Ki-67、CyclinD-1及STAT3蛋白在乳腺叶状肿瘤组织中的表达及其临床意义分析

目的探究Ki-67、细胞周期蛋白D-1(CyclinD-1)及信号转导子和转录激活子3(STAT3)蛋白在乳腺叶状肿瘤组织中的表达及其临床意义。方法回顾性选取2019年1月至2022年1月琼海市人民医院收治的75例乳腺叶状肿瘤患者纳入研究,经术后病理检查结果为恶性的68例患者入组。取患者肿瘤组织及其癌旁组织,采用免疫组织化学法对患者的Ki-67、CyclinD-1在组织中的表达情况进行检测;并采用蛋白质印迹法对患者组织中的STAT3蛋白表达情况进行检测;分析患者肿瘤组织中Ki-67、CyclinD-1及STAT3蛋白表达情况与患者临床病理间的关系,并分析其表达对患者乳腺恶性叶状肿瘤的诊断价值。结果乳腺叶状肿瘤组织中Ki-67、CyclinD-1、STAT3蛋白表达阳性率分别为30.88%、72.06%、22.06%,均高于癌旁组织(4.41%、16.18%、2.94%),差异均有统计学意义(P<0.05)。Ki-67阳性表达在年龄大小、肿瘤部位、组织学分级、是否绝经等方面比较,差异均无统计学意义(P>0.05),但患者临床分期为Ⅲ期的阳性率为55.56%,明显高于临床分期为Ⅰ~Ⅱ期患者(14.63%),差异有统计学意义(P<0.05)。CyclinD-1阳性表达在是否绝经、肿瘤部位、年龄大小等方面比较,差异均无统计学意义(P>0.05),但患者临床分期为Ⅲ期、组织学分级为3级的阳性率分别为82.22%、83.78%,均明显高于临床分期为Ⅰ~Ⅱ期、组织学分级为1~2级患者(52.17%、58.06%),差异均有统计学意义(P<0.05)。STAT3蛋白阳性表达在年龄大小、肿瘤部位、绝经情况等方面比较,差异均无统计学意义(P>0.05),但临床分期为Ⅲ期、组织学分级为3级的阳性率分别为54.55%、40.00%,均明显高于临床分期为Ⅰ~Ⅱ期、组织学分级为1~2级患者(6.52%、11.63%),差异均有统计学意义(P<0.05)。Ki-67、CyclinD-1、STAT3蛋白阳性表达诊断患者乳腺叶状肿瘤的受试者工作特征(ROC)曲线下面积(AUC)分别为0.732、0.879和0.721,95%CI分别为0.538~0.746、0.699~0.898、0.500~0.743。结论乳腺组织中Ki-67、CyclinD-1、STAT3蛋白过表达可能与患者乳腺叶状肿瘤有关,对其诊断具有一定价值。

Elevated retinol binding protein 4 levels are associated with atherosclerosis in diabetic rats via JAK2/STAT3 signaling pathway

BACKGROUND Atherosclerosis is a major cause of mortality worldwide and is driven by multiple risk factors,including diabetes,which results in an increased atherosclerotic burden,but the precise mechanisms for the occurrence and development of diabetic atheroscerosis have not been fully elucidated.AIM To summarize the potential role of retinol binding protein 4(RBP4) in the pathogenesis of diabetic atheroscerosis,particularly in relation to the RBP4-Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway.METHODS Male Wistar rats were randomly divided into three groups,including a control group(NC group),diabetic rat group(DM group),and diabetic atherosclerotic rat group(DA group).The contents of total cholesterol(TC), high-density lipoprotein cholesterol(HDL-c), triglycerides(TG), low-density lipoprotein cholesterol(LDLc), fasting insulin(FINS),fasting plasma glucose,and hemoglobin A1 c(HbA1 c)were measured.Moreover,the adipose and serum levels of RBP4,along with the expression levels of JAK2, phosphorylated JAK2(p-JAK2), STAT3,phosphorylated STAT3(p-STAT3), B-cell lymphoma-2(Bcl-2), and Cyclin D1 in aortic tissues were also measured.Besides,homeostasis model assessment of insulin resistance(HOMA-IR) and atherogenic indexes(AI) were calculated.RESULTS Compared with the NC and DM groups,the levels LDL-c,TG,TC,FINS,HOMAIR,RBP4,and AI were upregulated,whereas that of HDL-c was downregulated in the DA group(P <0.05);the mRNA levels of JAK2,STAT3,Cyclin D1,and Bcl-2 in the DA group were significantly increased compared with the NC group and the DM group;P-JAK2,p-JAK2/JAK2 ratio,p-STAT3,p-STAT3/STAT3 ratio,Cyclin D1,and Bcl-2 at protein levels were significantly upregulated in the DA group compared with the NC group and DM group.In addition,as shown by Pearson analysis,serum RBP4 had a positive correlation with TG,TC,LDL-c,FINS,HbA1 C,p-JAK2,p-STAT3,Bcl-2,Cyclin D1,AI,and HOMA-IR but a negative correlation with HDL-c.In addition,multivariable logistic regression analysis showed that serum RBP4,p-JAK2,p-STAT3,and LDL-c were predictors of the presence of diabetic atherosclerosis.CONCLUSION RBP4 could be involved in the initiation or progression of diabetic atherosclerosis by regulating the JAK2/STAT3 signaling pathway.

Role of the Notch Signaling Pathway in Fibrosis of Denervated Skeletal Muscle

In order to investigate the role of the Notch signaling pathway in skeletal muscle fibrosis after nerve injury, 60 Sprague-Dawley rats were selected and divided randomly into a control and two experimental groups. Group A served as controls without any treatment. Rats in groups B were injected intraperitoneally with 0.2 mL PBS and those in group C were injected intraperitoneally with 0.2 mL PBS+100 ymol/L, 0.2 mL N-[N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT, a gamma-secretase inhibitor that suppresses Notch signaling) respectively, on postoperative days 1, 3, 7, 10, and 14 in a model of denervation-induced skeletal muscle fibrosis by right sciatic nerve transection. Five rats from each group were euthanized on postoperative days 1, 7, 14, and 28 to collect the right gastrocnemii, and hematoxylin and eosin (HE) staining, immunohistochemistry test, real-time PCR, and Western blotting were performed to assess connective tissue hyperplasia and fibroblast density as well as expression of Notch 1, Jagged 1, and Notch downstream molecules Hes 1 and collagen I (COL I) on day 28. There was no significant difference in HE-stained fibroblast density between group B and C on postoperative day 1. However, fibroblast density was significantly higher in group B than in group C on postoperative days 7, 14, and 28. Notch 1, Jagged 1, Hes 1, and COL I proteins in the gastrocnemius were expressed at very low levels in group A but at high levels in group B. Expression levels of these proteins were significantly lower in group C than in group B (P<0.05), but they were higher in group C than in group A (P<0.05) on postoperative day 28. We are led to conclude that locking the Notch signaling pathway inhibits fibrosis progression of denervated skeletal muscle. Thus, it may be a new approach for treatment of fibrosis of denervated skeletal muscle.

Correlation of serum STMN1 and Cath-D levels with the invasive growth of cervical cancer

Objective:To study the correlation of serum stathmin 1 (STMN1) and cathepsin D (Cath-D) levels with the invasive growth of cervical cancer.Methods: The patients with cervical cancer and those with uterine fibroids who underwent surgical resection in the Second People's Hospital of Yichang between January 2013 and November 2017 were selected and enrolled in the malignant group and the benign group of the study respectively;the serum was collected before operation to detect the contents of STMN1 and Cath-D;the cervical cancer lesion tissues were collected from the malignant group and the normal cervical tissues were collected from the benign group to measure the expression of STMN1, Cath-D, invasion genes and invasion signal molecules.Results:STMN1 and Cath-D contents in serum as well as STMN1 and Cath-D mRNA expression in lesion tissue of malignant group were significantly higher than those of benign group, and serum STMN1 and Cath-D contents of malignant group were positively correlated with STMN1 and Cath-D mRNA expression in lesion tissue;RACK1, Twist, MMP9, mTOR, S6K1, Wnt andβ-catenin mRNA expression in lesion tissue of malignant group were significantly higher than those of benign group whereas E-cadherin, GRIM19 and GSK-3β mRNA expression were significantly lower than those of benign group, and serum STMN1 and Cath-D contents of malignant group were positively correlated with RACK1, Twist, MMP9, mTOR, S6K1, Wnt andβ-catenin mRNA expression in lesion tissue, and negatively correlated with E-cadherin, GRIM19 and GSK-3β mRNA expression. Conclusion:The abnormally elevated STMN1 and Cath-D in serum of patients with cervical cancer can evaluate the invasive growth of the lesion.

NIMA related kinase 2 promotes gastric cancer cell proliferation via ERK/MAPK signaling

BACKGROUND NIMA related kinase 2(NEK2) is closely related to mitosis, and it is currently considered to be over-expressed frequently in many poorly prognostic cancers.However, the effect of the up-regulated NEK2 on cellular signaling in tumors,such as gastric cancer(GC), is con-fusing.AIM To determine the role of the up-regulation of NEK2 in GC.METHODS To investigate the pathological significance of NEK2 in GC, the expression pattern of NEK2 in GC was investigated based on the 'Oncomain' database and compared between 30 pairs of cancer samples and adjacent tissues. The coexpression of NEK2 and ERK in GC was analyzed using The Cancer Genome Atlas(TCGA) database and confirmed in clinical samples by quantitative realtime PCR(qRT-PCR), and the survival curve was also plotted. Western blot or qRT-PCR was used to analyze the effect of NEK2 on the phosphorylation levels of ERK and c-JUN in two GC cell lines(BGC823 and SGC7901) with NEK2 overexpression, and the expression of the downstream effector cyclin D1.Furthermore, CCK8, EdU incorporation assay, and flow cytometry were used to detect the proliferative ability of BGC823 and SGC7901 cells with stably silenced ERK.RESULTS NEK2 was significantly up-regulated in human GC tissues. ERK was significantly associated with NEK2 expression in human clinical specimens, and combined overexpression of NEK2 and ERK potentially forecasted a poor prognosis andsurvival in GC patients. NEK2 knockdown in GC cells inhibited ERK and c-JUN phosphory-lation and reduced the transcription of cyclin D1. More interestingly,NEK2 can rescue the inhibition of cellular viability, proliferation, and cell cycle progression due to ERK knockdown.CONCLUSION Our results indicate that NEK2 plays a carcinogenic role in the malignant proliferation of GC cells via the ERK/MAPK signaling, which may be important for treatment and improving patient survival.

Brucine Inhibits Bone Metastasis of Breast Cancer Cells by Suppressing Jagged1/Notch1 Signaling Pathways

Objective： To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand（RANKL）-induced osteoclastogenesis. Methods： The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL（50 ng/m L） and macrophage-colony stimulating factor（50 ng/m L） were added to this system, followed by treatment with brucine（0.02, 0.04 and 0.08 mmol/L）, or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1（TGF-β1）, nuclear factor-kappa B（NF-κB） and Hes1 were determined by enzyme-linked immunosorbent assay. Results： Compared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells（P 〈0.01）. Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1（P〈0.05 or P〈0.01）. Conclusion： Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Hedgehog信号通路在食管癌中的异常活化

目的:探讨Hedgehog(Hh)信号通路与食管鳞状细胞癌(ESCC)的关系。方法:采用免疫组织化学随机分析30例ESCC及癌旁组织样本的Hh信号通路中主要组分Smo和Gli2及靶蛋白CyclinD1表达。构建分泌型配体ShhN的条件培养液,利用条件培养液及Hh信号通路激动剂Purmorphamine或Hh通路抑制剂环杷明及GANT61处理CaEs-17细胞后,MTT法检测细胞生存率的变化。结果:ESCC组织中的Smo、Gli2和CyclinD1表达普遍高于癌旁组织。含有ShhN的条件培养液能有效激活Hh信号通路下游靶基因CCND1的表达并明显促进食管癌CaEs-17细胞的生存率,提示食管癌中Hh信号通路以配体依赖的方式激活。直接作用于Smo的Hh信号通路激动剂Purmorphamine促进食管癌CaEs-17细胞的生长;而Smo特异性抑制剂环杷明有效地抑制CaEs-17细胞生存率。Gli1/Gli2的抑制剂GANT61比环杷明更为有效地抑制CaEs-17细胞生存率。结论:Hh信号通路在ESCC中异常活化,将可能成为新的治疗食管癌的药物靶点。

Alagille syndrome associated with total anomalous pulmonary venous connection and severe xanthomas:A case report

BACKGROUND Alagille syndrome(ALGS)is an autosomal dominant genetic disorder caused by mutations in the JAG1 or NOTCH2 gene.It is characterized by decreased intrahepatic bile ducts associated with a variety of abnormalities in many other organ systems,such as the cardiovascular,skeletal,and urinary systems.CASE SUMMARY We report a rare case of ALGS.A 1-month-old male infant presented with sustained jaundice and had a rare congenital heart disease:Total anomalous pulmonary venous connection(TAPVC).Sustained jaundice,particularly with cardiac murmur,caught our attention.Laboratory tests revealed elevated levels of alanine aminotransferase,aspartate aminotransferase,gamma-glutamyl transpeptidase,total bilirubin,and total bile acids,indicating serious intrahepatic cholestasis.Imaging confirmed the presence of butterfly vertebra at the seventh thoracic vertebra.This suggested ALGS,which was confirmed by genetic testing with a c.3197dupC mutation in the JAG1 gene.Ursodiol was administered immediately after confirmation of the diagnosis,and cardiac surgery was performed when the patient was 1.5 month old.He recovered well after treatment and was discharged at the age of 3 mo.At the age of two years,the patient returned to our clinic because multiple cutaneous nodules with xanthomas appeared,and their size and number increased over time.CONCLUSION We report a unique case of ALGS associated with TAPVC and severe xanthomas.This study has enriched the clinical manifestations of ALGS and emphasized the association between JAG1 gene and TAPVC.

miRNA-34b对子宫内膜癌细胞凋亡、侵袭、迁移的影响及作用机制

目的探究miR-34b对子宫内膜癌(endometrial cancer)凋亡、迁移、侵袭的影响及对Jag1/Cyclin D1信号通路的影响。方法将人子宫内膜癌细胞AN3CA细胞分为对照组、NC组和miR-34b组。通过流式细胞技术、Transwell实验分别检测各组细胞凋亡、迁移能力和侵袭能力;通过定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)及Westernblot检测各组细胞Jagged-1蛋白(Jag1)及其靶基因G1/S-特异性周期蛋白-D1(Cycling D1)mRNA和蛋白的表达水平;通过裸鼠荷瘤实验分析miR-34b对肿瘤生长的影响。结果对照组、NC组和miR-34b组细胞凋亡比例分别为5.6%±0.12%,5.80%±0.22%和19.4%±0.51%。miR-34组细胞凋亡比例高于对照组,差异具有统计学意义(t=8.325,P<0.01);对照组、NC组和miR-34b组细胞迁移数分别为322.15±13.71个,327.67±9.14个和162.19±7.49个,miR-34b组细胞迁移数低于对照组,差异具有统计学意义(t=7.945,P<0.01);对照组、NC组和miR-34b组细胞侵袭数分别为357.6±14.11个,364.05±16.12个和157.58±8.77个,miR-34b组细胞侵袭数低于对照组,差异具有统计学意义(t=7.643,P<0.01);qPCR结果表明对照组、NC组和miR-34b组Jag1 mRNA表达为2.75±0.21,2.67±0.14和1.19±0.19,miR-34b组低于对照组,差异具有统计学意义(t=8.434,P<0.01);对照组、NC组和miR-34b组蛋白表达为1.71±0.11,1.77±0.24和0.69±0.16,miR-34b组低于对照组,差异具有统计学意义(t=7.895,P<0.01);对照组、NC组和miR-34b组Cyclin D1mRNA表达为1.29±0.13,1.32±0.11和0.59±0.13,miR-34b组低于对照组,差异具有统计学意义(t=9.124,P<0.01);对照组、NC组和miR-34b组蛋白表达为1.81±0.18,1.79±0.14和1.29±0.16,miR-34b组低于对照组,差异具有统计学意义(t=8.227,P<0.01);裸鼠荷瘤实验表明过表达miR-34b裸鼠肿瘤生长曲线与对照组差异有统计学显著性意义,并且肿瘤体积显著低于对照组(t=8.184,P<0.01)。结论miR-34b可以促进人子宫内膜癌细胞的凋亡,抑制细胞迁移和侵袭能力,其机制与Jag1/Cyclin D1信号通路活性的抑制相关。