CAJ | 학술논문

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suppresser of fused (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SW1990细胞,应用蛋白质印迹法检测SW1990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600 bp,经测序并将其序列与NCBI网站Blast序列对比分析发现,在SUFU mRNA isoforml(NM_016169.3)的第10外显子和第1 1外显子之间插入了一个长为126 bp的新外显子.通过对SW1990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SW1990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.
목적 분석인이선암조직화세포중Hedgehog신호통로주요성원SUFU단백적전접체.방법 용반전록화3 'cDNA말단쾌속확증(3'RACE)법확증suppresser of fused (SUFU)편단,측서발현일개신적외현자,응용RT-PCR방법확증전장함신외현자적SUFU(nSUFU).채용지질체법장nSUFU급SUFU전염SW1990세포,응용단백질인적법검측SW1990세포급이선암조직신전접체편마적단백질적표체.결과 통과3'RACE법적제이륜소식PCR확증산물약600 bp,경측서병장기서렬여NCBI망참Blast서렬대비분석발현,재SUFU mRNA isoforml(NM_016169.3)적제10외현자화제1 1외현자지간삽입료일개장위126 bp적신외현자.통과대SW1990세포적험증,포함신외현자적완정적신전접체편단장약1400bp.전염nSUFU적SW1990세포강표체nSUFU단백,이선암조직기표체SUFU단백,우표체nSUFU단백.결론 인류이선암조직화세포중존재일충신적가이편마SUFU단백적전접체.
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 transfection 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.