Different Level of Zinc Oxide.

A total of 200 26-day-old crossbred weaning piglets ((Yorkshire × Landrace) × Duroc; 6.55 ± 0.62 kg) were used in a 6-week experiment to evaluate the effects of adding probiotics complex supplementation (Syner-ZymeF10) with high and low ZnO diets on the performance of weaning pigs in 42 days. Pigs were randomly allotted to a 2 × 2 factorial arrangement and they were supplemented with two concentration level of ZnO with 3000 ppm and 300 ppm and probiotics complex supplementation with 0 and 0.1%. There were ten replicate pens per treatment with five pigs per pen (two gilts and three barrows). Pigs fed diets with 3000 ppm ZnO had a higher BW during the overall period and ADG during d 8-21, d 22-42, and overall period than pigs receiving 300 ppm ZnO diets (p < 0.05), as well as a G: F which tended to increase on d 8-21 and overall period (p < 0.1) and decreased tendency on faecal gas emission of methyl mercaptans and acetic acid concentration (p < 0.1). Dietary probiotics complex supplementation had decreased the E. coli count (p < 0.05) and tended to increase the Lactobacillus count (p < 0.1). Dietary probiotics complex supplementation and different level of ZnO supplementation had no significant effect on the nutrition digestibility and faecal score (p > 0.05). In conclusion, probiotic supplementation reduced the fecal E. coli counts and tended to improve Lactobacillus counts. There were no interactive effects between ZnO and probiotic complex supplementation on all the measured parameters.

Clinical Characteristics, Quality of Life, and Risk Factors of Amputation Stump Skin Disease and Stump Fungal Infection in Adult Amputees in Shanghai, China.

Introduction: The stump site of amputees is clinically vulnerable and prone to various skin diseases. Data regarding the impact on quality of life (QoL) of amputees with amputation stump skin disease (ASSD) and risk factors of ASSD and stump fungal infection in the Shanghai area are yet unknown. Objective: This study aims to evaluate the QoL of amputees with ASSD and explore the risk factors of ASSD and stump fungal infection in the Shanghai area. Methodology: A total of 104 amputees from Shanghai Hebin Rehabilitation Hospital, Otto Bock (China) Industries Co., Ltd., Shanghai Tongji Hospital, and Shanghai Rehabilitation and Vocational Training Center for the Disabled were enrolled in this study. We collected demographic, clinical, and skin fungal examination data from these amputees from April 2015 to May 2021. Dermatology life quality index (DLQI) questionnaire was used to evaluate the QoL. The risk factors for ASSD and fungal skin infection were analyzed by the univariate analyses. Results: The median age of the 104 amputees was 57.9 ± 11.9 years with an average amputation time of 17.7 ± 15.1 years, and 73% of cases were men. The mean DLQI score of amputees with ASSD was13.6, suggesting the severe impairment of QoL. Among amputees, 41 (39.4%) had confirmed ASSD, of whom 24 (58.5%) suffered from fungal skin infection and the remaining were subjected to intertriginous dermatitis and eczema (22%), cutaneous keratosis (12.2%), and others (7.3%). Aspergillus (50.0%) was the most common species. The other fungal organisms included Trichophyton rubrum (33.3%), Candida krusei (8.3%), T. mentagrophytes (4.2%), and C. albicans (4.2%). ASSD rather than non-ASSD was more common in men (80.4%) and summer (46.3%). Summer (OR = 3.31, 95% CI = 1.19-9.17) was an established risk factor for ASSD compared to spring. The daily artificial limb wearing time > 8 h was associated with stump fungal infection. Conclusion: The QoL of amputees with ASSD was severely affected and the ASSD was characterized by fungal infection (tinea), intertriginous dermatitis, eczema, and skin keratosis. Summer and daily prosthesis wearing > 8 h was a risk factor for ASSD. Aspergillus was the most common fungal species, especially when the stump was exposed in summer.

miR-15a and miR-20b sensitize hepatocellular carcinoma cells to sorafenib through repressing CDC37L1 and consequent PPIA downregulation.

Sorafenib is a classical targeted drug for the treatment of advanced hepatocellular carcinoma (HCC), but intrinsic resistance severely limited its therapeutic effects. In the present study, we aimed to identify crucial genes in HCC cells that affect sorafenib resistance by a CRISPR/Cas9 genome-scale screening. The results indicated that the deficiency of miR-15a and miR-20b contributed to sorafenib resistance, whereas exogenous expression of miR-15a and miR-20b enhanced sorafenib sensitivity of HCC cells by cell viability, colony formation, and flow cytometry analyses. Further analyses revealed that cell division cycle 37 like 1 (CDC37L1) as a common target of miR-15a and 20b, was negatively regulated by the two miRNAs and could enhance sorafenib resistance of HCC cells in vitro and in vivo. Mechanistically, CDC37L1, as a cochaperone, effectively increased the expression of peptidylprolyl isomerase A (PPIA) through strengthening the binding between heat shock protein 90 (HSP90) and PPIA. The results from immunohistochemical staining of a HCC tissue microarray revealed a positive association between CDC37L1 and PPIA expression, and high expression of CDC37L1 and PPIA predicted worse prognosis of HCC patients after sorafenib therapy. Taken together, our findings reveal crucial roles of miR-15a, miR-20b, CDC37L1, and PPIA in sorafenib response of HCC cells. These factors may serve as therapeutic targets and predict prognosis for HCC treated with sorafenib.

A Genome-Scale CRISPR Knock-Out Screen Identifies MicroRNA-5197-5p as a Promising Radiosensitive Biomarker in Colorectal Cancer.

Radioresistance is one of the main reasons causing unsatisfactory curative effects of ionizing radiation (IR) against colorectal cancer (CRC). However, its underlying mechanisms remain unclear yet. In the present study, we applied a genome-scale CRISPR knockout screen in combination of NGS sequencing upon CRC cell lines to explore regulatory factors involved radioresistance of CRC, and 3 candidate genes were identified. Cytotoxicity of IR was determined by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and apoptosis assay, and microRNA-5197-5p (miR-5197) was found to significantly enhance the cytotoxicity of IR to CRC cells. By further mechanistic investigation, we demonstrated that miR-5197 directly targeted CDK6 and inhibited its expression in RKO cells, which induced cell cycle arrest at G1/S phase and inhibited cell division, thereby radiosensitivity was enhanced by miR-5197. Our findings revealed that miR-5197 might be a critical factor regulating CRC cell radiosensitivity and provided novel insights into the development of therapeutic strategies for CRC patients who are resistant to IR.

Genome-scale CRISPRa screening identifies MTX1 as a contributor for sorafenib resistance in hepatocellular carcinoma by augmenting autophagy.

Sorafenib is the standard first-line drug for the treatment of advanced hepatocellular carcinoma (HCC), however, its therapeutic efficacy is not satisfactory due to primary or secondary resistance of HCC cells. In the present study, we identified Metaxin 1 (MTX1) as a new regulator of sorafenib resistance in HCC through genome-scale CRISPR activation (CRISPRa) screening. We found that MTX1 was frequently upregulated in HCC tissues and overexpression of MTX1 promoted HCC cell proliferation in vitro and in vivo. As well, MTX1 overexpression increased cell growth rate and decreased cell apoptosis upon sorafenib treatment. Consistently, the resistance induced by MTX1 was also observed in subcutaneous xenograft tumor model. Clinically, high expression of MTX1 was closely related with poor outcomes in HCC patients who received sorafenib treatment. Mechanistically, overexpression of MTX1 could promote HCC cell autophagy via interacting with and inhibiting CDGSH iron sulfur domain 1 (CISD1), an autophagy negative regulator. Taken together, our findings suggest that MTX1 is upregulated in HCC and contributes to sorafenib resistance via a possible mechanism involving CISD1 mediated autophagy.

CSNK2B contributes to colorectal cancer cell proliferation by activating the mTOR signaling.

The function of Casein kinase 2 beta (CSNK2B) in human malignancies has drawn increasing attention in recent years. However, its role in colorectal cancer (CRC) remains unclear. In the present study, we aimed to explore the expression and biological functions of CSNK2B in CRC. Public gene expression microarray data from online database and immunohistochemistry analysis demonstrated that CSNK2B was highly expressed in CRC tissues than in normal tissues. In vitro and in vivo cellular functional experiments showed that increased CSNK2B expression promoted CRC cell viability and tumorigenesis of CRC. Further western blots and rescue experiments confirmed that CSNK2B promoted CRC cell proliferation mainly by activating the mTOR signaling pathway. These findings identified CSNK2B as a novel oncogene contributing to the development of CRC.

Enumeration of viable CD34+ cells in cord blood using a novel stem cell enumeration kit.

OBJECTIVE: To assess the detection performance of the hematopoietic stem cell enumeration kit developed by BD Biosciences. METHODS: Cord blood samples were prepared using a hematopoietic stem cell enumeration kit developed by BD Biosciences and Stem-Kit reagents from Beckman Coulter. CD34+ cells were enumerated using a BD FACSCanto instrument and FACSDiva software. RESULTS: A total of 519 samples were analyzed in this study. The hematopoietic stem cell enumeration kit developed by BD Biosciences yielded absolute counts of CD34-positive cells that were on average 8.7% lower than Beckman Coulter Stem-Kit reagents (range: -5.7% to-14.7%). The BD Biosciences kit yielded relative counts that were on average 9.9% higher compared with Beckman Coulter Stem-Kit reagents (range: -2.1% to +13.8%). The intraclass correlation coefficients for absolute and relative counts of CD34-positive cells were 0.9967 (95% confidence interval [CI]: 0.9961-0.9972) and 0.9512 (95% CI: 0.9423-0.9587) for the BD Biosciences and Beckman Coulter kits, respectively. CONCLUSIONS: The hematopoietic stem cell enumeration kit developed by BD Biosciences can be used to enumerate CD34-positive stem cells from cord blood samples.

Overexpression of NELFE contributes to gastric cancer progression via Wnt/ß-catenin signaling-mediated activation of CSNK2B expression.

BACKGROUND: Accumulating evidence has highlighted the importance of negative elongation factor complex member E (NELFE) in tumorigenesis. However, the relationship between NELFE and gastric cancer (GC) remains unclear. This study aimed to explore the expression pattern and specific function of NELFE in GC. METHODS: NELFE expression was evaluated by immunohistochemistry and qRT-PCR in GC tissues, respectively. Cell proliferation, migration and invasion were measured by CCK-8, colony formation, transwell assays, and nude mice model. Bioinformatics analysis was performed to search potential target genes of NELFE, and a Cignal Finder 10-Pathway Reporter Array was used to explore potential signaling pathways regulated by NELFE. Dual-luciferase reporter assays, qRT-PCR and western blotting were conducted to verify their regulatory relationship. The expression correlations among NELFE, ß-catenin and CSNK2B were further explored by immunohistochemistry on consecutive resections. RESULTS: NELFE was significantly overexpressed in GC tissues both in protein and mRNA level and negatively correlated with the prognosis of GC patients. Gain- and loss-of-function experiments showed that NELFE potentiated GC cell proliferation and metastasis in vitro and in vivo. CSNK2B was identified as a downstream effector of NELFE. Wnt/ß-catenin signaling may mediate the regulation of CSNK2B by NELFE. In addition, NELFE, ß-catenin and CSNK2B were all remarkably upregulated in tumor tissues compared with adjacent normal tissues, and their expression levels in GC were positively correlated with each other. CONCLUSION: Our findings reveal a new NELFE-Wnt/ß-catenin-CSNK2B axis to promote GC progression and provide new candidate targets against this disease.