1.Function and mechanism of TRIM22 targeting eIF4E in the process of NB4 cells differentiation
Yang HAN ; Guanhua SONG ; Jing TIAN ; Qiong LIAO ; Lianlian LI ; Xiaoyu ZHANG ; Hongyan LIU ; Zhiyong ZHANG ; Guosheng JIANG
Journal of International Oncology 2017;44(4):251-256
Objective To investigate the function of tripartite motif protein 22 (TRIM22) and the interaction with eukaryotic translation initiation factor-4E (eIF4E) in the differentiation of NB4 cells, one kind of acute promyelocytic leukemia cells, which elucidates the mechanism of TRIM22 targeting to regulate eIF4E.Methods The model of NB4 cells inducing differentiation was established in vitro.The expression changes of gene and protein of TRIM22 and eIF4E were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.In addition, the effect on cell function and protein expression level of eIF4E after adopting electroporation technology to depress or over-express TRIM22 was detected by CCK-8 and flow cytometry.Finally, the interaction of TRIM22 and eIF4E was verified by using co-immunoprecipitation (CO-IP).Results The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79 (F=280.700, P=0.000), and the protein relative expression level was gradually increasing from 0.22±0.03 to 0.51±0.05 (F=51.430, P=0.000) after the all-trans-retinoic acid (ATRA) induction for NB4 cell.However, the mRNA relative expression level of eIF4E was gradually decreasing from 1.01±0.09 to 0.47±0.06 (F=20.520, P=0.000), with the same trend, the protein relative expression level was gradually decreasing from 0.97±0.02 to 0.64±0.09 (F=14.700, P=0.001).The expression level of PE-CD11b in the TRIM22 over-expression group with ATRA detected by flow cytometry [(78.80±2.00)%] was higher than that in the transfection group of empty vetor with ATRA [(58.70±2.70)%] (t=9.535, P=0.000) and the cotransfection group with ATRA [(61.60±3.80)%] (t=8.187, P=0.000).Meanwhile, the protein level of eIF4E changed reversely after over-expressing the gene level of TRIM22 (t=4.985, P=0.007).The CO-IP experiment was used to verify the interaction of TRIM22 and eIF4E.ConclusionTRIM22 is able to promote the cell differentiation during the process of NB4 cells differentiation.Furthermore, eIF4E is a target of TRIM22 for binding with, which plays an important role in depressing the expression of eIF4E.
2.Changes of intestinal macrophage subsets at different stages in mice of inflammatory bowel disease
Lianlian TIAN ; Yan LIN ; Shibo GUO ; Hongfang ZHAO ; Chunyan DU ; Yalong ZHANG ; Yaxing HUI ; Wei ZHANG ; Baoxi WANG ; Xun JIANG
Chinese Journal of Applied Clinical Pediatrics 2021;36(16):1262-1266
Objective:To establish a mice model of inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS), and to analyze the changes in intestinal inflammation and macrophage subsets at different stages, so as to find a new target for the treatment of IBD.Methods:Thirty male C57BL/6 mice of 6-8 weeks were randomly divided into control group, activation stage group and resolution stage group.The latter 2 groups were given 25 g/L DSS for 5 consecutive days to establish the IBD model.After 5 days, the mice were given filtered and sterilized water and sacrificed on the 10 th and 15 th day, respectively.Colon inflammation in mice was evaluated, including body weight, disease activity index (DAI) score, changes in colon length, histopathology and histopathological score.Then the expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β in colon tissues were detected by quantitative real-time PCR(qPCR). Finally, the changes of intestinal macrophage subsets were detected by flow cytometry. Results:The colon inflammation of mice in the activation stage group was significantly more severe than that in the control group, while the colon inflammation of mice in the resolution stage group was reduced.The colon length of mice in the activation stage group was (5.94±0.40) cm, which was significantly shorter than that in the control group [(7.25±0.29) cm], and the situation was slightly improved in the resolution stage with the colon length of [(6.87±0.95) cm], and the differences were statistically significant (all P<0.05). The mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the activation stage were 53.40±6.58, 117.69±30.78 and 2.52±0.25, respectively, which were significantly higher than those in the control group (1.00±0.13, 1.00±0.39, 1.00±0.10); the mRNA expression levels of IL-1β, IL-6 and TNF-α in the resolution stage were 2.51±0.13, 5.43±0.51 and 1.73±0.14, respectively, which were significantly lower than those in the activation stages(all P<0.05). The expression level of anti-inflammatory cytokine TGF-β in the resolution stage was 2.41±0.17, which was significantly higher than that in the activation stage (0.94±0.12), and the diffe-rence was statistically significant ( P<0.05). During the progression of IBD, there were 3 groups of macrophages in the lamina propria of intestinal mucosa of mice, of which the number of F4/80 lowCD 64-MHCⅡ - macrophage subset with the lowest maturity increased significantly in the activation stage of IBD, accounting for (10.68±4.62)%, and it decreased and returned to the normal level in the resolution stage, accounting for (4.63±1.06)%, and the difference was statistically significant ( P<0.05). Conclusions:Macrophages play an important role in the progression of IBD, the hindrance of maturation and development may be the main cause of inflammatory injury in the activation stage of IBD, and the transformation of macrophage subsets may become a new target for the treatment of IBD.
3.Establishment of a prognostic model of Wnt signaling pathway related genes in gastric cancer
Lianlian TIAN ; Jun ZHU ; Qian MA ; Han PENG ; Yiran ZHANG ; Zhaoxi WANG ; Rui CHEN
Journal of Xi'an Jiaotong University(Medical Sciences) 2022;43(2):252-257
【Objective】 To confirm the role of Wnt signaling pathway in the occurrence and development of gastric cancer (GC), establish a prognostic model composed of Wnt pathway related genes, and then evaluate the predictive value of the model. 【Methods】 We downloaded the gene expression data and survival data of GC in TCGA database, and used GSEA enrichment analysis to verify the enrichment of Wnt pathway in GC and para-cancer samples. In this study, univariable COX regression analysis and survival curve analysis were used to select the prognosis-related genes of GC. Then the multivariate COX proportional hazard regression model was used to obtain the prognostic model of Wnt signaling pathway related genes. Then, receiver operating characteristic (ROC) curve and forest plot were used to verify the clinical predictive value of the model. The model was then validated in GEO external database. Finally, by utilizing quantitative real-time PCR (qPCR), we detected the expressions of Wnt signaling pathway related genes in 8 pairs of clinical GC and para-cancer samples. 【Results】 We downloaded 32 samples of normal para-cancer samples and 375 cancer samples and their corresponding clinical data. GSEA enrichment showed that compared with normal samples, Wnt pathway was significantly enriched in GC samples (P<0.05). The results of univariate COX analysis showed that 13 Wnt pathway genes were closely related to the prognosis of GC patients. Multivariate COX determined that the model was multiplied and accumulated by ETV2, SERPINE1, CPZ, VPS35 and IGFBP1 and their corresponding coefficient β. The survival curve and ROC curve showed that the model could accurately predict the prognosis of GC patients, and the 1-year, 3-year, and 5-year areas under the curve (AUC) were 68.0%, 69.4% and 78.5%, respectively. Clinical univariate and multivariate COX analyses showed that the model could become an independent prognostic factor other than TNM system of GC. The external data set (GSE84437) validation results of GC showed that the model could better predict the prognosis of GC patients. qPCR results indicated that ETV2, SERPINE1, CPZ, VPS35 and IGFBP1 expressions were upregulated in GC samples compared with para-cancer samples. 【Conclusion】 This study further confirmed that Wnt pathway plays an important role in the progress of GC from the perspective of bioinformatics, and we have established a prognosis-related risk model, providing a new perspective for clinical genetic testing, targeted therapy and individualized therapy.