Long noncoding RNA SOX2OT maintains the stemness of pancreatic cancer cells by regulating DEK via interacting with miR-200a/141.

The article "Long noncoding RNA SOX2OT maintains the stemness of pancreatic cancer cells by regulating DEK via interacting with miR-200a/141, by C.-S. Liu, Q. Zhou, Y.-D. Zhang, Y. Fu, published in Eur Rev Med Pharmacol Sci 2020; 24 (5): 2368-2379-DOI: 10.26355/eurrev_202003_20504-PMID: 32196588" has been withdrawn from the authors stating that "to validate the effect of lncRNA-SOX2OT, we constructed SOX2OT overexpressed cells using lentiviral vector with -IRES2-EGFP to easily visualize the transfection efficiency. Therefore, the stably transfected cells were carrying the green fluorescence. However, during our Flow cytometry assay, we took PC cells (Control) with no fluorescence to standardize our equipment and measured the negative control (NC) and overexpressed-Sox2ot (oe-Sox2ot) according to the manufacturer's guidance, without removing the disturbance that the green fluorescence caused. This means that, despite having performed the standard assay, the results obtained in Figure 2D were wrong and unscientific". The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20504.

Mettl3 regulates the proliferation, migration and invasion of glioma cells by inhibiting PI3K/Akt signaling pathway.

OBJECTIVE: Methyltransferase-like 3 (Mettl3), one of "writers" for N6-methyladenosine RNA methylation is determined to participate in a variety of cell biological functions. However, the functions of Mettl3 on tumor growth of glioma remain unknown. Here, we conducted a research to explore the contribution of Mettl3 in the progression of glioma. PATIENTS AND METHODS: To detect the expression level of RNAs, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed. To access the relative level of proteins, Western blot was conducted. The proliferative ability of glioma cells was detected by CCK-8 assay and colony formation assay. The migration and invasion of glioma cells were determined by wound healing assay and transwell invasion assay. RESULTS: The expression of Mettl3 was significantly downregulated in tumor tissues compared to the adjacent normal tissues. The downregulation of Mettl3 led to the enhancement of glioma cell proliferation, migration, and invasion in vitro, and promoted the tumor growth of glioma cells in vivo. In addition, further investigation confirmed that Mettl3 plays critical roles in the development of glioma by targeting PI3K/Akt pathway. CONCLUSIONS: Our study proves that Mettl3 plays a critical role in the proliferation, migration, and invasion of glioma cells by inactivating PI3K/Akt signaling pathway, providing a novel mechanism of glioma tumorigenesis and raising a new target for the treatment of glioma.

Long noncoding RNA SOX2OT maintains the stemness of pancreatic cancer cells by regulating DEK via interacting with miR-200a/141.

OBJECTIVE: Long noncoding RNA sex determination region of Y chromosome (SRY)-related HMG-box (SOX) is involved in the development of various cancers. However, the molecular mechanism of SOXOT, an overlapping transcript of SOX, in pancreatic cancer (PC) is still undefined. We aimed to explore the epigenetic function of SOX2OT and its downstream factors in advanced PC. PATIENTS AND METHODS: The levels of SOX2OT, miRNA, and DEK proto-oncogene (DEK) in pancreatic cancer tissues and cell lines were evaluated by quantitative polymerase chain reaction (qPCR). The log-rank test was applied to evaluate the role of high SOX2OT levels in shortening the overall survival of pancreatic cancer patients. The Chi-squared test was made to assess the relation between SOX2OT expression and clinicopathological features of PC patients. Colony assay tested the cell proliferation of PC cells with SOX2OT knockdown. Flow cytometry and Western blotting were used to determine the stemness of tumor cells in vitro. The underlying regulatory mechanism between SOX2OT and miR-200a/141 was predicted by bioinformatics and verified by RNA transfection, qPCR, and Western blotting. Mice xenograft models were applied to determine the promoting effects of SOX2OT on PC in vivo. RESULTS: The expression of SOX2OT in PC tissues and cell lines is strongly elevated. High levels of SOX2OT expression are more likely to present in patients with advanced TNM stage, positive CD44, and poor overall survival. SOX2OT overexpression promotes proliferation and stemness maintaining of PC cells in vitro and boosts tumor growth in vivo. Furthermore, SOX2OT upregulates DEK expression by binding to miR-200a/141 as a competing endogenous RNA. CONCLUSIONS: DEK induced by SOXOT- miR-200a/141 axis may markedly promote stem cell property of PC, resulting in an advanced stage and inferior survival. These findings suggest the SOX2OT-DEK axis as a novel therapeutic target in PC.

Effect of miR-124 on neuronal apoptosis in rats with cerebral infarction through Wnt/ß-catenin signaling pathway.

OBJECTIVE: The aim of this study was to observe the influence of micro ribonucleic acid (miR)-124 on neuronal apoptosis in rats with cerebral infarction (CI), and to further investigate the underlying mechanism of miR-124 in CI occurrence and development. MATERIALS AND METHODS: A total of 60 adult male Wistar rats were randomly divided into the Sham group, the CI group and the CI + miR-124 mimics group using a random number table. The focal CI model was established using the suture-occluded method. After successful modeling, miR-124 mimics were stereotactically injected into the lateral ventricle of rats. 24 h after operation, the neurological function of rats in each group was scored using modified neurological severity score (mNSS). Meanwhile, the infarction area of brain tissues was evaluated by the triphenyl tetrazolium chloride (TTC) method. The protein expression levels of apoptosis-related genes, including B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), C-Caspase and T-Caspase, were detected via Western blotting. The expression and location of caspase-3 in brain tissues were detected via immunofluorescence staining. Moreover, the level of apoptosis in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In addition, the expression levels of the Wnt/ß-catenin signaling pathway-related proteins were detected via Western blotting. RESULTS: Polymerase Chain Reaction (PCR) results revealed that the expression level of miR-124 in the CI group was significantly decreased when compared with that of the Sham group (p<0.05). The mNSS and TTC staining results manifested that injection of miR-124 mimics could significantly reduce the CI-induced neurological deficits and CI area (p<0.05). At the same time, the levels of Bax and C-caspase/T-Caspase were significantly decreased, whereas the expression of Bcl-2 was remarkably increased after the injection of miR-124 mimics (p<0.05). Besides, the number of apoptotic cells in the CI + miR-124 mimic group was remarkably decreased (p<0.05). In addition, miR-124 mimics significantly activated the expression levels of Wnt and ß-catenin (p<0.05). CONCLUSIONS: The inhibitory effect of miR-124 on neuronal apoptosis in CI rats is probably related to the activation of the Wnt/ß-catenin signaling pathway. Furthermore, miR-124 is expected to be a target drug for the clinical treatment of CI.

Long non-coding RNA H19 regulates the development of gliomas through the Wnt/ß-catenin signaling pathway.

OBJECTIVE: We investigated the effects of long non-coding RNA (lncRNA) H19 on glioma cell proliferation, invasion, migration, and apoptosis and the underlying mechanisms. PATIENTS AND METHODS: H19 expression in glioma tissues, para-carcinoma tissues, and glioma cell lines was analyzed by Real-time polymerase chain reaction (RT-PCR). After transfecting U251 and U87MG cells with siRNA-H19, cell proliferation was detected by the cell counting kit-8 (CCK8) assay. Invasion and migration were detected by a transwell assay; cell cycle distribution and apoptosis were measured by flow cytometry analysis; Dvl2, GSK-3ß, cyclin D1, and ß-catenin expressions were detected by RT-PCR and Western blotting. RESULTS: H19 expression in glioma tissues was higher than that in para-carcinoma tissues and associated with poor prognosis in glioma patients. Cell proliferation, invasion, and migration significantly decreased, the percentage of glioma cells in G0/G1 significantly increased, the percentage of glioma cells in the S phase significantly decreased, and apoptosis significantly increased in U251 and U87MG cells transfected with siRNA-H19 compared to those in the siRNA-NC group. Downregulation of H19 decreased DVL2, cyclin D1, and ß-catenin expression and increased GSK-3ß expression. The inhibitory effects of downregulation of H19 on glioma cell proliferation, invasion, and migration were reversed by SKL2001 via the activation of the Wnt/ß-catenin signal pathway, which was further enhanced by inhibition of the Wnt/ß-catenin signal pathway by XAV939. CONCLUSIONS: H19 was overexpressed in glioma tissues and glioma cell lines. Downregulation of H19 inhibited cell proliferation, invasion, and migration, arrested cell cycle progression in the G0/G1 phase, and induced cell apoptosis by restraining activation of the Wnt/ß-catenin signaling pathway in glioma cells. Therefore, H19 is a potential therapeutic target for glioma therapy.

Differentiation of focal organising pneumonia and peripheral adenocarcinoma in solid lung lesions using thin-section CT-based radiomics.

AIM: To evaluate the predictive role of radiomics based on computed tomography (CT) in discriminating focal organising pneumonia (FOP) from peripheral lung adenocarcinoma (LA). MATERIALS AND METHODS: Institutional research board approval was obtained for this retrospective study. One hundred and seventeen patients with FOP and 109 patients with LA who underwent thin-section CT from January 2011 to August 2017 were reviewed systematically and analysed. The clinical and radiological features were established as model A and multi-feature-based radiomics as model B. The diagnostic performance of model A, model B, and model A+B were evaluated and compared via receiver operating characteristic (ROC) curve analysis and logistic regression analysis. RESULTS: Sex, symptoms, necrosis, and the halo sign were identified as independent predictors of LA. The area under the ROC curve (Az value), accuracy, sensitivity, and specificity of model A were 0.839, 75.7%, 82.6%, and 69.2% respectively. Model B showed significantly higher accuracy than model A (83.6% versus 75.7%, p=0.032). The top four best-performing features, WavEnLH_s-3, WavEnHH_s-3, Teta3, and Volume, performed as independent factors for discriminating LA. Regression analysis indicated that model B had superior model fit than model A with Akaike information criterion (AIC) values of 73.6% versus 59.1%, respectively. Combining model A with model B is useful in achieving better diagnostic performance in discriminating FOP from LA: the Az value, accuracy, sensitivity, and specificity were 0.956, 87.6%, 85.3%, and 89.7% respectively. CONCLUSIONS: Radiomics based on CT exhibited better diagnostic accuracy and model fit than clinical and radiological features in discriminating FOP from LA. Combination of both achieved better diagnostic performance.

Two sphingomyelin synthase homologues regulate body weight and sphingomyelin synthesis in female brown planthopper, N. lugens (Stål).

Although sphingomyelins known to be are lipid constituents of the plasma membrane in vertebrates, much remains obscure about the metabolism of sphingomyelins in insects. With ultra performance liquid chromatography-time-of-flight-tandem mass spectrometry analysis, we revealed for the first time that sphingomyelins are abundant in Nilaparvata lugens (Stål), the brown planthopper (BPH), and their biosynthesis is carried out by sphingomyelin synthase-like protein 2 (SMSL2), which is homologous to sphingomyelin synthase-related protein (SMSr). Unlike other insect species, high concentrations of sphingomyelins rather than ceramide phosphoethanolamines exist in the BPH. Two putative genes, which are homologous to SMSr, are named Nilaparvata lugens SMS-like 1 (NlSMSL1) and 2 (NlSMSL2). Knockdowns of both NlSMSL2 and NlSMSL1 were conducted but only the first decreased concentrations of sphingomyelins in the BPH, indicating that NlSMSL2 plays a role in the biosynthesis of sphingomyelins. Real-time quantitative PCR analysis revealed both NlSMSL1 and NlSMSL2 are highly expressed in BPH adults, with NlSMSL1 specifically highly expressed in reproductive organs (ovaries and testes) whereas NlSMSL2 was highly expressed in the malpighian tubules. The knockdown of NlSMSL1 or NlSMSL2 increased BPH female body weight but not that of males, suggesting sex-specific roles for SMSLs in influencing BPH body weight. The results suggest that NlSMSL2 catalyses the synthesis of sphingomyelins and maintains female BPH body weight through alteration of sphingolipid content.

Quantitative evaluation of diffusion-kurtosis imaging for grading endometrial carcinoma: a comparative study with diffusion-weighted imaging.

AIM: To evaluate the diagnostic performance of histogram analysis of diffusion kurtosis magnetic resonance imaging (DKI) and standard diffusion-weighted imaging (DWI) in discriminating tumour grades of endometrial carcinoma (EC). MATERIALS AND METHODS: Seventy-three patients with EC were included in this study. The apparent diffusion coefficient (ADC) value from standard DWI, apparent diffusion for Gaussian distribution (Dapp), and apparent kurtosis coefficient (Kapp) from DKI were acquired using a 3 T magnetic resonance imaging (MRI) system. The measurement was based on an entire-tumour analysis. Histogram parameters (Dapp, Kapp, and ADC) were compared between high-grade (grade 3) and low-grade (grade 1 and 2) tumours. The diagnostic performance of imaging parameters for discriminating high- from low-grade tumours was analysed using a receiver operating characteristic curve (ROC). RESULTS: The area under the ROC curve (AUC) of the 10th percentile of Dapp, 90th percentile of Kapp and 10th percentile of ADC were higher than other parameters in distinguishing high-grade tumours from low-grade tumours (AUC=0.821, 0.891 and 0.801, respectively). The combination of 10th percentile of Dapp and 90th percentile of Kapp improved the AUC to 0.901, which was significantly higher than that of the 10th percentile of ADC (0.810, p=0.0314) in differentiating high- from low-grade EC. CONCLUSION: Entire-tumour volume histogram analysis of DKI and standard DWI were feasible for discriminating histological tumour grades of EC. DKI was relatively better than DWI in distinguishing high-grade from low-grade tumour in EC.

Influence of Treg cells and HBV genotype on sustained response and drug resistance in the treatment with nucleoside drugs

We aimed to investigate the influence of regulatory T cells including CD4+CD25+, CD8+CD28- and hepatitis B virus (HBV) genotype on sustained virological response and tolerance of nucleoside drugs. One hundred and thirty-seven patients were enrolled. Lamivudine was administered to 84 patients. Entecavir was administered to the other 53 patients. Before treatment, biochemical tests, HBV DNA load, HBV serum level, HBV genotype, PB CD3+, CD4+, CD8+, CD4+CD25+/CD3+, and CD8+CD28-/CD3+ frequencies were measured. Based on HBV DNA loads after 4 weeks of therapy, patients were divided into response group and suboptimal response group. The lamivudine group received treatment continuously, and then patients were categorized into non-resistance group and resistance group. Compared with the suboptimal response and resistance groups for lamivudine, CD4+CD25+/CD3+ levels were higher in the response and non-resistance groups (t=4.372, P=0.046; t=7.262, P=0.017). In the non-resistance group, CD8+CD28-/CD3+ frequency was lower than in the resistance group (t=5.527, P=0.037). Virus load and hepatitis B E antigen (HBeAg)-positive rate were significantly lower than in the response and resistance group (t=2.164, P=0.038; X2=4.239, P=0.040; t=2.015, P=0.044; X2=16.2, P=0.000). Incidence of drug resistance was high in patients with virogene type C. For the virological response to entecavir, CD8+CD28-/CD3+ level was significantly lower than that of the suboptimal response group (t=6.283, P=0.036). Response and suboptimal response groups were compared in CD3+, CD4+, CD8+, CD4+CD25+/CD3+ and virus genotype, and differences were not statistically significant (P>0.05). Baseline regulatory T cells including CD4+CD25+/CD3+ and CD8+CD28-/CD3+ frequencies have a relationship with the incidence of rapid virological response and the resistance to nucleoside drugs. Patients with HBV genotype C receiving lamivudine more often underwent drug resistance. Antiviral efficacy and the resistance to lamivudine were closely correlated with baseline factors; the same cannot be found for entecavir.

The approach of retroperitoneal laparoscopic partial dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels to the treatment of chyluria.

OBJECTIVE: To compare the clinical effect of two surgical methods of treating chyluria, namely, retroperitoneal laparoscopic partial dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels and retroperitoneal laparoscopic complete dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels. PATIENTS AND METHODS: Thirty-eight cases have been divided into A and B groups. Retroperitoneal laparoscopic partial dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels has been performed on Group A patients and retroperitoneal laparoscopic complete dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels has been performed on Group B cases, and then their respective clinical efficacy has been compared. RESULTS: All the operations for the 38 cases were successful. The average operation time for Group A was 76.35 ± 23.11 min, and that for Group B was 97.35 ± 16.20 min. The average post-operative length of stay for Group A was 5.43 ± 1.21 days, and that for Group B was 7.22 ± 1.34 days. No complications were found in both groups, and all cases were tested negative for chyluria when discharged. No recurrences were reported. CONCLUSIONS: Retroperitoneal laparoscopic ligation of renal pedicle lymphatic vessels is a reliable method of treating chyluria. Compared with complete dissection of adipose renal capsule plus ligation of renal lymphatic vessels, partial dissection of adipose renal capsule plus ligation of renal pedicle lymphatic vessels boasts the advantages of shorter operation time, less bleeding, shorter term of hospitalization, and no renal pedicle torsion.

Efficacy of an opposite position aspiration on resolution of pneumothorax following CT-guided lung biopsy.

OBJECTIVE: To evaluate the efficacy of aspiration in an opposite position to deal with pneumothorax after CT-guided lung biopsy. METHODS: A retrospective study was developed involving 210 patients with pneumothorax who had undergone CT-guided percutaneous core biopsies from January 2012 to March 2014 for various pulmonary lesions. Asymptomatic patients with minimal pneumothorax were treated conservatively. Simple manual aspiration was performed for symptomatic patients with minimal pneumothorax and for all patients with moderate to large pneumothorax. An opposite position aspiration was performed when simple manual aspiration failed. The efficacy of simple manual aspiration and the opposite position aspiration was observed. RESULTS: Among 210 patients with pneumothorax, 128 (61.0%) asymptomatic patients with minimal pneumothorax were treated conservatively. The remaining 82 were treated with attempted simple manual aspiration. Out of these 82 patients, simple manual aspiration was successful in 58 (70.7%, 58/82) cases. The complete and partial regression rates were 17.2% (10/58) and 82.8% (48/58), respectively. In the other 24 patients (29.3%, 24/82), simple aspiration technique was ineffective. An opposite position (from prone to supine or vice versa) was applied, and a new biopsy puncture site was chosen for reaspiration. This procedure was successful in 22 patients but not in 2 patients who had to have a chest tube insertion. The complete and partial regression rates were 25.0% (6/24) and 66.7% (16/24), respectively. Applying the new method, the total effective rate of aspiration improved significantly from 70.7% (58/82) to 97.6% (80/82). CONCLUSION: The opposite position aspiration can be safe, effective and minimally invasive treatment for CT-guided lung biopsy-induced pneumothorax thus reducing the use of chest tube significantly. ADVANCES IN KNOWLEDGE: (1) Opposite position aspiration can elevate the success rate of aspiration significantly (from 70.7% to 97.6% in our study); (2) this procedure is a safe, effective and minimally invasive treatment for pneumothorax caused by biopsy; and (3) opposite position aspiration is a useful technique to reduce the use of chest tube, which has clinical significance.

Varicocele anatomy during subinguinal microsurgical varicocelectomy in Chinese men.

Knowledge of subinguinal microsurgical varicocelectomy is of fundamental importance to ensure that varicocele is resolved and testicular function is preserved. Our study aimed to describe the number of veins, arteries and lymphatics in the subinguinal spermatic cord and to clarify their differences between two sides, between patients with different complaints and between varicoceles with different clinical grades. A total of 102 consecutive patients underwent 162 primary subinguinal microsurgical varicocelectomies, during which the number of vessels with different diameters was recorded. A mean number of 12.9 internal spermatic veins, 0.9 external spermatic veins, 1.8 internal spermatic arteries and 2.9 lymphatics were identified per cord. 88.2% of the internal spermatic arteries were surrounded by a dense complex of adherent veins. The external spermatic vein or veins were found in 49.4% of the cases. The mean number of medium (1-3 mm in diameter) internal spermatic veins on the left was larger than that on the right (P < 0.001). The mean number of medium internal spermatic veins in grade III varicocele was larger than that in grade I or grade II (P < 0.015). There was no significant anatomical difference between the men presenting for infertility, chronic testicular pain and both the two complaints.

Screening of differentially expressed genes of middle cerebral artery occlusion with DNA microarray.

OBJECTIVES: To screen differentially expressed genes of different days after cerebral artery occlusion and drug treatment, and identify related small drug molecules. MATERIALS AND METHODS: The gene expression profile GSE35338 of cerebral artery occlusion was downloaded from Gene Expression Omnibus database, including a total of 14 samples. 5 samples are 1 day after cerebral artery occlusion (control), 3 samples are 7 days after cerebral artery occlusion and 3 samples are under lipopolysaccharide (LPS) treatment. Differentially expressed genes (DEGs) between different days after cerebral artery occlusion were screened (p < 0.05, FDR < 0.05, |logFC| > 1). The DEGs were then entered into the CMAP database and related small drug molecules were retrieved, followed by calculation of co-expression score of the genes and construction of co-expression-drug network. FuncAssociate software and DAVID were used to obtain the functional clusters of genes with p-value < 0.05 and FDR < 0.05. RESULTS: Compared with the control group, 825, 1445, 218 DEGs and 4, 3, 2 most-related small drug molecules were respectively identified from 3, 7 days after cerebral artery occlusion and LPS treated group. Co-expression network was constructed and functional clusters were found to be 161, 146, and 6 in each group. CONCLUSIONS: Our study provides some underlying biomarkers for cerebral artery occlusion under varied conditions and potential small drug molecules for treatment of cerebral artery occlusion.

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer

AIM: Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer. METHODS: RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n = 99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan-Meier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. RESULTS: RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P = 0.008) and high UICC stage (P = 0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P = 0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator. CONCLUSION: These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer (AU)

Abnormal endothelial function in ED patients with normal nocturnal penile tumescence and rigidity: is it the role of psychogenic factors?

Underlying endothelial dysfunction (EnD) may present in the early stage of ED or psychogenic ED. We retrospectively evaluated 191 ED patients with effective nocturnal penile tumescence and rigidity (NPTR) recording, including detailed medical and psychosexual history, International Index of Erectile Function-5 and vascular parameter. All patients were allocated into psychogenic and organic groups according to the NPTR test. Brachial artery flow-mediated dilation (FMD) was used to diagnose EnD, and ED patients were classified into two groups: non-EnD (FMD10) and EnD (FMD<10). General and vascular parameters were compared between psychogenic and organic groups, and non-EnD and EnD groups with ED were compared in terms of NPTR parameters. In all, 48.7% and 51.3% patients were diagnosed as psychogenic and organic ED, respectively. 73.1% of the psychogenic patients had EnD and 39.8% organic patients had normal endothelial function. In all parameters, only the FMD value showed significant difference between psychogenic and organic ED groups (8.26±2.57 vs 9.16±2.76, P=0.020). No statistical difference was founded in NPTR parameters between non-EnD and EnD groups (P>0.05). In conclusion, NPTR cannot effectively identify the underlying vasculogenic ED from psychogenic ED. Psychogenic causes may cause or aggravate EnD in these ED patients with normal NPTR.

Histone demethylase Kdm4b functions as a co-factor of C/EBPß to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes.

CCAAT/enhancer-binding protein (C/EBP) ß is required for both mitotic clonal expansion (MCE) and terminal adipocyte differentiation of 3T3-L1 preadipocytes. Although the role of C/EBPß in terminal adipocyte differentiation is well defined, its mechanism of action during MCE is not. In this report, histone demethylase Kdm4b, as well as cell cycle genes Cdc45l (cell division cycle 45 homolog), Mcm3 (mini-chromosome maintenance complex component 3), Gins1 (GINS complex subunit 1) and Cdc25c (cell division cycle 25 homolog c), were identified as potential C/EBPß target genes during MCE by utilizing promoter-wide chromatin immunoprecipitation (ChIP)-on-chip analysis combined with gene expression microarrays. The expression of Kdm4b is induced during MCE and its induction is dependent on C/EBPß. ChIP, Electrophoretic Mobility Shift Assay (EMSA) and luciferase assay confirmed that the promoter of Kdm4b is bound and activated by C/EBPß. Knockdown of Kdm4b impaired MCE. Furthermore, Kdm4b interacted with C/EBPß and was recruited to the promoters of C/EBPß-regulated cell cycle genes, including Cdc45l, Mcm3, Gins1, and Cdc25c, demethylated H3K9me3 and activated their transcription. These findings suggest a novel feed forward mechanism involving a DNA binding transcription factor (C/EBPß) and a chromatin regulator (Kdm4b) in the regulation of MCE by controlling cell cycle gene expression.