The role of tazarotene-induced gene 1 in carcinogenesis:is it a tumor suppressor gene or an oncogene?

Tazarotene-induced gene 1(TIG1)is induced by a derivative of vitamin A and is known to regulate many important biological processes and control the development of cancer.TIG1 is widely expressed in various tissues;yet in many cancer tissues,it is not expressed because of the methylation of its promoter.Additionally,the expression of TIG1 in cancer cells inhibits their growth and invasion,suggesting that TIG1 acts as a tumor suppressor gene.However,in some cancers,poor prognosis is associated with TIG1 expression,indicating its protumor growth characteristics,especially in promoting the invasion of inflammatory breast cancer cells.This review comprehensively summarizes the roles of the TIG1 gene in cancer development and details the mechanisms through which TIG1 regulates cancer development,with the aim of understanding its various roles in cancer development.

The clinical association of programmed cell death protein 4(PDCD4) with solid tumors and its prognostic significance:a meta-analysis

Background: Programmed cell death protein 4(PDCD4) is a novel tumor suppressor protein involved in pro?grammed cell death. Its association with cancer progression has been observed in multiple tumor models, but evidence supporting its association with solid tumors in humans remains controversial. This study aimed to determine the clinical signiicance and prognostic value of PDCD4 in solid tumors.Methods: A systematic literature review was performed to retrieve publications with available clinical informa?tion and survival data. The eligibility of the selected articles was based on the criteria of the Dutch Cochrane Centre proposed by the Meta?analysis Of Observational Studies in Epidemiology group. Pooled odds ratios(ORs), hazard ratios(HRs), and 95% conidence intervals(CIs) for survival analysis were calculated. Publication bias was examined by Begg's and Egger's tests.Results: Clinical data of 2227 cancer patients with solid tumors from 23 studies were evaluated. PDCD4 expression was signiicantly associated with the diferentiation status of head and neck cancer(OR 4.25, 95% CI 1.87–9.66) and digestive system cancer(OR 2.87, 95% CI 1.84–4.48). Down?regulation of PDCD4 was signiicantly associated with short overall survival of patients with head and neck(HR: 3.44, 95% CI 2.38–4.98), breast(HR: 1.86, 95% CI 1.36–2.54), digestive system(HR: 2.12, 95% CI 1.75–2.56), and urinary system cancers(HR: 3.16, 95% CI 1.06–9.41).Conclusions: The current evidence suggests that PDCD4 down?regulation is involved in the progression of several types of solid tumor and is a potential marker for solid tumor prognoses. Its clinical usefulness should be conirmed by large?scale prospective studies.

Bioinformatics Analysis Revealed Potential Tumor Suppressors (KLF4/CGN), Oncogenes (SHH/LIF) and Biomarkers of Asian Stomach Adenocarcinoma

Stomach adenocarcinoma (STAD) is the fifth most prevalent cancer and the third leading cause of cancer-related death in the world and is more common in Asia than in most Western countries. There is an urgent need to identify potential novel oncogenes and tumor suppressor genes, and biomarkers for STAD. 6652 differentially expressed genes were identified between STAD and normal samples based on the transcriptome data analysis of the TCGA and GEO databases. 13 key modules were identified in STAD by WGCNA analysis. 293 potential STAD associated genes were identified from intersection by Venn Diagram. The 293 intersected genes were enriched in cell cortex and infection by GO and KEGG analysis. 10 hub genes were identified from PPI and Cytoscape analyses of the intersected genes. KLF4/CGN low and SHH/LIF high expression were associated with short overall survival of Asian STAD patients. Bioinformatics analysis revealed potential novel tumor suppressors (KLF4/CGN), oncogenes (SHH/LIF) and biomarkers for diagnosis, therapy and prognosis of STAD, specifically for Asian patients.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization

Objective YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors;differentiating between these roles may depend on the YAP1 phosphorylation pattern.The specific function of YAP1 in B cell acute lymphoblastic leukemia(B-ALL),however,is currently unclear.Thus,in the present study,the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets.Methods The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting,quantitative real-time polymerase chain reaction,flow cytometry,immunostaining,and nude mouse subcutaneous tumorigenesis experiments.Gene expression levels of Hippo pathway-related molecules before and after verteporfin(VP)treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells.Results Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels.YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells;YAP1 was distributed in the nuclei in NALM6 cells.Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase.Before and after VP treatment,the expression of the upstream gene LATS1 was upregulated;its overexpression promoted YAP1-Ser127 phosphorylation.Further,YAP1 was distributed in the plasma.Conclusion LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function;thus,VP,which targets this axis,may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor

BACKGROUND Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.Tyrosine kinase inhibitors,such as imatinib,have been used as first-line therapy for the treatment of GISTs.Although these drugs have achieved considerable efficacy in some patients,reports of resistance and recurrence have emerged.Extracellular signal-regulated kinase 1/2(ERK1/2)protein,as a member of the mitogen-activated protein kinase(MAPK)family,is a core molecule of this signaling pathway.Nowadays,research reports on the important clinical and prognostic value of phosphorylated-ERK(P-ERK)and phosphorylated-MAPK/ERK kinase(P-MEK)proteins closely related to raf kinase inhibitor protein(RKIP)have gradually emerged in digestive tract tumors such as gastric cancer,colon cancer,and pancreatic cancer.However,literature on the expression of these downstream proteins combined with RKIP in GIST is scarce.This study will focus on this aspect and search for answers to the problem.AIM To detect the expression of RKIP,P-ERK,and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease.Try to establish a new prognosis evaluation model using RKIP and PERK in combination with analysis and its prognosis evaluation efficacy.METHODS The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information.These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020.Immunohistochemical method was used to detect the expression of RKIP,PERK,and P-MEK proteins in GIST tissue samples from these patients.Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data.A Nomogram was used to represent the new prognostic evaluation model.The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors,based on two risk classification systems[the new risk grade model vs the modified National Institutes of Health(NIH)2008 risk classification system].Receiver operating characteristic(ROC)curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems.RESULTS In GIST tissues,RKIP protein showed positive expression in the cytoplasm and cell membrane,appearing as brownish-yellow or brown granules.The expression of RKIP was related to GIST tumor size,NIH grade,and mucosal invasion.P-ERK protein exhibited heterogeneous distribution in GIST cells,mainly in the cytoplasm,with occasional presence in the nucleus,and appeared as brownish-yellow granules,and the expression of P-ERK protein was associated with GIST tumor size,mitotic count,mucosal invasion,and NIH grade.Meanwhile,RKIP protein expression was negatively correlated with P-ERK expression.The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis.However,RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance.Furthermore,we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model.ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system.CONCLUSION Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size,NIH 2008 staging,and tumor invasion,and P-ERK expression was also related to mitotic count.The expression of the two proteins had a certain negative correlation.The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients.The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

Function of apoptosis and expression of the proteins Bcl-2,p53 and C-myc in the development of gastric cancer

INTRODUCTIONIn China ,the incidence and mortality of gastric cancer rank the second among all cancers. Recent development of cancer [1-20].The aim of this study was investigat the insight of apoptosis and bcl-2, p53 and C-myc protein expression in the development of gastric cancer .

AU-rich element-binding proteins in colorectal cancer

Trans-acting factors controlling mRNA fate are critical for the post-transcriptional regulation of inflammation-related genes, as well as for oncogene and tumor suppressor expression in human cancers. Among them, a group of RNA-binding proteins called "Adenylate-Uridylate-rich elements binding proteins"(AUBPs)control mRNA stability or translation through their binding to AU-rich elements enriched in the 3'UTRs of inflammation-and cancer-associated mRNA transcripts. AUBPs play a central role in the recruitment of target mRNAs into small cytoplasmic foci called Processing-bodies and stress granules(also known as P-body/SG). Alterations in the expression and activities of AUBPs and Pbody/SG assembly have been observed to occur with colorectal cancer(CRC)progression, indicating the significant role AUBP-dependent post-transcriptional regulation plays in controlling gene expression during CRC tumorigenesis.Accordingly, these alterations contribute to the pathological expression of many early-response genes involved in prostaglandin biosynthesis and inflammation,along with key oncogenic pathways. In this review, we summarize the current role of these proteins in CRC development. CRC remains a major cause of cancer mortality worldwide and, therefore, targeting these AUBPs to restore efficient post-transcriptional regulation of gene expression may represent an appealing therapeutic strategy.

Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis

Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of mefalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy.

Bioinformatic analysis for structure and function of TCTP from Spirometra mansoni

Objective:To predict structure and function of translationally controlled tumor protein(TCTP)from Spirometraa mansoni by bioinformatics technology,and to provide a theoretical basis for further study.Methods:Open reading frame(ORF)of EST sequence from Spirometra mansoni was obtained by ORE finder and was translated into amino acid residue by DNAclub.The structure domain was analyzed by Blast.By the method of online analysis tools:Protparam,InterProScan,protscale.SignalP-3.0,PSORTⅡ,BepiPred,TMHMM,VectorNT1 Suite 9 packages and Phyre2,the structure and function of the protein were predicted and analyzed.Results:The results showed that the EST sequence was Sm TCTP with 173 amino acid residues,theoretical molecular weight was 19 872.0 Da.The protein has the closest evolutionary status with Clonorchis sinensis.Schistosoma mansoni,and Schistosoma japonicum.Then it had no signal peptide site and transmembrane domain.Secondary structure of TCTP contained twoα-helices and eightβ-strands.Conclusions:Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule anti drug target.

Clinical and prognostic significance of raf kinase inhibitory protein expression in gastrointestinal stromal tumors

AIM To detect the expression of Raf kinase inhibitory protein(RKIP) in gastrointestinal stromal tumors(GISTs) and to analyze its relationship with clinicopatholgical characteristics and prognosis of this disease.METHODS Sixty-three patients with pathologically diagnosed GISTs who underwent surgical resection at the Shengjing Hospital of China Medical University from January 2011 to January 2015 and had complete clinical,pathological,and follow-up data were included. Immunohistochemical method was used to detect the expression of RKIP in GIST tissue samples from these patients. KaplanMeier method was used to calculate the survival rate of 60 patients with complete follow-up data,and Cox regression analysis was performed to identify factors affecting the prognosis of patients GISTs to evaluate further the diagnostic and prognostic value of RKIP in GISTs.RESULTS In GIST tissues,RKIP positive signals,manifesting as brownish yellow or brown granules,were located in the cytoplasm or on the membrane. Of 63 tissue samplesincluded in this study,34(54%) were positive and 29(46%) were negative for RKIP expression. Statistical analysis showed that RKIP expression in GISTs was significantly associated with tumor size,National Institutes of Health(NIH) risk grade,and mucosal invasion,but had no significant association with age,gender,tumor location,or the number of mitotic figures. Univariate Kaplan-Meier analysis revealed that the 1-,3-,and 5-year survival rates were 94.4%,89.2%,and 80.5% for patients with positive RKIP expression,and 88.6%,68.2%,and 48.2% for patients with negative RKIP expression,suggesting that patients with high RKIP expression had significantly higher survival rates than those with low expression(Log-rank test,P = 0.0015). Cox regression analysis demonstrated that NIH risk grade was significantly associated with the prognosis of GISTs(P = 0.037),suggesting that NIH risk grade is a significant predictor of the prognosis of GISTs. RKIP expression had a tendency to predict the survival of GISTs(P = 0.122),suggesting that RKIP expression may have appreciated value to predict the prognosis of GISTs.CONCLUSION This study demonstrated that:(1) RKIP expression in GISTs is associated with tumor size,NIH risk grade,and mucosal invasion,and low or no expression of RKIP predicts a high malignancy potential;(2) high RKIP correlates positively with the survival of patients with GISTs; and(3) RKIP expression has appreciated value for predicting the survival of patients with GISTs,although it is not an independent prognostic factor in GISTs.

Science Letters:IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

Insulin-like growth factor binding-protein-7 （IGFBP7） was obtained from our previous colonic adenocarcinoma （CRC） and normal mucosa suppression subtraction hybridization （SSH） cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR （MSP） and bisulfite sodium PCR （BSP）, we found that its expression was associated with DNA hypomethylation of exonl. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2＇-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1.

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 &#215;His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia

AIM： To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. METHODS： Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate. RESULTS： The histological examination did not demonstrate dysplasia; while MIB-1 （basal） showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 （26.7%） specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas （15/15） as well as 9 of the peritumoral samples （9/15） were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate （diploid： P 〈 0.0001; tetraploid： P = 0.001）, grading of malignancy according to Boecking （P 〈 0.0001） and the 5c- （P 〈 0.0001）, 7c-（P 〈 0.0001）, and 9c- （P = 0.0001） exceeding rate with progredient DNA alterations in the respective order. CONCLUSION： The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.

虎杖苷调节Akt/MDM2/p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响

目的探讨虎杖苷(PD)调节蛋白激酶B/原癌基因MDM2/抑癌基因p53信号通路对胆囊癌细胞增殖、迁移和细胞周期的影响。方法以人胆囊癌细胞株(GBC-SD)为研究对象,体外培养人胆囊癌细胞株(GBC-SD),使用浓度为10~160 mmol/L的虎杖苷处理细胞24、48、72 h,采用CCK-8法检测细胞的增殖能力,确定最佳实验浓度。将GBC-SD细胞分为对照组(Control组)、虎杖苷低、中、高浓度组(PD-L组、PD-M组、PD-H组)、虎杖苷+Akt激活剂组(PD+SC79组),Transwell小室法评价细胞的迁移能力,Hoechst染色观察细胞的凋亡,流式细胞术检测细胞周期与细胞凋亡,Western blot检测Akt、MDM2、p53磷酸化水平,建立荷瘤小鼠模型评价虎杖苷对胆囊癌肿瘤生长的影响。结果浓度为10~160 mmol/L的虎杖苷处理细胞24 h,可显著抑制GBC-SD细胞的增殖活性,选择10、20、40 mmol/L的虎杖苷进行后续实验;与Control组比较,PD-L组、PD-M组、PD-H组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著降低,G0/G1期细胞比例、P-p53蛋白表达显著升高,且呈浓度依赖性(P<0.05);与PD-H组比较,PD+SC79组GBC-SD细胞的迁移数、细胞凋亡率、G2/M期细胞比例及S期细胞比例、P-Akt、P-MDM2蛋白表达显著升高,G0/G1期细胞比例、P-p53蛋白表达显著降低(P<0.05);虎杖苷干预治疗后,小鼠移植瘤的生长速度显著降低(P<0.05)。结论虎杖苷可以通过调节Akt/MDM2/p53信号通路使细胞周期阻滞,抑制胆囊癌细胞增殖、迁移。

基于网络药理学方法和动物实验探讨五福健膝方治疗膝骨关节炎的作用机制

目的:探讨五福健膝方治疗膝骨关节炎(knee osteoarthritis,KOA)的作用机制。方法:①网络药理学研究。采用网络药理学方法筛选五福健膝方治疗KOA的靶点。②动物实验。将18只10周龄SPF级雄性C57BL/6小鼠随机分为3组,每组6只。模型组和五福健膝方组小鼠采用内侧半月板失稳术在右后肢构建KOA模型,假手术组小鼠仅切开对应区域皮肤和关节囊后缝合。造模手术后第2天,五福健膝方组小鼠以五福健膝方汤剂(生药浓度1.1 g·mL^(-1))灌胃,每次0.6 mL,每天1次,连续灌胃8周;模型组和假手术组小鼠以等量生理盐水灌胃。药物干预结束后,收集小鼠右后肢膝关节,分别进行股骨远端Micro-CT扫描、组织病理学观察(阿尔新蓝-苏木素/橙黄G染色)及免疫组织化学染色。免疫组织化学染色主要针对Ⅱ型胶原蛋白与网络药理学研究确定的核心靶点蛋白,测量Ⅱ型胶原蛋白表达阳性区域的厚度(视为软骨厚度),同时计算核心靶点蛋白阳性细胞百分比。结果:①网络药理学研究结果。通过网络药理学研究确定的五福健膝方治疗KOA的关键靶点基因15个,其中TP53为核心靶点基因,其对应的蛋白为P53蛋白。②动物实验结果。股骨远端Micro-CT检查结果显示,模型组较假手术组骨表面积骨体积比值降低(P=0.028)、骨体积分数升高(P=0.003),五福健膝方组较模型组骨表面积骨体积比值升高(P=0.004)、骨体积分数降低(P=0.048)。膝关节软骨组织阿尔新蓝-苏木素/橙黄G染色显示,模型组小鼠软骨缺失明显,五福健膝方组小鼠软骨丢失较模型组明显改善。免疫组织化学染色结果显示,模型组小鼠膝关节软骨厚度较假手术组减小(P=0.001),五福健膝方组小鼠膝关节软骨厚度较模型组增加(P=0.048);模型组小鼠膝关节软骨组织中P53阳性细胞百分比较假手术组增加(P=0.000),五福健膝方组小鼠膝关节软骨组织中P53阳性细胞百分比较模型组减少(P=0.000)。结论:五福健膝方能够有效延缓KOA模型小鼠的软骨退变,其机制可能与其抑制P53蛋白表达有关。

鉴定LTBP1作为胃癌预后的生物标志物及其与肿瘤免疫微环境的相关性

目的探索潜在转化生长因子β结合蛋白1(latent transforming growth factor beta binding protein 1,LTBP1)在胃癌发生发展及免疫微环境中的生物学功能。方法在TCGA数据库中分析LTBP1在泛癌中的表达,然后通过胃癌组织和癌旁组织进行验证。通过回归比例分析法分析LTBP1表达与临床病理变量之间的相关性。Cox回归分析和Kaplan-Meier图用于评估LTBP1在胃癌中的预后价值。通过TIMER分析LTBP1的表达水平与胃癌中免疫细胞浸润之间的相关性。免疫组织化学染色检测LTBP1蛋白在胃癌组织及癌旁组织中的表达水平。结果与正常胃组织相比,胃癌组织中LTBP1表达显著上调。其表达与病理TNM分期显著相关。LTBP1高表达的胃癌患者的总体生存率(overall survival,OS)缩短。免疫组化结果显示,与癌旁组织相比,LTBP1在胃癌组织中显著高表达。TIMER检测发现LTBP1的表达与3种免疫细胞浸润呈正相关。结论LTBP1可能是胃癌预后的一个潜在生物标志物,并影响癌症的肿瘤免疫微环境。

TP53BP2基因真核表达载体的构建及在人胚肾Expi293F细胞中蛋白表达、纯化及活性鉴定

目的构建人肿瘤抑制因子p53结合蛋白2(tumor suppressor p53-binding protein 2,TP53BP2)的重组真核表达载体,转染人胚肾Expi293F细胞,获得高纯度的重组人全长TP53BP2蛋白并对其进行生物学活性鉴定。方法利用UniProt网站查询TP53BP2基因序列,并进行Expi293F表达系统序列优化,通过同源重组连接至pcDNA3.1(+)-P2Ae GFP载体并进行双酶切和测序鉴定,通过转染试剂聚乙烯亚胺(polyethylenimine,PEI)将pcDNA3.1(+)-P2A-eGFPTP53BP2质粒瞬时转染至Expi293F细胞,荧光显微镜观察转染效率,收集实验组及对照组细胞,利用免疫印记试验(Western blot,WB)检测TP53BP2重组蛋白表达水平。通过His标签纯化试剂盒及Superdex 20010/300GL层析柱进行蛋白纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDSPAGE)对纯化后重组蛋白进行鉴定。利用免疫共沉淀(Co-immunoprecipitation,Co-IP)检测重组人全长TP53BP2蛋白与p65蛋白结合情况。利用免疫荧光(immunofluorescence,IF)检测重组人全长TP53BP2蛋白与p65蛋白共定位。利用表面等离子体共振(surface-plasmon resonance,SPR)技术,检测纯化后的重组人全长TP53BP2蛋白与TP53BP2抗体的相互作用。结果经测序和双酶切鉴定,重组质粒pcDNA3.1(+)-P2A-eGFP-TP53BP2构建成功。经荧光显微镜观察结果显示转染效率约为60%,WB结果表明TP53BP2蛋白在Expi293F细胞中过表达,证明转染成功。SDS-PAGE结果表明纯化后重组蛋白纯度在90%以上,证明纯化成功。Co-IP结果表明,TP53BP2重组蛋白可与p65蛋白相互作用。IF结果表明,His标签蛋白、TP53BP2蛋白及p65蛋白存在共定位,表明三者之间存在相互作用。SPR结果表明,纯化的重组人TP53BP蛋白与TP53BP2抗体具有较好的结合活性。以上结果均证明重组人全长TP53BP2蛋白具有生物学活性。结论成功构建了TP53BP2基因真核表达载体并在人胚肾Expi293F细胞中成功表达出具有生物学活性的重组人全长TP53BP2蛋白,为进一步研究TP53BP2的结构和功能奠定了基础。