Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Nerve growth factor pretreatment against glutamate-induced hippocampal neuronal injury Action mechanism of phosphatase and tensin homologue deleted on chromosome 10

BACKGROUND： Nerve growth factor （NGF） attenuates glutamate-induced injury to hippocampal neurons, and the human tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 （PTEN） promotes neuronal apoptosis. However, effects of PTEN in NGF-mediated neuroprotection against glutamate excitotoxicity remain poorly understood. OBJECTIVE： To investigate the relationship between NGF inhibition of glutamate-induced injury and PTEN. DESIGN, TIME AND SE＇I＇rlNG： The randomized, controlled, in vitro study was performed at the Department of Pathophysiology, Medical School of Nantong University, China from October 2007 to March 2008. MATERIALS： Glutamate, NGF, 4, 6-diamidino-2-phenyl-indolediacetate, 3-[4, 5-dimethylthiazol-2-yl]- 2, 5-diphenyl tetrazoliumbromide （M-I-F）, and lactate dehydrogenase kit （Sigma, USA）, fluorescence microscope and inverted phase contrast microscope （Olympus, Japan） were used in this study. METHODS： Hippocampal neurons were obtained from newborn （〈 24 hours） Sprague Dawley rats and cultured for 7 days. The control group was not treated with any intervention factor, the glutamate group was treated with glutamate （0.2 mmol/L）, and NGF groups were treated with NGF （10, 50, 100, and 200 μg/L, respectively） prior to glutamate treatment. MAIN OUTCOME MEASURES： The MTT and lactate dehydrogenase assays were applied to evaluate viability of hippocampal neurons. Morphological changes in hippocampal neurons were observed using an inverted phase-contrast microscope, and neuronal apoptosis was detected by 4, 6-diamidino-2- phenyl-indolediacetate staining. PTEN mRNA and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot analysis, respectively. RESULTS： Glutamate （0.2 mmol/L） induced significantly decreased neuronal viability and greater lactate dehydrogenase efflux compared with the control group （P 〈 0.01）. However, compared with the glutamate group, cell viability significantly increased and lactate dehydrogenase efflux decreased in the NGF group with increasing NGF concentrations （P 〈 0.05 or P 〈 0.01）. The apoptotic ratio and PTEN mRNA and protein expression decreased in the NGF group compared with the glutamate group （P 〈 0.01）. CONCLUSION： Pretreatment with NGF exerted neuroprotective effects against glutamate-induced injury, partially through inhibition of PTEN expression and neuronal apoptosis.

Expression of phosphatase and tensin homolog deleted on chromosome ten in liver of athymic mice with hepatocellular carcinoma and the effect of Fuzheng Jiedu Decoction

AIM: To explore the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in liver of athymic mice with hepatocellular carcinoma (HCC) and the effect of Fuzheng Jiedu Decoction (FJD). METHODS: Forty eight male BALB/c athymic mice models were built by Bel-7402 with an indirect method. After 24 h of postoperation, the 48 athymic mice were distributed randomly into 4 groups: A, B, C, D, each group had 12 athymic mice. Group A were were treated by intragastric administration with FT207 (Tegafur) for 4 wk. Group B, C and D were treated by intragastric administration with FJD (complex prescription of Chinese crude drug) that had been delegated into 3 kinds of density as the low, middle, and high for 4 wk. At last, athymic mice were put to death, live time, volume of tumors, exponent of tumors and the tumor metastasis in livers were observed; and PTEN was detected in hepatic tissue, latero-cancer tissue and cancer tissue by immunohistochemistry.RESULTS: Four weeks later, the total survival rate in treatment group (A + B + C) was 50% and higher than the control group (0%) treated by FT207, (P < 0.01). The survival rate in group A, B, C was higher than in group D, and except group A with D, there was significant differentces (Fisher's Exact Test P = 0.05 or 0.01). And no differences were observed between the treatment groups and the control group in volume of tumors and exponent of tumors (P > 0.05). Tumor metastasis in livers of the treatment group was less than the controls (Fisher's Exact Test, P = 0.021). The result of immunohistochemistry showed that the intensity of PTEN in latero-cancer tissue was the highest, and then the hepatic tissue, the lowest was cancer tissue (Kruskal-Wallis test, χ2 = 60.67, P = 0.000). It also showed that the intensity of PTEN in treatment groups (A, B, C) was higher than the control group (D) (F = 5.90, P = 0.002 in hepatic tissue and F = 15.99, P = 0.000 in latero-cancer tissue and χ2 = 26.08, P = 0.000 in cancer tissue), and group B is the highest in the treatment groups (P < 0.05, r = 0.01. respectively). However, there was no significant statistic difference between group A and group C (P > 0.05).CONCLUSION: FJD can prolong the survival time and decrease tumor metastasis in livers of these experimental mice. Mechanisms of FJD healing HCC may partially be explained by enhancing the expression of PTEN in liver.

Rapid construction of phosphatase and tensin homolog-deleted on chromosome ten gene recombinant adenovirus using the AdEasy system

Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.

miR-21低表达对垂体瘤细胞系RC-4BC增殖、凋亡的影响及与PTEN靶向关系

目的观察微小RNA-21(miR-21)低表达对垂体瘤细胞系RC-4BC增殖、凋亡的影响,并分析其与第10号染色体丢失的张力蛋白同源磷酸酶基因(PTEN)的靶向关系。方法取对数生长期的RC-4BC细胞分为两组,沉默组转染miR-21抑制物miR-21 inhibitor,阴性对照组转染抑制物阴性对照NC-inhibitor,采用RT-PCR法检测miR-21、第10号染色体丢失的张力蛋白同源磷酸酶基因(PTEN)mRNA,采用CCK8实验观察两组细胞增殖能力(以OD值表示),采用平板克隆实验观察两组细胞集落形成能力(以集落形成数表示),采用流式细胞术观察两组细胞凋亡率并观察细胞周期分布情况。收集RC-4BC细胞制备单细胞悬液,分别将miR-21 mimics或NC-mimics与PTEN-WT或PTEN-MUT共转染至RC-4BC细胞,转染后细胞标记为miR-21 mimics+PTEN-WT组、NC-mimics+PTEN-WT组、miR-21 mimics+PTEN-MUT组、NC-mimics+PTEN-MUT组,采用双荧光素酶报告基因实验验证miR-21与PTEN的靶向关系。结果沉默组RC-4BC细胞中miR-21、PTEN mRNA相对表达量分别为0.30±0.08、2.89±0.14,阴性对照组RC-4BC细胞中miR-21、PTEN mRNA相对表达量分别为1.01±0.02、0.99±0.03,两组相比,P均<0.05。沉默组RC-4BC细胞24 h、48 h、72 h时OD值均低于阴性对照组(P均<0.05)。沉默组RC-4BC细胞集落形成数低于阴性对照组(P<0.05)。沉默组RC-4BC细胞凋亡率高于阴性对照组(P<0.05)。沉默组RC-4BC细胞G0/G1期占比65.65%±7.82%、S期占比19.25%±3.70%,阴性对照组RC-4BC细胞G0/G1期占比45.62%±5.03%、S期占比35.72%±4.67%,两组相比,P均<0.05。miR-21 mimics+PTEN-WT组、NC-mimics+PTEN-WT组、miR-21 mimics+PTEN-MUT组、NC-mimics+PTEN-MUT组细胞的相对荧光素酶活性分别为0.39±0.07、1.02±0.03、1.01±0.04、1.00±0.03,其中miR-21 mimics+PTEN-WT组相对荧光素酶活性与其他各组相比,P均<0.05。结论沉默miR-21能够移至垂体瘤细胞系RC-4BC的增殖、促进其凋亡,其机制可能与靶向调控PTEN基因有关。

Upregulated DJ-1 Promotes Renal Tubular EMT by Suppressing Cytoplasmic PTEN Expression and Akt Activation

Recently,phosphatase and tensin homolog deleted on chromosome 10（PTEN） is suggested as a new agent in the fighting against fibrogenesis.In tumor,DJ-1 is identified as a negative regulator of PTEN.But the expression of DJ-1 and the regulation of PTEN in fibrosis are unclear.Renal fibrosis was induced in 5/6 subtotal nephrectomy rat model.Human proximal tubular epithelial cells（HKC） were treated with transforming growth factor-beta 1（TGF-β1）,or transfected with DJ-1 or PTEN.Confocal microscope was used to investigate the localization of DJ-1 and PTEN.The selective phosphoinositide-3 kinase（PI3K） inhibitor,LY294002,was administered to inhibit PI3K pathway.The DJ-1 and PTEN expression,markers of epithelial-mesenchymal transition（EMT） and Akt phosphorylation were measured by RT-PCR,Western blotting or immunocytochemistry.In vitro,after HKC cells were stimulated with 10 ng/mL TGF-β1 for 72 h,the expression of DJ-1 was increased,and that of PTEN was decreased.In vivo,the same results were identified in 5/6-nephrectomized rats.In normal HKC cells,most of DJ-1 protein localized in cytoplasm,and little in nucleus.TGF-β1 upregulated DJ-1 expression in both cytoplasma and nuclei.In contrary,TGF-β1 emptied cytoplasmic PTEN protein into nucleus.Overexpression of DJ-1 decreased the expression of PTEN,promoted the activation of Akt and the expression of vimentin,and also led to the loss of cytoplasmic PTEN.Contrarily,overexpression of PTEN protected HKC cells from TGF-β1-induced EMT.In conclusion,DJ-1 is upregulated in renal fibrosis and DJ-1 mediates EMT by suppressing cytoplasmic PTEN expression and Akt activation.

Relationsip between PTEN and VEGF Expression and Clinicopathological Characteristics in HCC

To investigate the expressions and significance of the tumor suppressor gene phosphatase and tensin homlog deleted on chromosome ten protein （PTEN） and vascular endothelial growth factor （VEGF） in hepatocellular carcinoma （HCC）, and to analyze the relationship between their expressions and the tumor＇s invasion and their pericarcinomatous tissues, the correlation of their expressions with the tumor＇s clinicopathological characteristics and invasion potential were studied. Our study showed that the expression level of PTEN in HCC was remarkably lower than that in pericarcinomatous liver tissues, while the expressions of both VEGF and MVD were higher than that in pericarcinomatous liver tissues. Correlation analysis revealed that the expression of PTEN was negatively related to the progression of the pathological differentiation and invasion of tumor, whereas the expressions of VEGF and MVD were positively related. Moreover, there was a negative relationship between the expression of PTEN and the expressions of VEGF and MVD, and a positive one between VEGF and MVD. The expressions of PTEN and VEGF may reveal the degree of differentiation and the invasive potential of HCC tissues. The mechanism by which the lack of PTEN expression probably induces abnormal hyperexpression of VEGF may play an important role in the invasion and metastasis of HCC.

PTEN and Ki67 expression is associated with clinicopathologic features of non-small cell lung cancer

Phosphatase and tensin homolog deleted on chromosome 10（PTEN） and the proliferating antigen Ki67 have been widely studied in several tumors.However,their role as indicator in non-small cell lung cancer（NSCLC）remains unknown.Here,we investigated the expression of PTEN and Ki67 in NSCLC tissues and paired normal lung tissues to identify whether these proteins are associated with lung cancer development and survival.Immunohistochemistry for PTEN and Ki67 was performed on 67 lung cancer tissues and 41 paired adjacent normal lung tissues to detect the expression of these two proteins.The expression of PTEN in NSCLC tissues（32.8%） was significantly lower than that in normal tissues（82.9%,P 〈 0.05）.In contrast,the expression of Ki67 in NSCLC tissues（76.1%） was significantly higher than that in normal tissues（27.3%,P 〈 0.05）.Expression of both PTEN and Ki67 were strongly associated with tumor histology,clinical stage,lymph node metastasis,differentiation and4-year postoperative survival rate（P 〈 0.05）.However,PTEN expression was negatively correlated with Ki67 expression（r =-0.279,P 〈 0.05）.In conclusion,low PTEN expression and Ki67 overexpression are associated with malignant invasion and lymph node metastasis of NSCLC.These proteins may serve as diagnostic and prognostic biomarkers of NSCLC.

血清TLR4和PTEN与特发性膜性肾病患者临床病理特征及预后的相关性

目的 探究血清Toll样受体4(TLR4)、第10号染色体缺失性磷酸酶和张力蛋白同源物基因(PTEN)与特发性膜性肾病(IMN)患者临床病理特征及预后的相关性。方法 选取2019年1月至2022年1月在河北以岭医院就诊治疗的IMN患者224例为IMN组,同时根据其预后结果将其分为预后良好组174例、预后不良组50例;另选取同期在本院体检健康者100名作为对照组。采用酶联免疫吸附法(ELISA)检测研究对象血清TLR4、PTEN水平;采用pearson分析IMN患者血清TLR4水平和PTEN水平相关性;多因素Logistic回归分析IMN患者预后不良的影响因素;受试者工作特征曲线(ROC)分析血清TLR4和PTEN在评估IMN患者预后中的价值。结果 IMN组血清TLR4、PTEN水平较对照组均显著升高,差异有统计学意义(t=12.918,13.249,P<0.05);预后不良组血清TLR4、PTEN水平较预后良好组均显著升高,差异有统计学意义(t=11.608,12.965,P<0.05);IMN患者血清TLR4与PTEN水平呈正相关;TLR4、PTEN、IgG、合并高血压均为IMN患者预后不良的独立危险因素(P<0.05);血清TLR4、PTEN水平预测IMN患者预后是否不良的曲线下面积(AUC)分别为0.878、0.868,两者联合预测的AUC为0.941,高于两者单独预测(z=1.874、2.065,P<0.05),且特异度为0.89,灵敏度为0.88。结论 血清TLR4、PTEN水平与IMN患者分期,肾小球硬化,二者对IMN预后具有一定的预测价值。

Colonic manifestations of PTEN hamartoma tumor syndrome: Case series and systematic review

AIM:To investigate our clinical experience with the colonic manifestations of phosphatase and tensin homolog on chromosome ten(PTEN)hamartoma tumor syndrome(PHTS)and to perform a systematic literature review regarding the same.METHODS:This study was approved by the appropriate institutional review board prior to initiation.A clinical genetics database was searched for patients with PHTS or a component syndrome that received gastrointestinal endoscopy or pathology interpretation at our center.These patient’s records were retrospectively reviewed for clinical characteristics(including family history and genetic testing),endoscopy results and pathology findings.We also performed a systematic review of the literature for case series of PHTS or component syndromes that reported gastrointestinal manifestations and investigations published after consensus diagnostic criteria were established in 1996.These results were compiled and reported.RESULTS:Eight patients from our institution met initial inclusion criteria.Of these,5 patients underwent4.2 colonoscopies at mean age 45.8±10.8 years.All were found to have colon polyps during their clinical course and polyp histology included adenoma,hyperplastic,ganglioneuroma and juvenile.No malignant lesions were identified.Two had multiple histologic types.One patient underwent colectomy due to innumerable polyps and concern for future malignant potential.Systematic literature review of PHTS patients undergoing endoscopy revealed 107 patients receiving colonoscopy at mean age 37.4 years.Colon polyps were noted in92.5%and multiple colon polyp histologies were reported in 53.6%.Common polyp histologies included hyperplastic(43.6%),adenoma(40.4%),hamartoma(38.3%),ganglioneuroma(33%)and inflammatory(24.5%)polyps.Twelve(11.2%)patients had colorectal cancer at mean age 46.7 years(range 35-62).Clinical outcomes secondary to colon polyposis and malignancy were not commonly reported.CONCLUSION:PHTS has a high prevalence of colon polyposis with multiple histologic types.It should be considered a mixed polyposis syndrome.Systematic review found an increased prevalence of colorectal cancer and we recommend initiating colonoscopy for colorectal cancer surveillance at age 35 years.

急性脑梗死患者血清CXCL1、PTEN mRNA水平与病情严重程度及预后的关系

目的探讨急性脑梗死患者血清CXC趋化因子配体1(CXCL1)、第10染色体同源丢失性磷酸酶张力蛋白基因(PTEN)mRNA水平与病情严重程度及预后的关系。方法将2022年3月至2023年3月该院收治的102例急性脑梗死患者纳入研究作为试验组,另选取同期于该院进行体检的85例健康者作为对照组。收集纳入研究者的空腹静脉血血清标本。采用酶联免疫吸附法检测血清CXCL1水平。采用实时荧光定量PCR(qPCR)检测血清PTEN mRNA相对表达水平(下称水平)。根据美国国立卫生研究院脑卒中量表(NIHSS)评分将试验组患者分神经功能缺损程度不同的3组(重症组、中度组、轻度组),比较3组血清CXCL1、PTEN mRNA水平。根据计算机断层扫描(CT)或磁共振成像(MRI)评估脑梗死体积,将试验组患者分为小型梗死组、中型梗死组和大型梗死组,比较3组血清CXCL1、PTEN mRNA水平。根据改良Rankin量表(mRS)将试验组患者分为预后良好组和预后不良组,比较2组患者血清CXCL1、PTEN mRNA水平。采用Pearson相关分析急性脑梗死患者血清CXCL1、PTEN mRNA水平的相关性。采用多因素Logistics回归分析影响急性脑梗死患者预后的因素。结果试验组有糖尿病史、高血压史者占比及血清CXCL1、PTEN mRNA水平均高于对照组,差异均有统计学意义(P<0.05)。随着神经功能缺损程度的增加,血清CXCL1水平、PTEN mRNA水平均增加,重症组、中度组、轻度组间比较差异均有统计学意义(P<0.05)。随着梗死面积增加,血清中CXCL1、PTEN mRNA水平均增加,小型梗死组、中型梗死组和大型梗死组间比较差异均有统计学意义(P<0.05)。预后不良组有糖尿病史者占比、有高血压史者占比及血清CXCL1、PTEN mRNA水平均高于预后良好组(P<0.05)。急性脑梗死患者血清CXCL1水平和PTEN mRNA水平呈正相关(r=0.479,P<0.001)。血清CXCL1、PTEN mRNA水平及糖尿病史、高血压史均为急性脑梗死患者预后的影响因素(P<0.05)。结论急性脑梗死患者血清CXCL1、PTEN mRNA水平升高,可用于评估患者病情程度和预后。

MiR-106b-5p Inhibits Tumor Necrosis Factor-α-induced Apoptosis by Targeting Phosphatase and Tensin Homolog Deleted on Chromosome 10 in Vascular Endothelial Cells

Background： Apoptosis of endothelial cells （ECs） plays a key role in the development of atherosclerosis and there are also evidence indicated that phosphatase and tensin homolog deleted on chromosome 10 （PTEN） is a viable target in therapeutic approaches to prevent vascular ECs apoptosis. Aberrant miR-106b-5p expression has been reported in the plasma of patients with unstable atherosclerotic plaques. However, the role and underlying mechanism of miR-106-5p in the genesis of atherosclerosis have not been addressed. In this study, we explored the anti-apoptotic role of miR-106-5p by regulating PTEN expression in vascular ECs. Methods： Real-time reverse transcription polymerase chain reaction （RT-PCR） was performed to detect the expression levels of miR-106b-5p in human atherosclerotic plaques and normal vascular tissues. Human umbilical vein endothelial cells （HUVEC） were transfected with miR-106b-5p mimic or negative control mimic, and apoptosis was induced by serum starvation and tumor necrosis factor-α （TN F-α） treat. Western blotting and real-time RT-PCR experiments were used to detect PTEN expression levels and TN F-α-induced apoptosis was evaluated by the activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Results： The expression ofmiR-106b-5p was significantly downregulated in plaques than in normal vascular tissues. TNF-α significantly downregulated miR-106b-5p expression levels and upregulated activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Overexpression ofmiR-106b-5p with miR-106b-5p mimic inhibited PTEN expression and TNF-α-induced apoptosis in HUVEC. Luciferase reporter assays confirmed that miR-106b-5p binds to PTEN mRNA 3＇ untranslated region site, Conclusion： MiR-106b-5p could inhibit the expression of PTEN in vascular ECs, which could block TNF-α-induced activation of caspase-3, thus prevent ECs apoptosis in atherosclerosis diseases.

食管黏膜上皮癌变过程中与细胞骨架蛋白tensin同源的磷酸酯酶基因的表达及意义

目的探讨与细胞骨架蛋白tensin同源的磷酸酯酶基因(PTEN)在食管黏膜上皮癌变过程中的表达及意义。方法采用免疫组织化学法检测20例正常食管黏膜、20例食管上皮非典型增生、24例原位癌、44例食管鳞癌组织中PTEN表达情况,并探讨PTEN与食管鳞癌病理分级的关系。结果正常食管黏膜、非典型增生、原位癌及食管鳞癌组织中PTEN蛋白阳性表达率分别为100%、85.00%、70.83%和45.45%,原位癌、食管鳞癌组织中PTEN蛋白阳性率低于正常食管黏膜(P<0.05),食管鳞癌组织中PTEN蛋白阳性率低于原位癌和食管上皮非典型增生组织(P<0.05)。高分化、中分化及低分化食管鳞癌组织中在患者中PTEN蛋白阳性表达率分别为75.00%(15/20)、21.43%(3/14)、20.00%(2/10),高分化食管鳞癌组织中PTEN蛋白阳性表达率显著高于中分化和低分化食管鳞癌组织中(P<0.05),中分化和低分化食管鳞癌组织中PTEN蛋白阳性表达率无明显差异(P>0.05)。结论PTEN表达降低可能与食管鳞状上皮癌变有关,并可能在食管癌早期形成与发展中起有重要的作用。

miR-29a、PTEN、p53在育龄子宫内膜癌组织中的表达及与其术后复发转移的关系分析

目的探讨育龄子宫内膜癌(EC)患者癌组织中微小RNA-29a(miR-29a)、张力蛋白同源物(PTEN)、肿瘤抑制基因(p53)表达与术后复发转移的关系。方法回顾性分析因EC进行手术治疗42例病理组织标本作为观察组,选取21例正常子宫内膜组织作为对照组。根据术后3年复发转移情况将观察组分为复发转移组19例和未复发转移组23例。荧光定量PCR检测EC癌组织和正常子宫内膜组织中miR-29a相对表达量;免疫组织化学技术检测EC癌组织和正常子宫内膜组织中PTEN、p53阳性表达率。结果观察组miR-29a相对表达量、PTEN阳性表达率低于对照组,p53阳性表达率高于对照组(P<0.05);育龄EC患者术后复发转移与肿瘤直径、浸润深度、远处转移、淋巴结转移和淋巴脉管转移有关(P<0.05);复发转移组miR-29a相对表达量、PTEN阳性表达率低于未复发转移组,p53阳性表达率高于未复发转移组(P<0.05);远处转移、miR-29a<1.92、PTEN阳性和P53阳性均为影响育龄EC患者术后复发转移的独立危险因素(P<0.05)。结论miR-29a、PTEN、p53在育龄EC患者癌组织中呈现表达异常,其异常表达是患者术后复发转移的独立危险因素。

联合PTEN、Gleason评分与PSA在预测前列腺癌进展中的价值

目的研究第10号染色体缺失的磷酸酶和张力蛋白同源物基因(PTEN)、Gleason评分、前列腺特异性抗原(PSA)在预测前列腺癌进展中的价值。方法应用S-P法测定29例前列腺癌(Pca)与20例前列腺增生(BPH)组织切片中PTEN蛋白的表达,回顾性研究上述Pca、BPH患者Gleason评分及PSA资料。结果BPH与Pca两组中PTEN总体表达有差异性(P<0.05),两组PSA值总体差异性显著(P<0.05)。PTEN与临床资料关系中,术前PSA<4ng/ml与>10ng/ml两组间PTEN表达有差异(P<0.05),余无差异性。Gleason评分2～4分与5～7分两组PTEN表达无差异(P>0.05),两组与8～10分组PTEN表达比较差异均有显著性(P<0.01);高分化与低分化组PTEN表达有显著差异(P<0.01);临床A、B期与C、D期PTEN表达差异性显著(P<0.01)。结论联合PTEN、Gleason评分及PSA对诊断并预测Pca进展有一定的临床意义。

青蒿琥酯通过调节miR-21/PTEN/AKT轴对甲状腺癌细胞增殖、迁移及凋亡的影响

目的 观察青蒿琥酯对甲状腺癌细胞增殖、迁移及凋亡的影响及微小RNA(miRNA)-21在该过程中的作用,并探讨相关机制。方法 取TPC-1细胞,将miR-21过表达质粒miR-21 mimics转染至TPC-1细胞,随机分为mimics组、联合组,mimics组常规培养,联合组加入青蒿琥酯(320μg/mL)。另取TPC-1细胞分为空白组、青蒿琥酯组,空白组常规培养,青蒿琥酯组加入青蒿琥酯(320μg/mL)。实时荧光定量聚合酶链反应(Real-Time quantitative polymerase chain reaction,qRT-PCR)法检测细胞miR-21表达量;MTT法检测细胞增殖能力;流式细胞术检测细胞凋亡率;划痕实验检测细胞迁移能力;双荧光素酶实验验证miR-21与第十染色体同源丢失性磷酸酶和张力蛋白基因(PTEN)的靶向关系;免疫印迹法检测细胞PTEN、蛋白激酶B(AKT)、p-AKT蛋白表达。结果 与空白组比较,青蒿琥酯组miR-21表达量,24、48、72 h吸光度值,迁移率,p-AKT/AKT降低,凋亡率、PTEN蛋白表达量升高(P<0.05),mimics组miR-21表达量,24、48、72 h吸光度值,迁移率,p-AKT/AKT升高,凋亡率、PTEN蛋白表达量降低(P<0.05);与青蒿琥酯组比较,联合组miR-21表达量,24、48、72 h吸光度值,迁移率,p-AKT/AKT升高,凋亡率、PTEN蛋白表达量降低(P<0.05);与mimics组比较,联合组miR-21表达量,24、48、72 h吸光度值,迁移率,p-AKT/AKT降低,凋亡率、PTEN蛋白表达量升高(P<0.05);双荧光素酶结果显示,PTEN是miR-21的靶基因。结论 青蒿琥酯可抑制甲状腺癌细胞增殖、迁移,并促进其凋亡,其作用机制可能与下调miR-21进一步靶向调控下游PTEN/AKT轴表达有关。

MiR-200a and miR-200b target PTEN to regulate the endometrial cancer cell growth in vitro

Objective:To study whether miR-200a and miR-200b target PTEN gene expression to regulate the endometrial cancer cell growth in vitro. Methods:Endometrial cancer cells ECC-1 were cultured and transfected with the miR-200a and miR-200b mimics and inhibitors as well as the negative control mimics and inhibitors,and then the cell proliferation activity as well as the expression of PTEN and downstream genes in cells was determined; after transfection of miR-200a and miR-200b mimics as well as PTEN-3'UTR luciferase report gene plasmids,the fluorescence activity of luciferase reporter gene was determined. Results:12 h,24 h and 48 h after transfection,the cell proliferation activity of miR-200a mimics group and miR-200b mimics group were significantly higher than those of NC mimics group while the cell proliferation activity of mi R-200 a inhibitor group and miR-200b inhibitor group were significantly lower than those of NC inhibitor group; 48 h after transfection,PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200 a mimics group and miR-200b mimics group were significantly lower than those of NC mimics group while p-PI3K and p-Akt expression were significantly higher than those of NC mimics group; PTEN expression in cells and PTEN-3'UTR luciferase reporter gene fluorescence activity of miR-200 inhibitor group and miR-200b inhibitor group were significantly higher than those of NC inhibitor group while p-PI3K and p-Akt expression were significantly lower than those of NC inhibitor group. Conclusion:miR-200 a and miR-200b can promote the endometrial cancer cell growth in vitro by targeted inhibition of PTEN gene expression.

Anti-miRNA-221 sensitizes human colorectal carcinoma cells to radiation by upregulating PTEN

AIM:To investigate the regulative effect of miRNA(miR)-221 on colorectal carcinoma(CRC)cell radiosensitivity and the underlying mechanisms.METHODS:A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays(0,2,4,6 and 8 Gy).The total RNA and protein of the cells were extracted 24 h after irradiation,and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10(PTEN)gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction(PCR).The protein alteration of PTEN in the cells was detected by Western blotting.Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of premiR-221 or anti-miR-221 using Lipofectamine 2000.Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation.Ad-ditionally,PTEN 3′-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid.The luciferase reporter plasmid construct was then transfected into CRC cells together with premiR-221 or anti-miR-221,and the luciferase activity in the transfected cells was detected.RESULTS:The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner.The miR-221 expression level improved gradually with the increase in irradiation dose,while the PTEN protein expression level reduced gradually.miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups(P<0.01).Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells(P<0.01).Moreover,the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA,suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN.A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221(P<0.01).CONCLUSION:Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.

Increased susceptibility of aging gastric mucosa to injury:The mechanisms and clinical implications

This review updates the current views on aging gastric mucosa and the mechanisms of its increased susceptibility to injury.Experimental and clinical studies indicate that gastric mucosa of aging individuals-"aging gastropathy"-has prominent structural and functional abnormalities vs young gastric mucosa.Some of these abnormalities include a partial atrophy of gastric glands,impaired mucosal defense(reduced bicarbonate and prostaglandin generation,decreased sensory innervation),increased susceptibility to injury by a variety of damaging agents such as ethanol,aspirin and other non-steroidal anti-inflammatory drugs(NSAIDs),impaired healing of injury and reduced therapeutic efficacy of ulcer-healing drugs.Detailed analysis of the above changes indicates that the following events occur in aging gastric mucosa:reduced mucosal blood flow and impaired oxygen delivery cause hypoxia,which leads to activation of the early growth response-1(egr-1)transcription factor.Activation of egr-1,in turn,upregulates the dual specificity phosphatase,phosphatase and tensin homologue deleted on chromosome ten(PTEN)resulting in activation of pro-apoptotic caspase-3 and caspase-9 and reduced expression of the anti-apoptosis protein,survivin.The imbalance between pro-and anti-apoptosis mediators results in increased apoptosis and increased susceptibility to injury.This paradigm has human relevance since increased expression of PTEN and reduced expression of survivin were demonstrated in gastric mucosa of aging individuals.Other potential mechanisms operating in aging gastric mucosa include reduced telomerase activity,increase in replicative cellular senescence,and reduced expression of vascular endothelial growth factor and importin-α-a nuclear transport protein essential for transport of transcription factors to nucleus.Aging gastropathy is an important and clinically relevant issue because of:(1)an aging world population due to prolonged life span;(2)older patients have much greater risk of gastroduodenal ulcers and gastrointestinal complications(e.g.,NSAIDs-induced gastric injury)than younger patients;and(3)increased susceptibility of aging gastric mucosa to injury can be potentially reduced or reversed pharmacologically.

Curcumin cytotoxicity is enhanced by PTEN disruption in colorectal cancer cells

AIM:To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10(PTEN) deficiency on the cytotoxicity of chemotherapeutic agents toward colorectal cancer cells.METHODS:PTEN-deficient colorectal cancer(CRC) cells were generated by human somatic cell gene targeting using the adeno-associated virus system. The cytotoxic effects of compounds including curcumin,5-fluorouracil(5-FU),dihydroartemisinin(DHA),irinotecan(CPT-11)and oxaliplatin(OXA) on cancer cells were determined using the MTT assay. Enhanced cytotoxicity of curcumin in PTEN-deficient CRC cells was observed,and this was confirmed using clonogenic assays. Apoptosis and cell cycle progression were analyzed by flow cytometry.Levels of apoptosis and cell cycle-related proteins were examined by Western blotting.RESULTS:We developed an isogenic set of CRC cell lines that differed only in their PTEN status. Using this set of cell lines,we found that disruption of the PTEN gene had no effect on the sensitivity of CRC cells to5-FU,CPT-11,DHA,or OXA,whereas PTEN disruption increased the sensitivity of CRC cells to curcumin. Loss of PTEN did not alter the curcumin-induced apoptosis in CRC cells. However,PTEN deficiency led to an altered pattern of curcumin-mediated cell cycle arrest.In HCT116 PTEN+/+cells,curcumin caused a G2/M phase arrest,whereas it caused a G0/G1 phase arrest in HCT116 PTEN-/-cells. Levels of cell cycle-related proteins were consistent with these respective patterns of cell cycle arrest.CONCLUSION:Curcumin shows enhanced cytotoxicity toward PTEN-deficient cancer cells,suggesting that it might be a potential chemotherapeutic agent for cancers harboring PTEN mutations.