Thymoquinone affects hypoxia-inducible factor-1αexpression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways

BACKGROUND Pancreatic cancer(PC)is associated with some of the worst prognoses of all major cancers.Thymoquinone(TQ)has a long history in traditional medical practice and is known for its anti-cancer,anti-inflammatory,anti-fibrosis and antioxidant pharmacological activities.Recent studies on hypoxia-inducible factor-1α(HIF-1α)and PC have shown that HIF-1αaffects the occurrence and development of PC in many aspects.In addition,TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α.Therefore,we speculate whether TQ affects HIF-1αexpression in PC cells and explore the mechanism.AIM To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1αexpression.METHODS Cell counting kit-8 assay,Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity,migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial(hTERTHPNE)cells.Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1αmRNA and protein in PC cells.The effects of TQ on the HIF-1αprotein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.RESULTS TQ significantly inhibited proliferative activity,migration,and invasion ability and promoted apoptosis of PANC-1 cells;however,no significant effects on hTERT-HPNE cells were observed.TQ significantly reduced the mRNA and protein expression levels of HIF-1αin PANC-1,AsPC-1,and BxPC-3 cells.TQ significantly inhibited the expression of the HIF-1αinitial expression pathway(PI3K/AKT/mTOR)related proteins,and promoted the ubiquitination degradation of the HIF-1αprotein in PANC-1 cells.TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1αprotein but affected the stability of the HIF-1αprotein by inhibiting the interaction between HIF-1αand HSP90,thus promoting its ubiquitination degradation.CONCLUSION The regulatory mechanism of TQ on HIF-1αprotein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1αprotein by inhibiting the interaction between HIF-1αand HSP90;Secondly,TQ reduced the initial expression of HIF-1αprotein by inhibiting the PI3K/AKT/mTOR pathway.

Low-temperature 3D-printed collagen/chitosan scaffolds loaded with exosomes derived from neural stem cells pretreated with insulin growth factor-1 enhance neural regeneration after traumatic brain injury

There are various clinical treatments for traumatic brain injury,including surgery,drug therapy,and rehabilitation therapy;howeve r,the therapeutic effects are limited.Scaffolds combined with exosomes represent a promising but challenging method for improving the repair of traumatic brain injury.In this study,we determined the ability of a novel 3D-printed collagen/chitosan scaffold loaded with exosomes derived from neural stem cells pretreated with insulin-like growth factor-1(3D-CC-INEXOS) to improve traumatic brain injury repair and functional recove ry after traumatic brain injury in rats.Composite scaffolds comprising collagen,chitosan,and exosomes derived from neural stem cells pretreated with insulin-like growth fa ctor-1(INEXOS) continuously released exosomes for 2weeks.Transplantation of 3D-CC-INExos scaffolds significantly improved motor and cognitive functions in a rat traumatic brain injury model,as assessed by the Morris water maze test and modified neurological seve rity scores.In addition,immunofluorescence staining and transmission electron microscopy showed that3D-CC-INExos implantation significantly improved the recove ry of damaged nerve tissue in the injured area.In conclusion,this study suggests that transplanted3D-CC-INExos scaffolds might provide a potential strategy for the treatment of traumatic brain injury and lay a solid foundation for clinical translation.

血清血管细胞黏附因子1和三叶因子3水平与晚期非小细胞肺癌化疗效果及预后的关系

目的研究血管细胞黏附因子1(VCAM1)、三叶因子3(TFF3)水平与晚期非小细胞肺癌(NSCLC)患者化疗效果及预后的关系。方法选取2019年2月至2021年2月江苏省肿瘤医院收治的112例接受铂类化疗初治的晚期NSCLC患者为观察组,另选取同期体检健康的70例受试者为对照组。检测受试者血清VCAM1、TFF3水平。根据化疗结束后的效果,将观察组患者分为化疗有效组和化疗无效组。随访1年,比较不同血清VCAM1、TFF3表达水平晚期NSCLC患者生存预后差异。采用多因素Cox回归模型分析影响晚期NSCLC患者生存预后的因素。结果观察组血清VCAM1、TFF3水平均高于对照组[(227±24)μg/L比(79±13)μg/L、(1.59±0.37)μg/L比(0.47±0.14)μg/L],差异均有统计学意义(均P<0.001)。晚期NSCLC患者血清VCAM1、TFF3水平与肿瘤分化程度及TNM分期有关(均P<0.05)。化疗无效组患者血清VCAM1、TFF3水平均高于化疗有效组,差异均有统计学意义(均P<0.001)。VCAM1高表达组1年总体生存率为26.9%(14/52),VCAM1低表达组为55.0%(33/60),组间比较差异有统计学意义(Log-rankχ^(2)=12.181,P<0.001)。TFF3高表达组1年总体生存率为27.8%(15/54),TFF3低表达组为55.2%(32/58),组间比较差异有统计学意义(Log-rankχ^(2)=14.146,P<0.001)。多因素Cox回归分析结果显示,肿瘤低分化程度、TNM分期Ⅳ期、VCAM1高表达、TFF3高表达是晚期NSCLC患者不良预后的独立危险因素(均P<0.001)。结论晚期NSCLC患者血清VCAM1、TFF3水平升高,二者与不良临床病理特征、化疗效果有关。

TFF3在结直肠癌发生发展中的价值

三叶因子3(trefoil factor 3,TFF3)作为三叶因子家族的成员之一,广泛存在于机体的黏膜上皮细胞中。在生理情况下,TFF3具有保护黏膜的功能。但当机体发生溃疡、炎症和恶性病变时,TFF3的表达水平会随着病理过程的进展而异常改变。特别是在结直肠癌中,TFF3的异常表达参与了多种生物学过程的调控,包括促进细胞的侵袭和转移、抑制细胞凋亡、调控EMT过程以及影响细胞自噬等。此外,TFF3作为一种分泌性多肽,能被检测到存在于血清中,并且在结直肠癌患者中明显升高,因此,作为一种新型生物标志物检测TFF3的表达水平在结直肠癌的诊断和预后评估中具有重要价值。

宫颈癌患者淋巴结转移的危险因素分析及血清TFF3、AIF-1、S100-A11、DKK1预测价值分析

目的 探讨宫颈癌患者淋巴结转移的危险因素分析及血清三叶因子3(trefoil factor 3,TFF3)、同种异体移植物炎性因子-1(allograft inflammatory factor-1,AIF-1)、S100钙结合蛋白-A11(S100 calcium-binding protein-A11,S100-A11)、Wnt通路抑制因子Dickkopf-1(Wnt pathway inhibitor Dickkopf-1,DKK1)的预测价值。方法 选择2021年1月—2023年1月本院收治的71例宫颈癌患者作为研究对象,根据患者有无盆腔淋巴结转移分为淋巴结转移阳性组(28例)和淋巴结转移阴性组(43例)两组。收集两组患者临床病理特征,并检测血清TFF3、AIF-1、S100-A11、DKK1水平。结果 单因素分析显示,FIGO分期、宫旁浸润、肌层浸润、血清TFF3、S100-A11、DKK1是患者发生宫颈癌淋巴结转移的影响因素(P<0.05)。与年龄、分化程度、肿瘤大小、组织学类型、脉管浸润及血清AIF-1无关(P>0.05);Logistic多元回归分析显示,FIGO分期、宫旁浸润、肌层浸润及血清TFF3是影响宫颈癌淋巴结转移的独立因素(P<0.05);ROC曲线分析显示,TFF3对淋巴结转移有中等预测价值(AUC=0.649),明显大于机会参考线下面积(P<0.05)。而AIF-1、S100-A11、DKK1对淋巴结转移无预测价值(AUC分别为0.477、0.517、0.524),与机会参考线下面积比较无统计学意义(P>0.05)。结论 FIGO分期高、宫旁浸润、肌层浸润、血清TFF3异常升高是宫颈癌淋巴结转移的危险因素,血清TFF3有望成为预测宫颈癌淋巴结转移的新标志物;血清AIF-1、S100-A11、DKK1对宫颈癌淋巴结转移的预测效能不理想。

Apigenin ameliorates imiquimod-induced psoriasis in C57BL/6J mice by inactivating STAT3 and NF-κB

Psoriasis is a chronic autoimmune disease featured by patches on the skin.It is caused by malfunction of immune cells and keratinocytes with inflammation as one of its key features.Apigenin(API)is a natural flavonoid with anti-inflammatory and immunoregulatory properties.Therefore,we speculated that API can ameliorate psoriasis,and determined its effect on the development of psoriasis by using imiquimod(IMQ)-induced psoriasis mouse model.Our results showed that API attenuated IMQ-induced phenotypic changes,such as erythema,scaling and epidermal thickening,and improved splenic hyperplasia.Abnormal differentiation of immune cells was restored in API-treated mice.Mechanistically,we revealed that API is a key regulator of signal transducer activator of transcription 3(STAT3).API regulated immune responses by reducing interleukin-23(IL-23)/STAT3/IL-17A axis.Moreover,it suppressed IMQ-caused cell hyperproliferation by inactivating STAT3 through regulation of extracellular signal-regulated kinase 1/2 and nuclear factor-κB(NF-κB)pathway.Furthermore,API reduced expression of inflammatory cytokines through inactivation of NF-κB.Taken together,our study demonstrates that API can ameliorate psoriasis and may be considered as a strategy for psoriasis treatment.

Prognostic significance and relationship of SMAD3 phosphoisoforms and VEGFR-1 in gastric cancer:A clinicopathological study

BACKGROUND The TGF-β/SMAD3 and VEGFR-1 signaling pathways play important roles in gastric cancer metastasis.SMAD3 phosphorylation is a crucial prognostic marker in gastric cancer.AIM To determine the prognostic value and relationship of SMAD3 phospho-isoforms and VEGFR-1 in gastric cancer.METHODS This was a single-center observational study which enrolled 98 gastric cancer patients and 82 adjacent normal gastric tissues from patients aged 32-84 years(median age 65)between July 2006 and April 2007.Patients were followed up until death or the study ended(median follow-up duration of 28.5 mo).The samples were used to generate tissue microarrays(TMAs)for immunohistochemical(IHC)staining.The expressions of TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 in gastric cancer(GC)tumor tissue and normal tissue were measured by IHC staining using TMAs obtained from 98 GC patients.Prognosis and survival information of the patients was recorded by Outdo Biotech from May 2007 to July 2015.The relationship between TGF-β1,pSMAD3C(S423/425),pSMAD3L(S204),and VEGFR-1 protein expression levels was analyzed using Pearson's correlation coefficient.The relationship between protein expression levels and clinicopathological parameters was analyzed using the Chi-squared test.A survival curve was generated using the Kaplan-Meier survival analysis.RESULTS TGFβ-1 and VEGFR-1 expression was significantly upregulated in gastric cancer tissue compared to adjacent noncancerous tissue.The positive expression of phosphorylated isoforms of Smad3 varied depending on the phosphorylation site[pSMAD3C(S423/425):51.0%and pSMAD3L(S204):31.6%].High expression of pSMAD-3L(S204)was significantly correlated with larger tumors(P=0.038)and later N stages(P=0.035).Additionally,high expression of VEGFR-1 was closely correlated with tumor size(P=0.015)and pathological grading(P=0.013).High expression of both pSMAD3L(S204)and VEGFR-1 was associated with unfavorable outcomes in terms of overall survival(OS).Multivariate analysis indicated that high expression of pSMAD3L(S204)and VEGFR-1 were independent risk factors for prognosis in GC patients.VEGFR-1 protein expression was correlated with TGF-β1(r=0.220,P=0.029),pSMAD3C(S423/425)(r=0.302,P=0.002),and pSMAD3L(S204)(r=0.201,P=0.047),respectively.Simultaneous overexpression of pSMAD3L(S204)and VEGFR-1 was associated with poor OS in gastric cancer patients.CONCLUSION Co-upregulation of pSMAD3L(S204)and VEGFR-1 can serve as a predictive marker for poor gastric cancer prognosis,and pSMAD3L(204)may be involved in enhanced gastric cancer metastasis in a VEGFR-1-dependent manner.

桥本甲状腺炎合并甲状腺乳头状癌患者中Toll样受体3和核转录因子-κB的表达及相关性分析

目的分析桥本甲状腺炎(Hashimoto thyroiditis,HT)合并甲状腺乳头状癌(PTC)患者Toll样受体3(toll-like receptor 3,TLR3)和核转录因子-κB(nuclear transcription factor-κB,NF-κB)表达及相关性。方法收取邯郸市中心医院2020年3月~2022年3月收治的130例行手术切除的甲状腺标本,其中正常甲状腺组织标本43例,HT标本47例,HT合并PTC标本40例,分析TLR3和NF-κB在正常甲状腺组织、HT组、HT合并PTC组中的表达,分析HT合并PTC组中TLR3和NF-κB表达与临床病理参数关系,Pearson相关性分析TLR3和NF-κB的关系。结果TLR3在正常甲状腺组织、HT组、HT合并PTC组中的阳性表达率分别为0(0/43)、80.85%(38/47)、90.00%(36/40);NF-κB在以上三组中的阳性表达率分别为0(0/43)、68.09%(32/47)、85.00%(34/40)。TLR3和NF-κB在HT组、HT合并PTC组中的阳性表达率均高于正常甲状腺组织(P<0.05),TLR3和NF-κB表达与性别、年龄、HT合并PTC病理学特征、病灶类型、淋巴结转移、甲状腺包膜侵犯差异比较均无统计学意义(P均>0.05)。TLR3和NF-κB呈显著正相关(r=0.589,P<0.05)。结论TLR3和NF-κB在HT合并PTC组织中的阳性率高于正常甲状腺组织,且二者表达呈正相关。

从脑肠轴探讨黄芪建中汤对胃溃疡大鼠肝细胞生长因子及ERK1/2和TFF3蛋白表达的影响

目的观察黄芪建中汤对脾胃虚寒型胃溃疡大鼠胃组织中肝细胞生长因子(HGF)及下丘脑和海马区中磷酸化细胞外信号调节激酶1/2(p-ERK1/2)及肠三叶因子3(TFF3)蛋白表达的影响,探究黄芪建中汤治疗脾胃虚寒型胃溃疡的作用及可能机制。方法用随机数字表法将60只大鼠分为正常组、模型组、奥美拉唑组和黄芪建中汤组,每组15只。正常组隔日蒸馏水灌胃,每日不限饮食;其余组大鼠先以小承气汤结合饥饱失常法复制脾胃虚寒证模型,实验第11天采用冰醋酸法建立胃溃疡模型。之后模型组继续隔日上午给予小承气汤并当日禁食,次日恢复饮食,共持续20 d;奥美拉唑组和黄芪建中汤组大鼠除同模型组的每日处理外,每日下午分别给予4.2 mg/(kg·d)奥美拉唑和6.8 g/(kg·d)黄芪建中汤灌胃;正常组隔日上午及每日下午给予蒸馏水灌胃。实验结束后摘取各组大鼠胃,记录溃疡大小并计算胃溃疡指数,HE染色观察胃组织病理形态,免疫组化染色检测胃组织中HGF及下丘脑和海马区中p-ERK1/2、TFF3蛋白阳性表达情况。结果正常组大鼠胃黏膜正常,未见溃疡点及糜烂斑等;模型组大鼠胃黏膜皱襞存在中断,有点状溃疡、糜烂及大量炎性细胞浸润;黄芪建中汤组与奥美拉唑组胃黏膜损伤较模型组轻,有少量炎性细胞浸润,未见明显溃疡。奥美拉唑组和黄芪建中汤组大鼠的胃溃疡指数均明显低于模型组(P均<0.05),胃组织中HGF及下丘脑和海马区中p-ERK1/2、TFF3蛋白表达平均光密度均明显高于模型组(P均<0.05)。结论黄芪建中汤能促进脾胃虚寒型胃溃疡大鼠溃疡愈合,上调HGF、ERK1/2及TFF3的表达可能是其基于脑肠轴治疗脾胃虚寒型胃溃疡的作用机制之一。

Diagnostic value evaluation of trefoil factors family 3 for the early detection of colorectal cancer

AIM The purpose of this study was to evaluate the diagnostic value of trefoil factor family 3(TFF3) for the early detection of colorectal cancer(CC). METHODS Serum TFF3 and carcino-embryonic antigen(CEA) were detected in 527 individuals, including 115 healthy control(HC), 198 colorectal adenoma(CA), and 214 CC individuals in the training group. RESULTS Serum TFF3 showed no significant correlation with age, gender, or tumor location but showed significant correlation with the tumor stage. Serum TFF3 in the CC group was significantly higher than in the HC or CA group. The AUC values of TFF3 for discriminating between HC and CC and between CA and CC were 0.930(0.903, 0.958) and 0.834(0.796, 0.873). A multivariate model combining TFF3 and CEA was built. Compared to TFF3 or CEA alone, the multivariate model showed significant improvement(P < 0.001). For discriminating between HC and CC, HC and early stage CC, HC and advanced stage CC, CA and CC, CA and early stage CC, and CA and advanced stage CC in the training group, the sensitivities were 92.99%, 91.46%, 93.18%, 73.83%, 76.83%, and 81.82%, and the specificities were 91.30%, 91.30%, 93.91%, 88.38%, 77.27%, and 88.38%, respectively. After validation, the sensitivities were 89.39%, 85.71%, 90.79%, 72.73%, 71.43%, and 78.95%, and the specificities were 87.85%, 87.85%, 2.52%, 87.85%, 80.77%, and 87.50%, respectively. CONCLUSION The multivariate diagnostic model that included TFF3 and CEA showed significant improvement over the conventional biomarker CEA and might provide a potential method for the early detection of CC.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency.However, the proof of chondrogenic potential of the cells is scarce.Therefore,we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor(TGF)-p3 and bone morphogenetic protein(BMP)-6.After isolation of periodontal ligament stem cells(PDLSCs) from human periodontal ligament,the cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) with 20%fetal bovine serum(FBS).A mechanical force initiated chondrogenic differentiation of the cells.For chondrogenic differentiation,10μg·LTGF-β3 or 100μg·LBMP-6 and the combination treating group for synergistic effect of the growth factors.We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay,histology,immunohistochemistry and genetic analysis.PDLSCs showed mesenchymal stem cell properties proved by FACS analysis.Glycosaminoglycans contents were increased 217%by TGF-β3 and 220%by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281%compared to control.The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls,but also TGF-P3 or BMP-6 single treatment dramatically.The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions.The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis,which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

血清Stathmin、TFF3与上皮性卵巢癌患者初始肿瘤细胞减灭术治疗结局和术后复发的关系

目的研究血清微管解聚蛋白(Stathmin)、三叶因子3(TFF3)与上皮性卵巢癌患者初始肿瘤细胞减灭术治疗结局和术后复发的关系。方法选取2016年7月至2020年1月该院收治的177例上皮性卵巢癌患者,收集患者的临床资料,采用酶联免疫吸附试验检测Stathmin、TFF3水平。根据手术结局将患者分为满意组和不满意组,采用受试者工作特征(ROC)曲线分析血清Stathmin联合TFF3对治疗结局不满意的预测价值;统计手术治疗满意患者的复发情况,根据是否复发分为复发组和未复发组,采用单因素及多因素Logistic回归模型分析影响患者术后复发的危险因素,并分析血清Stathmin联合TFF3对术后复发的预测价值。结果手术治疗结局满意113例(满意组),不满意64例(不满意组)。满意组血清Stathmin、TFF3水平低于不满意组(t=-14.302、-8.405,P<0.05)。血清Stathmin联合TFF3预测治疗结局不满意的曲线下面积(AUC)为0.826,灵敏度为78.1%、特异度为83.2%,预测效能高于Stathmin、TFF3单独检测。复发患者47例(复发组),未复发66例(未复发组)。多因素Logistic回归分析显示,术前血清Stathmin、TFF3水平升高为患者术后复发的独立危险因素[OR(95%CI)=3.402(1.599~7.231)、1.031(1.014~1.049),P<0.05]。血清Stathmin联合TFF3联合预测术后复发的AUC为0.834,灵敏度为80.9%、特异度为83.3%,预测效能高于Stathmin、TFF3单独检测。结论血清Stathmin、TFF3水平在上皮性卵巢癌患者中异常升高且与初始肿瘤细胞减灭术治疗结局和术后复发有关。

Interplay between micro RNA-17-5p, insulin-like growth factor-Ⅱ through binding protein-3 in hepatocellular carcinoma

AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.

血清TFF3、AIF-1和S100-A11水平在宫颈癌早期诊断和预后评估中的临床价值

目的观察血清三叶因子3(TFF3)、同种异体移植物炎性因子-1(AIF-1)和S100钙结合蛋白-A11(S100-A11)水平在宫颈癌早期辅助诊断和预后评估中的临床价值。方法选取2019年1月至2020年6月在该院确诊的128例宫颈癌患者作为宫颈癌组,选取同期在该院确诊的75例宫颈上皮内瘤变(CIN)患者作为CIN组,选取同期在该院行健康体检的45例女性作为健康对照组。观察各组血清TFF3、AIF-1和S100-A11水平变化情况,血清TFF3、AIF-1和S100-A11水平在宫颈癌中的诊断效能,以及其与临床指标和预后的关系。结果宫颈癌组血清TFF3、AIF-1和S100-A11水平均明显高于CIN组和健康对照组,CIN组血清TFF3、AIF-1和S100-A11水平均明显高于健康对照组,差异均有统计学意义(P<0.05)。血清TFF3、AIF-1和S100-A11水平在诊断宫颈癌中具有较高的价值,3项指标联合检测的灵敏度为89.1%,特异度为97.3%,受试者工作特征曲线下面积为0.968,明显高于TFF3(Z=4.627,P<0.05)、AIF-1(Z=4.164,P<0.05)和S100-A11(Z=5.217,P<0.05)单独检测。低分化、人乳头瘤病毒(HPV)阳性、临床分期为Ⅱ期和有淋巴结转移宫颈癌患者血清TFF3、AIF-1和S100-A11水平均明显高于高中分化、HPV阴性、临床分期为Ⅰ期和无淋巴结转移宫颈癌患者,差异均有统计学意义(P<0.05);而不同年龄、病理类型、肿瘤最大径、浸润肌层深度、脉管浸润和神经浸润宫颈癌患者血清TFF3、AIF-1和S100-A11水平比较,差异均无统计学意义(P>0.05)。随访1年后死亡组患者血清TFF3、AIF-1和S100-A11水平均明显高于存活组,差异均有统计学意义(P<0.05)。结论血清TFF3、AIF-1和S100-A11水平在宫颈癌诊断和预后评估中具有较高的临床价值,3项指标联合检测有助于提高宫颈癌的辅助诊断效能。

血清ITF3、HMGB1水平对结直肠癌患者根治术后早期炎性肠梗阻的诊断价值

目的探讨血清肠三叶因子3(ITF3)和高迁移率族蛋白B1(HMGB1)水平对结直肠癌患者根治术后早期炎性肠梗阻(EPISBO)的诊断价值。方法选取2020年2月至2023年2月在我院确诊结直肠癌并进行手术的277例患者作为研究对象,选取同期在我院体检健康者277例为对照组。检测并分析两组患者血清ITF3、HMGB1水平。采用Logistic回归分析影响结直肠癌患者发生EPISBO的因素。采用Pearson分析EPISBO患者血清ITF3、HMGB1的相关性。受试者工作特征(ROC)曲线分析ITF3、HMGB1水平对结直肠癌患者EPISBO的诊断效能。结果研究组患者血清ITF3水平显著低于对照组(P<0.05),HMGB1水平显著高于对照组(P<0.05);Pearson等级相关分析显示,EPISBO患者血清ITF3、HMGB1水平呈现负相关性(r=-0.671,P<0.001)。Logistic回归结果显示,ITF3是结直肠癌患者术后发生EPISBO的独立保护因素(P<0.05),HMGB1是导致EPISBO的独立危险因素(P<0.05)。ROC曲线显示,血清ITF3、HMGB1联合应用诊断EPISBO的临床价值优于ITF3、HMGB1单独检测(Z_(二者联合-ITF3)=3.112、P=0.002,Z_(二者联合-HMGB1)=2.000、P=0.046)。结论结直肠癌术后发生EPISBO患者血清ITF3水平显著降低,HMGB1表达水平显著升高,二者对EPISBO的临床诊断预测具有重要意义。

Effect of Tumor Necrosis Factor-αon Resistin Expression in 3T3-L1 Adipocytes and Its Mechanism

Summary: In order to investigate the effect of tumor necrosis factor-α (TNFα) on resistin expression in 3T3-L1 adipocytes, and further explore its mechanisms, the differentiated 3T3-L1 adipocytes were incubated with 0, 1, 10, 100 ng/mL TNFα respectively for 24 h, and then the expression of resistin was determined. The differentiated 3T3-L1 adipocytes were incubated with 100 ng/mL TNFα for 3, 6, 24 h respectively, and then the expression of resistin mRNA was analyzed. 3T3-L1 adipocytes were induced to differentiate into mature adipocytes. The cells were randomly divided into 4 groups for culture. In the control group, no drugs were added. Cells of TNFα group were treated with 100 ng/mL TNFα. In Ro-31-8220 group, 5 μmol/L protein kinase C inhibitor Ro-31-8220 was added. With TNFα+Ro-31-8220 group, 100 ng/mL TNFα were added 1 h after the addition of 5 μmol/L Ro-31-8220. All adipocytes were cultured for 24 h. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to detect the expression of resistin gene. Our results showed that resistin protein and mRNA in 3T3-L1 adipocytes were inhibited by TNFα at different concentrations (P<0.01), and the inhibitory effect increased with the concentration (P<0.01). At the same concentrations, the inhibitory effect increased with time (P<0.01). Ro-31-8220 could inhibit its expression and the inhibitive effect remained unchanged with addition of TNFα(P>0.05). It was concluded that TNFα could inhibit the expression of resistin in 3T3-L1 adipocytes. The mechanism may be that the expression of resistin is partly controlled by protein kinase C signal conduction pathway.

Hsa_circRNA_102610 upregulation in Crohn’s disease promotes transforming growth factor-β1-induced epithelial-mesenchymal transition via sponging of hsa-miR-130a-3p

BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.

Epigallocatechin-3-gallate suppresses transforming growth factor-beta signaling by interacting with the transforming growth factor-beta typeⅡreceptor

AIM: To investigate the(-)-epigallocatechin-3-gallate(EGCG) binding to transforming growth factor-β(TGF-β) type Ⅱ receptor(TGFRⅡ).METHODS: The expression of α-smooth muscle actin(α-SMA) was used as a marker for fibrotic change inhuman lung fibroblast MRC-5 cells. The α-SMA expression level was determined by western blotting and immunohistological analysis. We examined whether the anti-fibrotic effects of EGCG on MRC-5 cells was dependent on antioxidant mechanism by using edaravone and N-acetylcysteine(NAC). The suppression effects of EGCG on Smad2/3 activation were studied by confocal fluorescence microscopy. The binding of EGCG to recombinant TGFRⅡ protein was analyzed by immunoprecipitation and affinity chromatography.RESULTS: When MRC-5 cells were treated with TGF-β, EGCG decreased the expression of α-SMA in a dose dependent manner, whereas catechin did not influence the α-SMA expression in the cells. Except for EGCG, antioxidant compounds(e.g., edaravone and NAC) had no effects on the TGF-β-induced α-SMA expression. Nuclear localization of phosphorylated Smad2/3 was observed after TGF-β treatment; however, EGCG treatment attenuated the nuclear transportation of Smad2/3 in the presence or absence of TGF-β. After a TGFRⅡ expression vector was introduced into COS-7 cells, cell lysates were untreated or treated with EGCG or catechin. The immunoprecipitation experiments using the lysates showed that EGCG dose-dependently bound to TGFRⅡ and that catechin did not at all. Affinity chromatography study indicated that EGCG would bind to TGFRⅡ.CONCLUSION: Our results demonstrate that EGCG interacts with TGFRⅡ and inhibits the expression of α-SMA via the TGF-β-Smad2/3 pathway in human lung fibroblast MRC-5 cells.