Y-box-binding protein 1 mediates sorafenib resistance via the extracellular signal regulated-protein kinase pathway in hepatoma cells / 中华肝脏病杂志

Objective: To investigate the effect and possible mechanism of Y-box-binding protein 1 (YB-1) on sorafenib resistance in hepatoma cells. Methods: Lentiviral vectors with YB-1 overexpression and knockdown were constructed, respectively, to stimulate human hepatoma cell lines (HepG2 and Huh7) alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups: overexpression control group (Lv-NC), YB-1 overexpression group (Lv-YB-1), overexpression control combined with sorafenib resistance group (Lv-NC+sorafenib), YB-1 overexpression combined with sorafenib resistance group (Lv-YB-1 + sorafenib). The knockdown part of the experiment was also divided into four groups: knockdown control group (Lv-shNC), YB-1 knockdown group (Lv-shYB-1), knockdown control combined with sorafenib resistance group (Lv-shNC + sorafenib), YB-1 knockdown combined with sorafenib resistance group (Lv-shYB-1 + sorafenib). The occurrence of cell apoptosis was detected by TUNEL. The protein expression levels of phosphorylated (p)-ERK and ERK, key proteins in the extracellular regulatory protein kinase (ERK) signaling pathway, were detected by Western blot and quantified by ImageJ software. Subcutaneous tumorigenesis experiments were performed in nude mice. The effect of YB-1 on the efficacy of sorafenib was verified in vivo. The comparison between the two sets of data was carried out by an independent sample t-test. One-way ANOVA was used for comparisons between the three groups of data above. Results: Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells, while YB-1 overexpression had inhibited cell apoptosis, and at the same time also inhibited the apoptosis-accelerating impact of sorafenib. On the contrary, YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis. Furthermore, sorafenib resistance had down-regulated p-ERK levels (HepG2: Lv-NC 0.685 ± 0.143, Lv-NC + sorafenib 0.315 ± 0.168, P < 0.05; Huh7: Lv-NC 0.576 ± 0.078, Lv-NC + sorafenib 0.150 ± 0.131, P < 0.01), whereas YB-1 overexpression had inhibited sorafenib resistance p-ERK reduction (HepG2: Lv-NC + sorafenib 0.315 ± 0.168, Lv-YB-1 + sorafenib 0.688 ± 0.042, P < 0.05; Huh7: Lv-NC + sorafenib 0.150 ± 0.131, Lv-YB-1 + sorafenib 0.553 ± 0.041, P < 0.05). YB-1 knockdown further increased sorafenib-induced p-ERK downregulation (HepG2: Lv-shNC + sorafenib 0.911 ± 0.252, Lv-shYB-1 + sorafenib 0.500 ± 0.201, P < 0.05; Huh7: Lv-shNC + sorafenib 0.577 ± 0.082, Lv-shYB-1 + sorafenib 0.350 ± 0.143, P < 0.05), which was further verified in naked mice (Lv-shNC + sorafenib 0.812 ± 0.279, Lv-shYB-1 + sorafenib 0.352 ± 0.109, P < 0.05). Conclusion: YB-1 mediates the occurrence of sorafenib resistance via the ERK signaling pathway in hepatoma cells.

Identification of differentially expressed genes between lung adenocarcinoma and squamous cell carcinoma using transcriber signature analysis / 南方医科大学学报

OBJECTIVE@#To analyze the differentially expressed genes (DEGs) between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) with bioinformatics analysis and search for potential biomarkers for clinical diagnosis of nonsmall cell lung cancer (NSCLC).@*METHODS@#The gene expression profiling datasets of LUAD and LUSC were acquired. The transcriptome differences between LUAD and LUSC were identified using R language processing and t-test analysis. The differential expressions of the genes were shown by Venn diagram. The DEGs identified by GEO2R were analyzed with DAVID and Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and biomarkers that could be used for differential diagnosis of LUAD and LUSC. The TCGA data and the biomarker expression data from clinical lung cancer samples were used to verify the differential expressions of the Osteoarthritis pathway and LXR/RXR between LUAD and LUSC. We further examined the differential expressions of miR-181 and its two target genes, and , in 23 clinical specimens of lung squamous cell carcinoma and the paired adjacent tissues.@*RESULTS@#GEO data analysis identified 851 DEGs (including 276 up-regulated and 575 down-regulated genes) in LUAD and 885 DEGs (including 406 up-regulated and 479 down-regulated genes) in LUSC. DAVID and IPA analysis revealed that leukocyte migration and inflammatory responses were more abundant in LUAD than in LUSC. Osteoarthritis pathway was inhibited in LUAD and activated in LUSC. IPA analysis showed that transcription factors (GATA4, RELA, YBX1, TP63 and MBD2), cytokines (WNT5A and IL1A) and microRNAs (miR-34a, miR-181b and miR-15a) differed significantly between LUAD and LUSC. miR-34a with IL-1A, miR-15a with YBX1, and miR-181b with WNT5A and MBD2 could serve as the paired microRNA and mRNA targets for differential diagnosis of NSCLC subtypes. Analysis of the clinical samples showed an increased expression of miR-181b-5p and the down-regulation of WNT5A, which could be used as molecular markers for the diagnosis of LUSC.@*CONCLUSIONS@#Through transcriptome analysis, we identified candidate genes, paired microRNAs and pathways for differentiating LUAD and LUSC, and they can provide novel differential diagnosis and therapeutic strategies for LUAD and LUSC.

Correlation of Twist and YB-1 up-regulation and epithelial-mesenchymal transition during tumorigenesis and progression of cervical carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the clinicopathological significance of Twist and YB-1 up-regulation in cervical cancer, and to correlate the expression of the two genes with E-cadherin, a marker of epithelial-mesenchymal transition (EMT).</p><p><b>METHODS</b>A total of 202 tissue samples were collected during January 2008 to December 2013, including 50 cases of normal cervical tissues, 100 cases of cervical intraepithelial neoplasia (CIN) and 52 cases of squamous cell carcinoma (SCC). Twist, YB-1 and E-cadherin expression was investigated by MaxVision.</p><p><b>RESULTS</b>Increased expression levels of Twist and YB-1 were found and correlated with the malignant transformation of cervical epithelium, histological progression and metastasis of cervical cancer. In addition, Twist and YB-1 overexpression was also associated with aberrant expression of E-cadherin. Regression analysis revealed that Twist expression was an independent factor for the histological progression of cervical cancer.</p><p><b>CONCLUSIONS</b>It is suggested that Twist and YB-1 overexpression is significantly linked to cervical cancer tumorigenesis and progression, likely related to EMT through (YB-1)-Twist-(E-cadherin) pathway. Twist and YB-1 may be markers for determining the metastatic potential of cervical cancer.</p>

YB-1 stabilizes HIV-1 genomic RNA and enhances viral production

HIV-1 utilizes cellular factors for efficient replication. The viral RNA is different from cellular mRNAs in many aspects, and is prone to attacks by cellular RNA quality control systems. To establish effective infection, the virus has evolved multiple mechanisms to protect its RNA. Here, we show that expression of the Y-box binding protein 1 (YB-1) enhanced the production of HIV-1. Downregulation of endogenous YB-1 in producer cells decreased viral production. YB-1 increased viral protein expression by stabilizing HIV-1 RNAs. The stem loop 2 in the HIV-1 RNA packaging signal was mapped to be the YB-1-responsive element. Taken together, these results indicate that YB-1 stabilizes HIV-1 genomic RNA and thereby enhances HIV-1 gene expression and viral production.

overview of therapeutic approaches to brain tumor stem cells

Primary and secondary malignant central nervous system [CNS] tumors are devastating invasive tumors able to give rise to many kinds of differentiated tumor cells. Glioblastoma multiform [GBM], is the most malignant brain tumor, in which its growth and persistence depend on cancer stem cells with enhanced DNA damage repair program that also induces recurrence and resists current chemo- and radiotherapies. Unlike non-tumor stem cells, tumor stem cells lack the normal mechanisms that regulate proliferation and differentiation, resulting in uncontrolled production and incomplete differentiation of tumor cells. In current paper recent developments and new researches in the field of brain tumor stem cells have been reviewed

Expression of YB-1 enhances production of murine leukemia virus vectors by stabilizing genomic viral RNA

Murine leukemia virus (MLV)-based retroviral vectors is widely used for gene transfer and basic research, and production of high-titer retroviral vectors is very important. Here we report that expression of the Y-box binding protein 1 (YB-1) enhanced the production of infectious MLV vectors. YB-1 specifically increased the stability of viral genomic RNA in virus-producing cells, and thus increasing viral RNA levels in both producer cells and virion particles. The viral element responsive to YB-1 was mapped to the repeat sequence (R region) in MLV genomic RNA. These results identified YB-1 as a MLV mRNA stabilizer, which can be used for improving production of MLV vectors.

Effect of regulation of Y-box protein 1 by RNA interference on the doxorubicin induced mdr1 gene expression in K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of YB-1 on the transcription of induced mdr1 gene expression in K562 cells.</p><p><b>METHODS</b>K562 cells were treated with doxorubicin (DOX) at different concentrations and times. Expression of mdr1 and YB-1 genes was examined by RT-PCR and P-glycoprotein (P-gp) by flow cytometry. Cyto/nuclear protein was extracted for YB-1 detection by Western blotting. The expression of YB-1 gene in K562 cells was inhibited by YB-1 gene specific RNA interference (RNAi), then the expression of mdr1 and P-gp in YB-1 gene silenced cells treated with DOX was detected.</p><p><b>RESULTS</b>The mdr1 gene as well as its corresponding protein P-gp was highly expressed in DOX exposed K562 cells. DOX up-regulated the expression of YB-1 gene, and promoted YB-1 protein nuclear translocation. On YB-1 gene silenced, the expressions of mdr1 gene and P-gp were obviously down-regulated in DOX treated K562 cells.</p><p><b>CONCLUSION</b>Doxorubicin can induce the expression of mdr1 gene in K562 cells, which may result from the transcription of mdr1 gene by activated YB-1.</p>

Mechanisms of preventive effect of tetrandrine on acquired multidrug resistance in K562 cells / 中国实验血液学杂志

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.

Influence of YB-1 protein on the biological behaviour in K562/A02 cells / 中国实验血液学杂志

The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.

Effects of changwelqing on nuclear translocation of Y-box binding protein-1 and expression of P-glycoprotein in human colon cancer cell line with drug-resistance induced by vincristine / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To evaluate the effects and molecular mechanism of action of Changweiqing (CWQ) in reversing multidrug resistance by observing its impacts on nuclear translocation of Y-box binding protein-1 (YB-1), multi-drug resistance gene (MDR1) expression and P-glycoprotein (P-gp) expression in human colon cancer cell line HCT8/V with drug-resistance induced by vincristine.</p><p><b>METHODS</b>Cultured HCT8/V cells were exposed to the CWQ-containing rat serum prepared by drug perfusion. YB-1 expressions in cell plasma and nuclei were examined by Western blot; the binding activity of YB-1 to MDR1 gene promoter sequences was detected by electrophoretic mobility shift assay (EMSA); the mRNA transcription levels of MDR1, YB-1 and multi-resistance related protein (MRP) were examined by RT-PCR; the expression of P-gp on cell membrane was determined by flow cytometry. Results Along with the increasing drug's concentration of CWQ-containing serum from 1.25% up to 2.5% and 5%, the expressions of YB-1 decreased in HCT8/V cell nuclear and increased in cytoplasm gradually; the binding activity of YB-1 to MDR1 gene promoter weakened (P < 0.01), MDR1 mRNA expression and fluorescence intensity of P-gp on cell membrane attenuated (P < 0.05 or P < 0.01), while YB-1 and MRP mRNA unchanged (P > 0.05).</p><p><b>CONCLUSION</b>CWQ could reverse the drug-resistance of colon cancer cells by influencing nuclear translocation of YB-1 and reducing the expression of MDR1/P-gp.</p>

Effect of YB-1 gene knockdown on human leukemia cell line K562/A02 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry.</p><p><b>RESULTS</b>The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3.</p><p><b>CONCLUSION</b>Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.</p>

Effects of acupuncture on expressions of transcription factors NF-E2, YB-1, LRG47 in the SAMP10 mouse / 中国针灸

<p><b>OBJECTIVE</b>To explore the mechanism of acupuncture in delaying aging.</p><p><b>METHODS</b>Using SAMP10 mice and normal control SAMR1 as model and applying RT-PCR and DIG probed Northern blot techniques to observe expression of NF-E2, YB-1, LRG47 genes in whole brain, cortex and hippocampus in the 8-month SAMR1 control group, 8-month SAMP10 control group, 8-month SAMP10 acupuncture group and 8-month SAMP10 non-point acupuncture group.</p><p><b>RESULTS</b>In the SAMP10 control group, the expression of NF-E2, YB-1 and LRG47 were down-regulated in the whole brain, cortex and hippocampus, and after acupuncture they were up-regulated and tended to normal.</p><p><b>CONCLUSION</b>Aging of the SAMP10 mouse brain is related with expression of NF-E2, YB-1 and LRG47 genes, and acupuncture can regulate the expression of NF-E2, YB-1 and LRG47 genes, improving the functions of erythrocyte series, increasing proliferation of cells and immune function of cells in anti-bacteria, hence anti-aging.</p>