Stat3信号转导通路调控Survivin表达促进结肠癌细胞凋亡

目的 探讨Stat3/Survivn信号转导通路调控结肠癌细胞凋亡的作用机制.方法 用阳离子脂质体介导Stat3反义寡核苷酸(20 mol/L)转染人结肠癌HT29细胞,噻唑蓝(MTY)法检测细胞增殖状态;流式细胞术检测细胞周期与凋亡;Western blot检测Stat3、p-Stat3、Survivin与bcl-2凋亡家族成员bcl-2、bcl-Xl于bax的表达.结果 Stat3反义寡核苷酸作用HT29细胞72 h后,G1期细胞比率由61.4%上升至75.6%,S期细胞比率由19.4%下降至8.6%,细胞增殖受抑制.凋亡细胞百分比由5.6%增加至22.1%,促进细胞凋亡.Stat3、p-Stag、Cychn D1与Survivin表达下降,bcl-2、bcl-Xl与bax变化不明显.结论 阻断Stat3通路可以抑制靶基因Survivin表达并诱导结肠癌细胞凋亡.     

表没食子儿茶素没食子酸酯下调胃癌细胞血管内皮生长因子表达的分子机制研究

目的探讨表没食子儿茶素没食子酸酯（EGCG）通过抑制转录活化因子（Stat3）活性下调胃癌细胞血管内皮生长因子（VEGF）表达的分子机制。方法采用Westernblot法检测AGS胃癌细胞分别在50μg／L IL-6加0、5、10、25、50μmol／LEGCG作用下,VEGF、总Stat3（tStat3）和活化Stat3（pStat3）的表达情况：检测Stat3信号通路抑制剂对IL-6诱导的VEGF蛋白表达水平的影响。酶联免疫吸附法检测肿瘤细胞培养液中VEGF蛋白水平。RT-PCR法检测胃癌细胞VEGFmRNA表达水平。制备细胞核提取液,WestemMot法检测肿瘤细胞核内pStat3表达;同时采用染色体免疫沉淀方法检测Stat3与DNA的结合活性。结果IL-6可以诱导AGS胃癌细胞VEGF表达．与未添加组比较,添加IL．6组胃癌细胞VEGF蛋白表达、分泌和mRNA表达分别增加了2．4、2．8和3．1倍（均P〈O．01）：EGCG剂量依赖性地抑制IL-6诱导的胃癌细胞VEGF蛋白表达、分泌和VEGFmRNA表达（P〈0．05）。EGCG和Stat3信号通路抑制剂AG490可以显著抑制IL-6诱导的VEGF表达（P〈0．01）：EGCG处理的胃癌细胞中IL-6诱导的pStat3表达剂量依赖性地减少（P〈O．05）,但EGCG不影响IL-6诱导的tStat3表达（P〉0．05）;同时,EGCG可以直接抑制胃癌细胞自身和IL-6诱导的Stat3核转位和Stat3-DNA结合活性（P〈0．05）。结论EGCG通过抑制Stat3活性．从而下调胃癌细胞VEGF表达。     

Cyclin E1 is a prognostic biomarker and potential therapeutic target in osteosarcoma

While amplified expressed cyclin E1 is a well-known tumorigenic factor and prognostic biomarker in several malignancies, its prognostic predictive potential and function in osteosarcoma is poorly understood. Here we reveal discrete expression pattern, correlation to clinicopathological characteristics and prognosis and overall function of cyclin E1 in osteosarcoma. Sixty-nine osteosarcoma patient tumor specimens were enrolled to construct a tissue microarray to evaluate cyclin E1 expression through immunohistochemical staining. Cyclin E1 expression in osteosarcoma cell lines and fresh tissues was assessed by Western blot. Cyclin E1 gene expression was evaluated using RNA sequencing data acquired from the public database. We correlated staining intensity to clinical characteristics. Cyclin E1 small interfering RNA was used to determine the effect of cyclin E1 silencing on osteosarcoma cell proliferation and chemotherapeutic sensitivity. Sixty-one percent of the osteosarcoma patient specimens in the tissue microarray had high cyclin E1 expression. Cyclin E1 gene was significantly highly expressed in osteosarcoma tissues and cell lines compared to normal tissues. The expression of cyclin E1 positively correlated with disease status, and inversely correlated to prognosis and response to neoadjuvant chemotherapy. The expression of cyclin E1 was an independent prognostic factor for osteosarcoma patients. In addition, silencing cyclin E1 expression in osteosarcoma cells significantly inhibited cell proliferation and increased sensitivity to chemotherapeutics. We conclude that cyclin E1 is overexpressed in osteosarcoma and is a promising biomarker for prognosis and chemotherapeutic response. We confirm aberrant cyclin E1 expression is a potent therapeutic target in osteosarcoma, and its selective inhibition is a rational treatment strategy for osteosarcoma.     

Stat3反义寡核苷酸联合化疗调控结肠癌细胞增殖与凋亡的分子机制

目的　探讨Stat3反义寡核苷酸 (Stat3AS ON)联合 5 氟尿嘧啶 (5 FU )治疗结肠癌的作用机制。方法　应用Stat3AS ON与 5 FU处理结肠癌细胞HCT116,Westernblot检测Stat3、p Stat3、CyclinD1与bcl xL表达 ,MTT法检测细胞增殖状态 ,流式细胞技术检测细胞周期与凋亡。结果　Stat3AS ON与 5 FU作用于HCT116细胞 72h后 ,G1期细胞比率由 63 .7%上升至 82 .2 % ,S期细胞比率分别由 17.6% ,下降至 9.9% ,凋亡细胞百分比由 6.1%增加至 3 2 .0 %。Stat3AS ON与 5 FU可以抑制结肠癌细胞增殖 ,促进结肠癌细胞凋亡 ,联合应用Stat3AS ON与 5 FU可以起协同作用 ,明显抑制结肠癌细胞Stat3信号转导通路活化。结论　选择性阻断细胞内信号转导通路可能为治疗结肠癌提供新途径     

Clinical and biological significance of PIM1 kinase in osteosarcoma

Osteosarcoma is the most prevalent histological form of primary malignant bone tumor. The majority of osteosarcoma patients have limited alternative therapeutic options and metastatic patients generally have a poor prognosis. Proto‐oncogene serine/threonine‐protein kinase PIM1 is associated with growth and survival of many kinds of tumor cells. However, the role of PIM1 in osteosarcoma remains largely unknown. In this study, we investigated the functional and therapeutic relevance of PIM1 as a putative target in osteosarcoma. We found PIM1 was highly expressed in various osteosarcoma cell lines and in tumor tissues from osteosarcoma patients. Tissue microarray and immunohistochemistry analysis showed that the overall and disease‐free survival rate of patients with high levels of PIM1 protein expression were significantly shorter than patients with low levels. High levels of PIM1 were also associated with present metastasis and can be considered as an independent prognostic factor in osteosarcoma patients. Knockdown of PIM1 expression by synthetic siRNA or shRNA greatly inhibited cell growth, migration, and invasion. Moreover, these changes accompanied with down‐regulation of anti‐apoptotic protein Bcl‐2. The similar results were obtained in osteosarcoma cells treated with PIM1 specific inhibitor (SMI‐4a). These results suggest that PIM1 kinase is critical for the growth and metastasis of osteosarcoma cells and can be a potential therapeutic target for osteosarcoma treatment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1185–1194, 2016.     

ROCK1 as a potential therapeutic target in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. Patients with localized disease are routinely treated with surgery and chemotherapy. Unfortunately, many of these patients eventually relapse even after high‐dose pre‐ and postoperative chemotherapy. Upon recurrence of the tumor locally or distantly, they have limited treatment options that are usually unsuccessful. Our prior studies screening lentiviral shRNA libraries, searching for kinases involved in osteosarcoma cell growth and proliferation have identified the Rho‐associated coiled‐coil containing protein kinase 1 (ROCK1) as a possible hit. We show in this study that ROCK1 is highly expressed in various tumor cell lines and tumor tissues from osteosarcoma patients. ROCK1 knockdown by synthetic siRNA decreases cell proliferation, viability and induces apoptosis in osteosarcoma cell lines KHOS and U‐2OS. Finally, we established the relationship between expression levels of ROCK1 and clinical prognosis in osteosarcoma patients by using immunohistochemistry. There were significant differences in overall survival between cohorts of patients with ROCK1 levels categorized as high‐staining, moderate‐staining, and low‐staining. High levels of ROCK1 were associated with poor outcomes in clinical osteosarcoma. These findings suggest that knockdown of ROCK1 inhibits proliferation and induces apoptosis in osteosarcoma cell lines. ROCK1 may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1259–1266, 2011     

Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway

BACKGROUND: Elevated expression of interleukin-6 (IL-6) is implicated in the progression of hormone refractory prostate cancer. Previous studies demonstrated that IL-6 promotes androgen-independent growth of prostate cancer cells. In this study, the effect of IL-6 on apoptosis induced by androgen deprivation was investigated. METHODS: The effect of IL-6 on apoptosis induced by androgen deprivation in LNCaP cells was examined by cell death ELISA and Western blot using cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9, as well as Bcl-xL and phosphorylated Bad. The Stat3 in IL-6-mediated anti-apoptosis in prostate cancer cells was examined using either dominant-negative or constitutively activated Stat3 mutants. RESULTS: Overexpression of IL-6 renders androgen sensitive LNCaP human prostate cancer cells more resistant to apoptosis induced by androgen deprivation. LNCaP cells undergo apoptosis after 72 hr of androgen deprivation, an outcome is largely absent in clones overexpressing IL-6 as measured by cell death ELISA and chromatin degradation assays. IL-6 over-expressing cells resulted in a significant decrease in the expression of cleaved PARP and cleaved caspase-9 as well as an increase in the expression of Bcl-xL and phosphorylated Bad. Addition of IL-6 antibody completely abolished the anti-apoptotic activity of IL-6. This protective effect of IL-6 was reversed by the expression of a dominant-negative Stat3 mutant, Stat3F. Furthermore, ectopic expression of a constitutively active Stat3 antagonized androgen deprivation-induced cell death of LNCaP cells. CONCLUSION: These results indicate that IL-6 protects androgen sensitive LNCaP cells from apoptosis induced by androgen deprivation, and Stat3 activation play an important role in IL-6-mediated anti-apoptosis in prostate cancer cells.     

Differential expression of microRNA (miRNA) in chordoma reveals a role for miRNA‐1 in Met expression

Emerging evidence suggests that microRNA (miRNA) expression signatures in cancer may have important diagnostic, prognostic, and therapeutic value, but there is no data on miRNA expression in chordoma. The purpose of this study was to identify the role of miRNAs in human chordoma. We analyzed miRNA expression in chordoma‐derived cell lines and chordoma tissue by using miRNA microarray technology with unsupervised hierarchical clustering analysis. The relative expression levels of these miRNAs were confirmed by real‐time quantitative RT‐PCR and Northern blot analysis. To characterize the potential role of miRNA‐1, miRNA‐1 was stably transfected into a chordoma cell line, UCH1. The expression of miRNA‐1 targeted gene Met in chordoma tissues was also studied. We observe that human chordoma tissues and cell lines can be distinguished from normal muscle tissue by comparing miRNA expression profiles. Several miRNAs were differentially expressed in chordoma cell lines compared to controls, and similar expression patterns were found in primary chordoma tissues. Importantly, we were able to show for the first time, to our knowledge, that expression of miRNA‐1 and miRNA‐206, two miRNAs implicated in a number of other cancer types, were markedly decreased in both chordoma tissues and cell lines. When chordoma cell lines were transfected with miRNA‐1, downregulation of known miRNA‐1 targets was observed. These targets included Met and HDAC4—two genes that were observed to be overexpressed in chordoma. Our results demonstrate that some miRNAs are differentially expressed in chordoma and, in particular, miRNA‐1 may have a functional effect on chordoma tumor pathogenesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:746–752, 2010     

Expression and Clinical Significance of High‐Mobility Group AT‐hook 2 (HMGA2) in Osteosarcoma

ObjectiveAlthough high‐mobility group AT‐hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma.MethodsSixty‐nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow‐up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos‐2, MNNG/HOS, and KHOS. HMGA2‐specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity.ResultsHMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos‐2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased.ConclusionOur study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.     

癌基因Stat3与结直肠癌临床病理特征的关系

目的探讨转录信号传导子与激活子 3(Stat3)在结直肠癌中的表达情况 ,分析Stat3的表达与结直肠癌临床病理特征的关系。方法应用Westernblot检测 4 5例结直肠癌组织及其邻近正常肠粘膜中Stat3蛋白的表达。结果结直肠癌组织中Stat3蛋白表达水平明显高于正常肠粘膜(P <0 0 5 ) ,平均为正常肠粘膜的 2 4 5倍 ;Stat3表达水平与Dukes分期、分化及淋巴结转移有关 (P <0 0 5 ) ;Stat3表达水平与肿瘤大小、远处转移、年龄、性别以及是否浸润浆膜无关 (P >0 0 5 )。结论Stat3的过量表达可能在结直肠癌的发生、发展过程中起重要作用。     

Targeting Cdk11 in osteosarcoma cells using the CRISPR‐cas9 system

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR‐Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U‐2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR‐Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR‐Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:199–207, 2015.

     

Stat3靶向RNA干扰重组体的构建及鉴定

目的设计针对Stat3的发夹样RNA干扰重组体,通过脂质体转染结肠癌细胞HCT116观察其干扰效果,为探索肿瘤基因治疗的新途径奠定基础。方法设计针对Stat3编码区的有短发夹结构的两条DNA序列,经退火成互补双链,克隆到载体Pavu6 27中构建重组体Pavu6 27-Stat3,再对重组质粒进行酶切鉴定、DNA测序分析。脂质体法转染重组体至大肠癌HCT116细胞株中,以空质粒(Pavu6 27)转染为对照;RT-PCR法观察Stat3基因表达改变。结果酶切鉴定和测序分析均提示Stat3靶向RNA干扰重组体的构建成功,Pavu6 27-Stat3转染后HCT116细胞Stat3基因表达较空载体Pavu6 27转染的对照组显著下降。结论Stat3靶向RNA干扰重组体成功构建,并能有效抑制HCT116细胞中Stat3基因表达。本实验为下一步利用RNAi技术沉默肿瘤细胞中Stat3基因的表达,诱导肿瘤细胞凋亡或抑制其增生,为探索肿瘤基因治疗的新途径打下基础。