Rad23在恶性肿瘤中的研究进展

1 Rad23家族蛋白简介辐射敏感蛋白23(radiation sensitivity proteins-23,Rad23)蛋白是一类多功能和多结构域的蛋白质,也可叫做HR23和Hhr23,最初在酵母体内被发现,在进化上比较保守[1]。Rad23蛋白具有4个结构域:2个结合泛素或泛素化蛋白的泛素相关结构域(ubiquitin-associated,UBA),1个与蛋白酶受体结合的泛素化样区域(ubiquitin-like,UBL)以及1个XPC/Rad4结合域[1]。Rad23蛋白在包括核苷酸切除修复、泛素化相关蛋白质降解等多种细胞过程中发挥重要作用[1]。

RAD23B基因rs10759225位点多态性与含铂类方案治疗非小细胞肺癌疗效和血液学毒性的关系

目的探讨RAD23B基因rs10759225位点多态性与含铂类方案一线治疗中晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的疗效及血液学毒性的关系。方法回顾性收集接受含铂类联合方案一线治疗的150例NSCLC患者的临床资料及外周血样本,采用改良多重高温连接酶检测反应技术对rs10759225进行基因分型,并分析其与患者疗效及血液学毒性的关系。结果 150例NSCLC患者的总有效率(ORR)为28.7%(43/150)。与携带G/G基因型患者比较,携带A等位基因型(G/A和A/A)患者接受含铂类方案治疗的ORR显著提高(OR校正=1.780,95%CI:1.110~2.884,P=0.042)。全组患者中位无进展生存期(mPFS)为5.6个月,各基因型患者的mPFS差异均无统计学意义(P>0.05)。以G/G基因型为参照,A/A基因型与患者化疗后发生Ⅲ/Ⅳ度白细胞减少的风险相关(OR校正=0.468,95%CI:0.204~0.711,P=0.008);A等位基因型分别与患者发生Ⅲ/Ⅳ度中性粒细胞减少(OR校正=0.502,95%CI:0.155~0.887,P=0.022)和≥Ⅰ度血小板减少的风险有关(OR校正=0.494,95%CI:0.101~0.833,P=0.047);而G/A基因型与患者发生Ⅱ/Ⅲ度贫血的风险有关(OR校正=0.504,95%CI:0.213~0.890,P=0.047)。结论 RAD23B基因rs10759225位点多态性与含铂类方案治疗NSCLC的疗效及血液学毒性有关,但与患者的预后无关。与携带G/G基因型患者比较,携带A等位基因型患者的疗效更好,但发生骨髓抑制的风险更高。

Genome-wide identification of the radiation sensitivity protein-23(RAD23)family members in apple(Malus×domestica Borkh.)and expression analysis of their stress responsiveness

Radiation sensitivity proteins-23 （RAD23） are DNA repair factors participate in the ubiquitin/proteasome system （UPS）. Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple （Malusxdomestica Borkh.）. We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses. Various members showed a ubiqui- tous pattern of expression in all selected apple parts. Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid （ABA） treatment and water deficiency, suggesting their possible roles in plant stress responses. These results provide essential information about RAD23 genes in apple and will contribute to further functional studies.

Protective effects of Rad23 protein on ultraviolet damage to HeLa cells

Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.

利用CRISPR/Cas9系统快速构建白念珠菌RAD23高表达菌株的研究

目的构建白念珠菌RAD23高表达菌株,探索其高表达对细胞耐受遗传毒性试剂胁迫的影响。方法采用瞬时CRISPR/Cas9系统,分别于体外扩增Cas9基因、sgRNA以及含MET3启动子的修复DNA,通过醋酸锂法转化白念珠菌野生型菌株,于SD-Ura-Met-Cys培养基筛选转化子,通过菌落PCR验证基因型,通过Western blot检测Rad23相对表达量,利用平板表型试验检测RAD23高表达对细胞耐受遗传毒性试剂的影响。结果成功构建了RAD23高表达菌株;与野生型菌株相比,高表达菌株中RAD23蛋白相对表达量约为野生型菌株的7倍(10.20 VS 1.46);在UV胁迫条件下,RAD23高表达菌株存活率显著下降(与野生型菌株相比,下降约1 000倍)。结论利用CRISPR/Cas9系统快速构建了白念珠菌RAD23高表达菌株,且RAD23高表达会抑制细胞对遗传毒性试剂UV的耐受能力。

日本血吸虫SjRAD23基因的克隆表达及基因重组抗原的免疫保护效果

辐射敏感蛋白23具有核苷酸切除修复功能,在泛素蛋白酶体途径中起到重要作用。本研究利用PCR技术克隆了日本血吸虫辐射敏感蛋白23(Sj RAD23)编码的c DNA序列,成功获得Sj RAD23的基因序列,其ORF为1 053 bp。构建Sj RAD23基因重组表达质粒p ET28a(+)-Sj RAD23,并在大肠杆菌BL21中成功诱导表达,重组蛋白在上清和沉淀中都有存在。利用免疫组化技术检测该蛋白在虫体的分布情况,该蛋白广泛分布在日本血吸虫虫体被膜。用重组蛋白免疫BALB/c小鼠后,免疫小鼠血清中检测到较高水平的特异性Ig G、Ig G1和Ig G2a。Western blotting分析显示重组蛋白能够被日本血吸虫成虫可溶性抗原免疫小鼠血清所识别。用重组蛋白r Sj RAD23免疫结果与206佐剂对照组比较,r Sj RAD23在BALB/c小鼠中诱导了35.94%减虫率,40.59%肝脏减卵率。结果表明Sj RAD23具有作为疫苗候选分子的潜力。