摘要
目的分析人胰腺癌组织和细胞中Hedgehog信号通路主要成员SUFU蛋白的剪接体。方法用反转录和3’cDNA末端快速扩增（3’RACE）法扩增suppresseroffused（SUFU）片段，测序发现一个新的外显子，应用RT—PCR方法扩增全长含新外显子的SUFU（nSUFU）。采用脂质体法将nSUFU及SUFU转染SWl990细胞，应用蛋白质印迹法检测SWl990细胞及胰腺癌组织新剪接体编码的蛋白质的表达。结果通过3’RACE法的第二轮巢式PCR扩增产物约600bp，经测序并将其序列与NCBI网站Blast序列对比分析发现，在SUFUmRNAisoforml（NM_016169．3）的第10外显子和第11外显子之间插入了一个长为126bp的新外显子。通过对SWl990细胞的验证，包含新外显子的完整的新剪接体片段长约1400bp。转染nSUFU的SWl990细胞强表达nSUFU蛋白，胰腺癌组织既表达SUFU蛋白，又表达nSUFU蛋白。结论人类胰腺癌组织和细胞中存在一种新的可以编码SUFU蛋白的剪接体。
Objective To investigate spliceosome of suppresser of fused（ SUFU）, a major member of hedgehog signaling pathway in human pancreatic cancer. Methods SUFU fragment was amplified by using reverse transcription and 3＇ RACE. After sequencing, a new exon was discovered, and then nSUFU was amplified by RT-PCR. nSUFU and SUFU were transfected into SW1990 by liposomes, and then the expressions of SUFU protein encoded by new spliceosome in SW1990 cells and pancreatic cancer tissues were detected by Western blot. Results PCR products by 3＇RACE were of 600 bp, after sequencing and comparison with Blast data of NCBI, it was detected that a new exon was inserted between SUFU mRNA isoforml （NM_016169.3） exon 10 and exon 11. After verification with SW1990, it was noted the entire new spliceosome containing new exon was of 1400 bp. SW1990 with nSUFU transfeetion strongly expressed nSUFU protein, and pancreatic cancer tissues expressed both SUFU and nSUFU protein. Conclusions A new spliceosome of SUFU, which can encode SUFU protein, is present in pancreatic cancer tissue and cell.
出处
《中华胰腺病杂志》
CAS
2013年第4期231-234,共4页
Chinese Journal of Pancreatology
基金
国家自然科学基金（30910103911,81272663）
上海市重点科技攻关项目（11441901800）
国家科技支撑计划（2006BA102A12）
作者简介
通信作者：高军，Emai：gaojunaaa@gmail．com，
李兆申，Email：zhsli@81890．net
