双启动子shRNA表达载体对hTERT和bcl-2基因表达的抑制作用

目的:研究双启动子shRNA表达载体对人端粒酶蛋白催化亚单位(hTERT)和B细胞淋巴瘤/白血病-2(bcl-2)基因表达的抑制作用。方法:分别从构建好的大肠杆菌DH5α菌体中提取含有双启动子shRNA的表达载体pdPRO-TB、单启动子对照pdPRO-T、pdPRO-B和阴性对照质粒pdPRO,通过脂质体介导瞬时转染Hela细胞,倒置显微镜下观察细胞形态。实时荧光定量RCR检测hTERT和bcl-2基因的表达水平。结果:4种菌体中均提取出5 000 bp左右的质粒,分别转染Hela细胞48 h后,可见转染pdPRO的Hela细胞形态无明显变化,转染pdPRO-T和pdPRO-B的Hela细胞有部分悬浮,转染pdPRO-TB的Hela细胞有大量悬浮。各载体质粒构建均成功,各目的基因相对管家基因的含量:T(hTERT)=39.8%,B(hTERT)=107.9%,TB(hTERT)=32.1%;T(bcl-2)=91.4%,B(bcl-2)=35.4%,TB(bcl-2)=31.4%。结论:双启动子shRNA表达载体构建成功,可同时抑制hTERT和bcl-2基因的表达。

Naa10基因与肿瘤相关性的研究进展

N-末端α位乙酰基转移酶基因10(Naa10)广泛存在于真核生物细胞中,其编码的Naa10p是NatA复合物的催化亚基。Naa10p修饰新生蛋白质N末α位氨基酸残基乙酰化,进而通过介导mTOR、Ca^(2+)/MLCK、DNMT1、PGK1、PIX-蛋白等信号通路调控蛋白降解、细胞凋亡、增殖、迁移、浸润、血管生成等生物学过程。根据乙酰化底物的不同,Naa10p还在细胞凋亡的调控中起双重作用。研究发现Naa10p在肝癌、结直肠癌、乳腺癌、肺癌、前列腺癌等多种癌组织中过表达,而在癌旁组织中不表达或低表达,因肿瘤差异其预后意义不同。因此,Naa10可作为部分肿瘤诊断和预后的新型潜在生物标志物及治疗靶点。

Genetic alterations in pancreatic cancer

The diagnosis of pancreatic patients and their relatives cancer is devastating for as the incidence rate is approximately the same as mortality rate. Only a small percentage, which ranges from 0.4% to 4% of patients who have been given this diagnosis, will be alive at five years. At the time of diagnosis, 80% of pancreatic cancer patients have unresectable or metastatic disease. Moreover, the therapeutic alternatives offered by chemotherapy or radiotherapy are few, if not zero. For all these reasons, there is an imperative need of analyzing and understanding the primitive lesions that lead to invasive pancreatic adenocarcinoma. Molecular pathology of these lesions is the key of our understanding of the mechanisms underlying the development of this cancer and will probably help us in earlier diagnosis and better therapeutic results. This review focuses on medical research on pancreatic cancer models and the underlying genetic alterations.

Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

醋酸阿比特龙联合多西他赛与泼尼松在转移性去势抵抗性前列腺癌患者中的应用及Naa10与治疗敏感性的关系

目的 探讨醋酸阿比特龙联合多西他赛与泼尼松在转移性去势抵抗性前列腺癌(mCRPC)患者中的应用及N-末端α位乙酰基转移酶基因10(acetyltransferase gene 10,Naa10)与治疗敏感性的关系。方法 回顾性选择2017年10月—2019年12月邢台医学高等专科学校第二附属医院收治的122例mCRPC患者为研究对象,入选患者均经过雄激素剥夺治疗。根据治疗方案不同将患者分为对照组和试验组,对照组患者静脉滴注多西他赛注射液,剂量75 mg·m-2,每3周1次;口服醋酸泼尼松片,1次1片,每天2次;在注射多西他赛注射液化疗前1天、当天和后1天均口服醋酸地塞米松片,1日2次,总剂量7.5 mg。试验组患者在对照组的基础上空腹口服醋酸阿比特龙片,每次1 g,每日1次,治疗4个周期(12周)。比较两组患者近期临床疗效、不良反应及远期生存率等情况,随访3年,计算患者的中位生存期。免疫组化法检测mCRPC癌组织中Naa10蛋白表达,将患者分成Naa10阳性表达组和阴性表达组,分析其临床病理参数,单因素和Cox回归分析确定影响mCRPC预后的因素,分析Naa10蛋白阳性表达与治疗敏感性的关系。结果 治疗4个周期后,试验组患者9例完全缓解、40例部分缓解、8例疾病稳定及4例疾病进展,治疗总有效率为80.33%;对照组患者2例完全缓解、34例部分缓解、20例疾病稳定及5例疾病进展,治疗总有效率为59.02%,两组间比较差异显著(χ^(2)=6.556,P=0.011)。试验组和对照组患者均出现骨髓抑制(χ^(2)=0.209,P=0.648)、肝功能损伤(χ^(2)=0.830,P=0.362)、胃肠道反应(χ^(2)=0.370,P=0.543)、水钠潴留(χ^(2)=0.209,P=0.648)及心脏毒性(χ^(2)=0.899,P=0.343)等3级以上不良反应,但两组间差异未见显著性。mCRPC癌组织中Naa10蛋白阳性表达与治疗周期(χ^(2)=9.106,P=0.003)、淋巴结转移(χ^(2)=5.055,P=0.025)、T分期(χ^(2)=4.391,P=0.036)、骨转移病灶数(χ^(2)=19.863,P=0.000)、内脏转移(χ^(2)=5.009,P=0.025)等具有显著相关性,但与年龄(χ^(2)=3.059,P=0.080)、化疗方案(χ^(2)=0.880,P=0.348)、EOCG评分(χ^(2)=3.453,P=0.178)、民族(χ^(2)=0.135,P=0.713)及Gleason评分(χ^(2)=0.837,P=0.360)等没有显著相关性。单因素分析结果显示,mCRPC患者中位生存期与EOCG评分(χ^(2)=7.464,P=0.006)、淋巴结转移(χ^(2)=6.114,P=0.013)、化疗方案(χ^(2)=7.049,P=0.030)、治疗周期(χ^(2)=5.051,P=0.038)、T分期(χ^(2)=1.196,P=0.035)、骨转移病灶数(χ^(2)=9.611,P=0.008)、内脏转移(χ^(2)=1.085,P=0.048)及Naa10蛋白阳性表达(χ^(2)=15.600,P=0.000)等指标具有显著相关性。Cox回归分析结果显示,骨转移病灶数(>10个)(OR=2.404,95%CI:1.424~4.056)、治疗周期(>8个疗程)(OR=0.458,95%CI:0.253~0.832)、化疗方案(试验组)(OR=0.360,95%CI:0.160~0.801)和Naa10蛋白阳性表达(OR=2.563,95%CI:2.106~3.117)是mCRPC患者预后生存的独立影响因素(P<0.05)。结论 醋酸阿比特龙联合多西他赛与泼尼松应用于mCRPC治疗,可有效提高近期临床疗效,延长生存期,且不增加不良反应,同时Naa10蛋白高表达是mCRPC患者预后不良的危险因素,临床宜监测指标,及时调整和评估临床治疗效果。