Clinical outcome and prognostic factors of T4N0M0 colon cancer after R0 resection:A retrospective study

BACKGROUND Paradoxically,patients with T4N0M0(stage II,no lymph node metastasis)colon cancer have a worse prognosis than those with T2N1-2M0(stage III).However,no previous report has addressed this issue.AIM To screen prognostic risk factors for T4N0M0 colon cancer and construct a prognostic nomogram model for these patients.METHODS Two hundred patients with T4N0M0 colon cancer were treated at Tianjin Medical University General Hospital between January 2017 and December 2021,of which 112 patients were assigned to the training cohort,and the remaining 88 patients were assigned to the validation cohort.Differences between the training and validation groups were analyzed.The training cohort was subjected to multi-variate analysis to select prognostic risk factors for T4N0M0 colon cancer,followed by the construction of a nomogram model.RESULTS The 3-year overall survival(OS)rates were 86.2%and 74.4%for the training and validation cohorts,respectively.Enterostomy(P=0.000),T stage(P=0.001),right hemicolon(P=0.025),irregular review(P=0.040),and carbohydrate antigen 199(CA199)(P=0.011)were independent risk factors of OS in patients with T4N0M0 colon cancer.A nomogram model with good concordance and accuracy was constructed.CONCLUSION Enterostomy,T stage,right hemicolon,irregular review,and CA199 were independent risk factors for OS in patients with T4N0M0 colon cancer.The nomogram model exhibited good agreement and accuracy.

Retinoblastoma binding protein 4 up-regulation is correlated with hepatic metastasis and poor prognosis in colon cancer patients

Background: Retinoblastoma binding protein 4 (RBBP4) plays an essential role in the development of multiple cancers. However, its relationship with prognosis in colon cancer and colon cancer hepatic metastasis has not been elucidated. The aim of this study was to explore the relationship between RBBP4 expression and prognosis of colon cancer patients and to evaluate RBBP4 as a new prognostic marker in these patients. Methods: Eighty colon cancer patients underwent surgical resection of the colon were enrolled. Among them, forty colon cancer patients suffered with hepatic metastasis. The colon cancer tissues, para-colon cancer tissues, and hepatic metastatic cancer tissues were collected from the pathological department for further analysis. The expression of RBBP4 proteins was examined by immunohistochemistry and correlated with clinicopathological parameters. The Cancer Genome Atlas (TCGA) database was used to validate the expression and explore its relationship with clinical characteristics. Results: RBBP4 was up-regulated in the colon cancer tissues compared with the para-colon cancer tissues. The analysis of TCGA database verified the upregulation of RBBP4 in the colon cancer tissues and RBBP4 overexpression was correlated with nerve invasion and poor outcomes of chemotherapy. Moreover, the positive rate of RBBP4 expression in 40 colon cancer patients with hepatic metastasis was higher in the hepatic metastatic cancer tissues (39/40, 97.5%) than in the colon cancer tissues (26/40, 65.0%). Our clinicopathological analysis showed that RBBP4 expression was significantly correlated with vascular invasion, hepatic metastasis, and lymph node involvement (all P < 0.05). Additionally, the survival analysis demonstrated that RBBP4 over-expression was correlated with poor prognosis. Conclusions: RBBP4 was upregulated in the colon cancer. RBBP4 may be a novel predictor for poor prognosis of colon cancer and colon cancer hepatic metastasis.

Smaller tumor size is associated with poor survival in T4b colon cancer

AIM: To hypothesize that in patients with colon cancer showing heavy intestinal wall invasion without distant metastasis(T4b N0-2M0), small tumor size would correlate with more aggressive tumor behaviors and therefore poorer cancer-specific survival(CSS).METHODS: We analyzed T4 b N0-2M0 colon cancer patients in the Surveillance, Epidemiology and End Results(SEER) database. A preliminary analysis of T4 b N0-2M0 colon cancer patients at the Fudan University Shanghai Cancer Center is also presented.RESULTS: A total of 1734 T4 b N0-2M0 colon cancer patients from the SEER database were included. Kaplan-Meier analysis revealed decreasing CSS with decreasing tumor size(P < 0.001). Subgroup analysis showed a significant association between poorer CSS with smaller tumor size in T4 b N0 patients(P = 0.024), and a trend of association in T4 b N1(P = 0.182) and T4 b N2 patients(P = 0.191). Multivariate analysis identified tumor size as an independent prognostic factor for CSS in T4 b N0-2M0 patients(P = 0.024). Preliminary analysis of Fudan University Shanghai Cancer Center samples suggested the 5-year CSS was 50.0%, 72.9% and 77.1% in patients with tumors ≤ 4.0 cm, 4.0-7.0 cm and ≥ 7.0 cm.CONCLUSION: Smaller tumor size is associated with poorer CSS in the T4 b N0-2M0 subset of colon cancer, particularly in the T4 b N0M0 subgroup.

SFRP4 expression correlates with epithelial mesenchymal transitionlinked genes and poor overall survival in colon cancer patients

BACKGROUND Colon cancer is among the most commonly diagnosed cancers in the United States with an estimated 97220 new cases expected by the end of 2018.It affects 1.2 million people around the world and is responsible for about 0.6 million deaths every year.Despite decline in overall incidence and mortality over the past 30 years,there continues to be an alarming rise in early-onset colon cancer cases(<50 years).Patients are often diagnosed at late stages of the disease and tend to have poor survival.We previously showed that the WNT“gatekeeper”gene,secreted frizzled-related protein 4(SFRP4),is over-expressed in early-onset colon cancer.SFRP4 is speculated to play an essential role in cancer by inhibiting the epithelial mesenchymal transition(EMT).AIM To investigate the correlation between SFRP4 expression and EMT-linked genes in colon cancer and how it affects patient survival.METHODS SFRP4 expression relative to that of EMT-linked genes and survival analysis were performed using the University of California Santa Cruz Cancer Browser interface.RESULTS SFRP4 was found to be co-expressed with the EMT-linked markers CDH2,FN1,VIM,TWIST1,TWIST2,SNAI1,SNAI2,ZEB1,ZEB2,POSTN,MMP2,MMP7,MMP9,and COL1A1.SFRP4 expression negatively correlated with the EMTlinked suppressors CLDN4,CLDN7,TJP3,MUC1,and CDH1.The expression of SFRP4 and the EMT-linked markers was higher in mesenchymal-like samples compared to epithelial-like samples which potentially implicates SFRP4-EMT mechanism in colon cancer.Additionally,patients overexpressing SFRP4 presented with poor overall survival(P=0.0293).CONCLUSION Considering the implication of SFRP4 in early-onset colon cancer,particularly in the context of EMT,tumor metastasis,and invasion,and the effect of increased expression on colon cancer patient survival,SFRP4 might be a potential biomarker for early-onset colon cancer that could be targeted for diagnosis and/or disease therapy.

Suppression of EphB4 Improves the Inhibitory Effect of mTOR shRNA on the Biological Behaviors of Ovarian Cancer Cells by Down-regulating Akt Phosphorylation

The aim of the present study was to examine the effects of suppression of EphB4 and/or mTOR on the biological behaviors of ovarian cancer cells, and the potential regulatory pathways. An-tisense EphB4 vectors and shRNA vectors targeting mammalian target of rapamycin （mTOR） were constructed and transfected into A2780 and SKOV3 cells （two ovarian cancer cell lines）. The effects of the antisense EphB4 vectors and the shRNA vectors on the proliferation, apoptosis and invasion of ovarian cancer cells were measured, and the expression of EphB4, mTOR and Akt detected. The results showed that transfection with mTOR shRNA could inhibit growth, induce apoptosis, and reduce invasive ability of ovarian cancer cells, which was accompanied by downregulation of EphB4, mTOR and Akt. The inhibitory effects on cell growth caused by mTOR shRNA alone were weaker than those by antisense pEGFP-C1-EphB4. In the antisense pEGFP-C1-EphB4-transfected cells, it was found that EphB4 knockdown could decrease the mTOR expression and slightly reduce the Akt phosphorylation. Significant suppressive effects on cell growth were observed in cells co-transfected with antisense pEGFP-C1-EphB4 and mTOR shRNA. In co-transfection group, the expression levels of EphB4, mTOR and Akt were distinctly lower than those in other groups. It was concluded that suppression of EphB4 may inhibit the growth of ovarian cancer cells by downregulation of the PI3K/Akt/mTOR pathway, and reverse Akt phosphorylation induced by mTOR shRNA. Inhibition of EphB4 and mTOR combined may cooperatively suppress the biological behaviors of ovarian cancer cells.

RBBP4 promotes colon cancer malignant progression via regulating Wnt/β-catenin pathway

BACKGROUND Our previous study demonstrated that RBBP4 was upregulated in colon cancer and correlated with poor prognosis of colon cancer and hepatic metastasis.However,the potential biological function of RBBP4 in colon cancer is still unknown.AIM To investigate the biological role and the potential mechanisms of RBBP4 in colon cancer progression.METHODS Real-time polymerase chain reaction and western blot analysis were used to detect the expression of RBBP4 in colon cancer cell lines.The cell proliferation and viability of SW620 and HCT116 cells with RBBP4 knockdown was detected by Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine staining.The transwell assay was used to detect the invasion and migration capabilities of colon cancer cells with RBBP4 knockdown.Flow cytometry apoptosis assay was used to detect the apoptosis of colon cancer cells.Western blotting analysis was used to detect the expression of epithelial-mesenchymal transition and apoptosis related markers in colon cancer.The nuclear translocation ofβ-catenin was examined by Western blotting analysis in colon cancer cells with RBBP4 knockdown.The TOPFlash luciferase assay was used to detect the effect of RBBP4 on Wnt/β-catenin activation.The rescue experiments were performed in colon cancer cells treated with Wnt/β-catenin activator LiCl and RBBP4 knockdown.RESULTS We found that RBBP4 was highly expressed in colon cancer cell lines.The 5-ethynyl-2’-deoxyuridine assay showed that knockdown of RBBP4 significantly inhibited cell proliferation.RBBP4 inhibition reduced cell invasion and migration via regulating proteins related to epithelial-mesenchymal transition.Knockdown of RBBP4 significantly inhibited survivin-mediated apoptosis.Mechanistically,the TOPFlash assay showed that RBBP4 knockdown increased activity of the Wnt/β-catenin pathway.Meanwhile,RBBP4 knockdown suppressed nuclear translocation ofβ-catenin.With Wnt/β-catenin activator,rescue experiments suggested that the role of RBBP4 in colon cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION RBBP4 promotes colon cancer development via increasing activity of the Wnt/β-catenin pathway.RBBP4 may serve as a novel therapeutic target in colon cancer.

Expression of EphB4 and HIF-1α in lung cancer and its clinical significance

Objective： To investigate the relationship between the expression of EphB4 and HIF-la in lung cancer and their biological significance. Methods： The expression of EphB4 and HIF-1α was detected in 54 specimens of lung cancer by using streptavidin-peroxidase （SP）immunohistochemical method, and 10 normal lung tissues as control. Results： Among the 54 cases of lung cancer, the positive rate of EphB4 was 50.0%; 42.6% of the tumors were positive for HIF-1α. The expression of these two kinds of proteins was related to gross types, differentiation and clinical stages （P〈0.05）, but not to histological classification, age, sex and lymphoid metastasis （P〉0.05）. A highly positive correlation was observed between the expression of EphB4 and HIF-1α （P〈0.01）. Conclusion： Overexpression of EphB4 and HIF-1α may play an important role in the pathogenesis, progression and malignant degree of lung cancer. Detection of EphB4 and HIF-1α expression might be helpful to predict prognosis of patients with lung cancer.

EphB4基因重组慢病毒载体的构建及在结肠癌细胞中的表达

目的有研究显示EphB4表达异常在结肠癌发生发展中具有重要意义。文中通过构建慢病毒介导稳定过表达EphB4基因的结肠癌细胞株,以期为结肠癌的基因治疗提供一定的实验依据。方法通过PCR反应将人工合成的EphB4基因序列装入pcDNA3.1(+)载体,并通过测序验证重组克隆插入片段的序列信息。将质粒中扩增的目的基因片断克隆装入慢病毒表达载体pLenti6.3-MCS-IRES2-EGFP并进行基因测序以确认慢病毒载体是否成功构建。携带EphB4和EGFP基因的慢病毒通过阳离子脂质体转染293T细胞,收集富含EphB4基因的病毒颗粒上清液,梯度稀释后测定病毒滴度并感染人结肠癌细胞株SW480和Coca-2,通过药物筛选及维持构建稳定过表达EphB4基因的结肠癌细胞株。荧光显微镜下观察各组细胞中绿色荧光蛋白(green fluorescence protein,GFP)表达情况,qPCR检测转染前后各组细胞中靶基因EphB4 mRNA,评价过表达效果。结果含有2964bp人EphB4基因编码区的慢病毒载体pLenti6.3-EphB4-IRES2-EGFP构建成功,基因测序结果完全正确,经慢病毒包装所得病毒上清液Lenti6.3-EphB4滴度为1×108TU/mL。稳转细胞株在荧光显微镜下可见GFP表达,qPCR实验证明转染后SW480及Coca-2细胞中EphB4 mRNA表达水平较转染前明显增加。结论成功扩增、克隆了EphB4基因,并建立其慢病毒表达系统,构建了稳定过表达EphB4基因的结肠癌细胞株,从而对于研究结肠癌的基因治疗提供了实验依据。

靶向EphB4的慢病毒干扰载体对结肠癌细胞中EphB4及EphrinB2表达的影响

目的有研究显示Eph B4在肿瘤发生发展中的作用与其配体Ephrin B2密切相关。文中主要通过慢病毒介导的RNA干扰技术干扰结肠癌细胞中Eph B4基因的表达,观察Eph B4基因表达改变对其配体Ephrin B2表达的影响。方法根据Eph B4基因序列设计并合成3对miRNA的oligo DNA,退火形成双链后与pc DNA6.2-GW/Em GFP-miRNA载体相连并瞬时转染HEK293细胞,通过荧光显微镜、q PCR筛选出干扰效率最高的质粒,将其与中间载体p DONR221和慢病毒载体p Lenti6/V5-DEST连接构建慢病毒表达载体p Lenti6/V5-DEST-Eph B4。用脂质体将慢病毒载体以及包装质粒(p LP1、p LP2和p LP/VSVG)共转染293FT细胞,收集上清Lenti-miR-Eph B4感染HEK293细胞株进行滴度鉴定。将获得的病毒液感染人结肠癌细胞株,通过荧光显微镜和q PCR检测结肠癌细胞中Eph B4及其配体Ephrin B2的表达变化。结果测序结果表明3对干扰载体SR1、SR2、SR3序列与参考序列一致,成功转染HEK293细胞后,各转染组细胞荧光显微镜下均可见到绿色荧光蛋白表达;q PCR显示载体SR-3的沉默效率最高;构建出来的慢病毒载体p Lenti6.3/V5-DEST-miR-Eph B4在293FT细胞中包装成功,所得慢病毒滴度为7×108TU/m L;Lenti-miR-Eph B4浸染后,SW480和Caco-2中Eph B4 mRNA相对表达量(0.49±0.02、0.62±0.04)均较原始SW480(1.00±0.00)和Caco-2降低(1.00±0.00),差异有统计学意义(P=0.000);SW480中Ephrin B2 mRNA表达量F(2.11±0.04)较原始SW480(1.00±0.00)明显升高(P=0.000);而Caco-2细胞中Ephrin B2 mRNA表达无明显改变(1.01±0.02),差异无统计学意义(P=0.315)。结论靶向Eph B4的慢病毒干扰载体能够有效降低SW480及Caco-2细胞中Eph B4 mRNA表达水平。Eph B4与其配体Ephrin B2之间的相互作用可能受到多种因素调控,有待后续深入研究。

PAD4介导H3cit促结肠癌细胞增殖、侵袭和上皮间质转化的机制研究

目的 探究肽基精氨酸脱亚胺酶4(PAD4)介导的瓜氨酸化组蛋白H3(H3cit)在结肠癌细胞增殖、侵袭和上皮间质转化中的作用及其相关机制。方法 收集我院30例结肠癌患者的血清和30例健康受试者的血清,ELISA检测血清中PAD4和H3cit水平,并分析两者相关性。将oe-NC、过表达PAD4(oe-PAD4)和sh-NC、sh-NLRP3载体转染至人结肠癌SW480细胞中,并设为oe-NC组、oe-PAD4组、oe-PAD4+sh-NC组、oe-PAD4+sh-NLRP3组。采用qRT-PCR检测PAD4、NLRP3、IL-1β、IL-18基因表达水平;Western blot检测PAD4、H3cit、NLRP3、E-cadherin、N-cadherin蛋白表达;CCK-8检测细胞增殖水平;Transwell检测细胞侵袭水平;ChIP-qPCR检测H3cit在NLRP3转录起始位点(TSS)的富集水平。结果 与健康受试者比较,结肠癌患者血清中PAD4和H3cit水平均升高,且两者呈正相关(P<0.05)。与oe-NC组比较,oe-PAD4组PAD4、NLRP3、IL-1β、IL-18基因表达水平升高,PAD4、H3cit、NLRP3、N-cadherin蛋白表达升高,E-cadherin蛋白表达降低,细胞增殖、侵袭水平升高,H3cit在NLRP3 TSS区富集增加(P<0.05);与oe-PAD4+sh-NC组比较,oe-PAD4+sh-NLRP3组NLRP3、IL-1β、IL-18基因表达水平降低,NLRP3和N-cadherin蛋白表达降低,E-cadherin蛋白表达升高,细胞增殖、侵袭水平降低(P<0.05)。结论 结肠癌患者体内PAD4和H3cit水平升高,PAD4介导的H3cit通过转录调控NLRP3的表达,促进结肠癌细胞增殖、侵袭和上皮间质转化。

结肠癌组织TLR4、CHI3L1表达与根治术后预后的关系

目的探讨结肠癌组织中Toll样受体4(TLR4)、壳多糖酶3样蛋白1(CHI3L1)的表达及其与患者根治术后预后的关系。方法选取2017年1月至2018年5月于我院行结肠癌根治术的152例患者为研究对象,根据患者术后5年生存情况分为预后良好组(n=97)和预后不良组(n=47)。采用免疫组织化学染色法检测结肠癌组织及癌旁组织中TLR4、CHI3L1的表达水平;分析结肠癌组织中TLR4和CHI3L1表达与患者预后的关系,并分析结肠癌患者预后的影响因素。结果结肠癌组织中TLR4和CHI3L1阳性表达率显著高于癌旁组织(P<0.05)。结肠癌组织中TLR4表达与患者肿瘤分化程度、临床分期和淋巴结转移有关(P<0.05),CHI3L1表达与结肠癌患者肿瘤直径、肿瘤分化程度、临床分期和淋巴结转移有关(P<0.05)。与预后良好组比较,预后不良组患者肿瘤低分化、临床分期Ⅲ期、淋巴结转移、TLR4阳性表达和CHI3L1阳性表达的占比较高(P<0.05)。TLR4阳性表达患者5年生存率为60.38%,低于TLR4阴性表达患者的86.84%(χ^(2)=9.104,P<0.05);CHI3L1阳性表达患者5年生存率为58.06%,低于CHI3L1阴性表达患者的84.31%(χ^(2)=10.935,P<0.05)。TLR4阳性、CHI3L1阳性、肿瘤低分化、临床分期Ⅲ期、淋巴结转移是结肠癌患者预后的独立危险因素(P<0.05)。结论TLR4和CHI3L1与结肠癌的发生和临床病理特征有关,且结肠癌组织中TLR4和CHI3L1阳性表达不利于患者预后,二者有望成为临床治疗靶点。

Suppression of human colon tumor growth by adenoviral vector-mediated NK4 expression in an athymic mouse model

AIM： To investigate the suppressive effects of adenoviral vector-mediated expression of NK4, an antagonist of hepatocyte growth factor （HGF）, on human colon cancer in an athymic mouse model to explore the possibility of applying NK4 to cancer gene therapy. METHODS： A human colon tumor model was developed by subcutaneous implantation of tumor tissue formed by LS174T cells grown in athymic mice. Fifteen tumorbearing mice were randomized into three groups （n= 5 in each group） at d 3 after tumor implantation and mice were injected intratumorally with phosphate-buffered saline （PBS） or with recombinant adenovirus expressing 13-galactosidase （Ad-LacZ） or NK4 （rvAdCMV/NK4） at a 6-d interval for total 5 injections in each mouse. Tumor sizes were measured during treatment to draw a tumor growth curve. At d 26 after the first treatment, all animals were sacrificed and the tumors were removed to immunohistochemically examine proliferating cell nuclear antigen （PCNA）, microvessel density （represented by CD31）, and apoptotic cells. In a separate experiment, 15 additional athymic mice were employed to develop a tumor metastasis model by intraperitoneal injection （ip） of LS174T cells. These mice were randomized into 3 groups （n = 5 in each group） at d 1 after injection and were treated by ip injection of PBS, or Ad-LacZ, or rvAdCMV/NK4 at a 6-d interval for total two injections in each mouse. All animals were sacrificed at d 14 and the numbers and weights of disseminated tumors within the abdominal cavity were measured. RESULTS： Growth of significantly suppressed human colon tumors were in the athymic mice treatedwith rvAdCMV/NK4 （2537.4± 892.3 mm^3） compared to those treated by either PBS （5175.2 ± 1228.6 mm^3） or Ad-LacZ （5578.8± 1955.7 mm^3） （P 〈 0.05）. The tumor growth inhibition rate was as high as 51%. Immunohistochemical staining revealed a similar PCNA labeling index （75.1% ± 11.2% in PBS group vs 72.8% ± 7.6% in Ad-LacZ group vs 69.3% ± 9.4% in rvAdCMV/ NK4 group） in all groups, but significantly lower microvessel density （10.7 ± 2.4 in rvAdCMV/NK4 group vs 25.6 ± 3.8 in PBS group or 21.3 ± 3.5 in Ad-LacZ group, P 〈 0.05）, and a markedly higher apoptotic index （7.3% ± 2.4% in rvAdCMV/NK4 group vs 2.6 4, 1.1% in PBS group or 2.1% ± 1.5% in Ad-LacZ group, P 〈 0.05） in the rvAdCMV/NK4 group compared to the two control groups. In the tumor metastasis model, the number and weight of disseminated tumors of mice treated with rvAdCMV/NK4 were much lower than those of the control groups （tumor number： 6.2 ± 3.3 in rvAdCMV/ NK4 group vs 22.9 ± 7.6 in PBS group or 19.8 ± 8.5 in Ad-LacZ group, P 〈 0.05; tumor weight： 324 ± 176 mg in rvAdCMV/NK4 group vs 962 ± 382 mg in PBS group or 1116 ± 484 mg in Ad-LacZ group, P 〈 0.05）. CONCLUSION： The recombinant adenovirus, rvAdCMV/ NK4, can attenuate the growth of colon cancer in vivo, probably by suppressing angiogenesis and inducing tumor cell apoptosis, but not by direct suppression of tumor cell proliferation. Moreover, rvAdCMV/NK4 may inhibit peritoneal dissemination of colon cancer cells in a murine tumor metastasis model. These findings indicate that NK4 gene transfer may be an effective tool for the treatment of colon cancer.

Effect of indomethacin on cell cycle proteins in colon cancer cell lines

AIM: To studythe effect of indomethacin (IN) on human colon cancer cell line SW480 with p53 mutant and SW480transfected wild-type p53 (wtp53/SW480) in vitro and investigate molecular mechanism of anti-tumor effect of IN on colon cancer.METHODS: SW480 cells and wtp53/SW480 cells were treated with different concentrations of IN respectively,the expressions of CDK2, CDK4 and p21WAF1/CIP1 protein were detected by Western blotting.RESULTS: IN gradually down-regulated the expression of CDK2, CDK4 protein of wtp53/SW480 cells in a dosedependent manner, and inhibitory effect reached the maximum level at 600 μmol/L; IN up-regulated the expression of p21WAF1/CIP1 protein in a dose-dependent manner at a certain concentration range, and the expression reached the maximum level at 400 μmol/L,and returned to the base level at 600 μmol/L. The expression of CDK2, CDK4 and p21WAF1/CIP1 protein of SW480cells did not change.CONCLUSION: IN exerts antitumor effect partly through down regulation of the expression of CDK2, CDK4 protein and up regulation of the expression of p21WAF1/PIC1.

吉西他滨联合放射对人胰腺癌CFPAC-1细胞株裸鼠移植瘤EphB4、Caspase3表达的影响

目的 探讨吉西他滨联合放射对胰腺癌发病机制的调节。方法 建立裸小鼠皮下移植瘤模型,模型建立后将36只裸小鼠随机分为6组[对照组(A),单体放疗组(B),25 mg/kg吉西他滨组(C),50 mg/kg吉西他滨组(D),25 mg/kg吉西他滨+放射组(E),50 mg/kg吉西他滨+放射组(F)]。RT-PCR测各组组织中EphB4、Caspase3基因表达,免疫组化测定组织EphB4、Caspase3的蛋白表达。MTT(3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenytetrazolium bromide)法分析CFPAC-1细胞的增殖水平。结果 与A组(抑瘤率为0%)比较,E、F组的抑瘤率明显高于B组(P<0.05)和C、D组(P<0.05)。采用RT-PCR检测新鲜组织,在EphB4和Caspase3基因中A组、B组、C组、D组、E组、F组依次为递减趋势和递增趋势。免疫组化检测显示EphB4、Caspase3抗体六组两两比较具有统计学意义(P<0.05)。MTT实验显示人胰腺癌CFPAC-1细胞株在联合组中增殖率最低。结论 通过本实验的研究,可以得出以下结论:(1)吉西他滨联合放射的作用优于吉西他滨单药以及单纯放射。(2)吉西他滨对人胰腺癌CFPAC-1细胞株裸鼠移植瘤有放射增敏作用,其可能的机制之一是通过下调EphB4和上调Caspase3的表达。(3)MTT法提示吉西他滨联合放射可能具有放射增敏作用。

结肠癌组织中miR-183-5p和THEM4的表达水平及临床意义

目的:研究结肠癌组织中微小RNA-183-5p(miR-183-5p)和硫酯酶超家族成员4(THEM4)的表达水平及其临床意义。方法:收集河北中石油中心医院96例结肠癌患者为研究对象,在进行根治切除术过程中收集患者结肠癌组织及癌旁正常组织。检测结肠癌组织及癌旁正常组织中miR-183-5p和THEM4 m RNA的相对表达水平。分析两者的相关性及其与预后的关系。Cox回归分析影响结肠癌患者预后的危险因素。结果:与癌旁正常组织相比,结肠癌组织中miR-183-5p表达水平升高(P<0.05),THEM4 m RNA表达水平降低(P<0.05)。结肠癌组织中miR-183-5p与THEM4 m RNA表达呈负相关(r=-0.529,P<0.05)。miR-183-5p高表达组生存率低于低表达组(P<0.05),THEM4高表达组生存率显著高于低表达组(P<0.05)。TNM分期(Ⅲ~Ⅳ)、miR-183-5p高表达、THEM4低表达是结肠癌患者不良预后的危险因素(P<0.05)。结论:结肠癌织中miR-183-5p表达水平升高,THEM4表达水平降低,两者均与患者临床病理特征及预后密切相关。

SOX4靶向miR-17对结肠癌细胞HCT-116生物学行为的影响

目的探讨性别决定区Y框蛋白4(SOX4)及miR-17在结直肠癌中的表达及对HCT-116细胞生物学行为的作用。方法收集2020年3月至2022年1月在河北北方学院一附院和张家口市第一医院的27例结直肠癌患者组织标本并检测SOX4及miR-17的表达水平,并分析与临床病理特征的关系。利用慢病毒转染技术干预HCT-116细胞的SOX4及miR-17表达。检测各组细胞中SOX4和miR-17的表达及细胞增殖、侵袭、迁移能力、细胞活性变化和相关蛋白表达。结果与正常组织比较,结直肠癌组织中SOX4表达水平升高。SOX4及miR-17在结直肠癌组织中的表达呈正相关(r=0.724,P<0.001),SOX4和miR-17的表达和病理分期、淋巴结转移、肝转移情况相关,差异有统计学意义(P<0.05),而与肿瘤的大小、分化程度无相关(P>0.05)。SOX4模拟组细胞增殖能力、迁移能力及细胞活性显著高于对照组及SOX4抑制剂组(P<0.05)。SOX4抑制剂组与对照组相比迁移及增殖相关蛋白表达差异有统计学意义(P<0.05)。结论SOX4在结直肠癌组织中表达上调,SOX4的表达可促进结肠癌细胞增殖、迁移和侵袭能力,miR-17可能是其下游调控靶点。

结肠癌组织中β-连环蛋白/转录因子4信号通路相关蛋白表达及其与患者术后复发转移的关系

目的探讨结肠癌组织中β-连环蛋白(β-catenin)/转录因子4(TCF4)信号通路相关蛋白表达及其与患者术后复发转移的关系。方法选择驻马店市中心医院2020年1月至2022年1月收治的结肠癌患者86例为研究对象,患者均接受手术切除。采用实时荧光定量聚合酶链式反应检测患者癌组织及癌旁组织中Wnt-1、β-catenin、TCF4 mRNA的表达,免疫组织化学法检测Wnt-1、β-catenin、TCF4蛋白的表达,Cox比例回归模型分析结肠癌患者术后复发转移的危险因素。结果结肠癌患者癌组织中Wnt-1、β-catenin、TCF4 mRNA相对表达量和蛋白表达阳性率显著高于癌旁组织(P<0.05)。Wnt-1、β-catenin、TCF4蛋白表达阳性率与肿瘤浸润程度、肿瘤分期以及淋巴结转移有关(P<0.05),Wnt-1、β-catenin、TCF4的蛋白表达阳性率与结肠癌患者年龄、性别、肿瘤大小、分化程度无关(P>0.05)。Cox回归分析结果显示,有淋巴结转移、Wnt-1、β-catenin、TCF4蛋白表达阳性是结肠癌患者术后复发转移的危险因素(P<0.05)。结论结肠癌组织中Wnt-1、β-catenin、TCF4 mRNA表达及蛋白表达阳性率均较高,Wnt-1、β-catenin、TCF4蛋白表达阳性率与肿瘤浸润程度、肿瘤分期以及淋巴结转移显著相关,是结肠癌术后复发转移的危险因素。

上调基因-4在结肠癌中的表达及临床意义

目的探讨上调基因-4(up-regulated gene-4,URG-4)在结肠癌及其癌旁组织中的表达及临床意义。方法收集2010年1-6月本院手术切除的82例结肠癌、癌旁组织样本及石蜡切片标本,用RT-PCR、免疫组化检测URG-4基因的转录和表达情况,分析其与临床病理参数的关系。结果结肠癌组织中的URG-4 mRNA阳性率为85.37%(70/82),显著高于癌旁组织中的36.59%(30/82)(P<0.01)。URG-4蛋白在结肠癌中的表达率为70.73%(58/82),癌旁组织为24.39%(20/82),两组比较差异非常显著(P<0.01);肿瘤组织中URG-4的表达与患者的年龄、性别、肿瘤的分化程度、有无远处转移无明显相关性(P>0.05),而与浸润深度及淋巴结转移显著相关(P<0.01)。结论与癌旁组织相比URG-4在结肠癌中高表达,并且URG-4表达强度与肿瘤浸润深度和淋巴结转移呈正相关,提示其可能在结肠癌的发生、发展中起重要作用。

酪酸梭菌培养上清液通过下调TLR4/NF-κB信号通路抑制SW-480细胞增殖

目的研究酪酸梭菌培养上清液(C.butyricum culture supernatant;C.b.cs)对结肠癌SW-480细胞增殖的抑制作用,并探讨C.b.cs中可能的活性成分及相关分子机制。方法用C.b.cs处理后SW-480细胞,CCK-8及FCM检测细胞的增殖和凋亡。HPLC检测C.b.cs中主要的有机酸含量。Western blot及RT-q PCR检测经过C.b.cs和丁酸处理后的TLR4的表达。用TLR4配体脂多糖(LPS)激活TLR4,再用C.b.cs和丁酸处理细胞,观察TLR4和NF-κB的表达。结果 C.b.cs呈浓度依赖性地抑制SW-480增殖,并能诱导凋亡和G0/G1期阻滞。HPLC检测C.b.cs中乙酸和丁酸的含量分别为9.27 g/L和4.53 g/L。C.b.cs和丁酸可下调TLR4的表达(P<0.01),并下调TLR4的下游基因NF-κB的表达(P<0.05)。LPS激活TLR4的表达(P<0.01)后,C.b.cs和丁酸下调TLR4和NF-κB的表达(P<0.05)。结论 C.b.cs通过下调TLR4/NF-κB通路抑制SW-480细胞增殖,丁酸可能是发挥抑制作用的活性成分之一。

奥曲肽对结肠癌SW480细胞Wnt通路β-catenin/TCF及下游靶基因表达的影响

目的研究不同浓度奥曲肽对结肠癌SW480细胞增殖以及对SW480细胞Wnt／β-catenin信号通路的影响，阐明其抗肿瘤的作用机制。方法采用MTT比色法测定不同浓度奥曲肽对SW480细胞增殖的抑制作用；应用Westernblot法分析不同浓度奥曲肽对SW480细胞核中β—catenin、TCF-4及Wnt信号通路下游靶分子C—myc、CyclinDl蛋白表达的影响。结果奥曲肽可抑制SW480细胞的增殖，并在10-10～10-8M浓度范围内呈剂量依赖性，在48h内呈时间依赖性，浓度为10-8M时抑制作用最强，浓度为10-11M以下对细胞增殖无抑制作用。浓度为10-6、10-8、10-10M的奥曲肽对β—catenin蛋白、TCF-4蛋白、C—myc蛋白及CyclinDl蛋白的表达有明显抑制作用，与未经奥曲肽处理的细胞比较有统计学意义（均P〈0．01）。结论奥曲肽对Wnt／β-catenin信号通路的抑制作用可能是通过减少β—catenin在细胞核内的聚集，抑制TCF-4的激活，从而抑制下游靶基因C—myc及CyclinDl。