探討p54nrb基因在非小細胞肺癌之上游調控區域

Abstract

p54nrb/NonO is a multiple function nuclear protein. It has been implicated in numerous processes within the nucleus including transcriptional regulation, DNA unwinding, nuclear RNA processing, DNA double-strand break repair and tumorigenesis. In vivo, p54nrb usually forms a heterodimer with its partner PSF as a RNA splicing factor, but its roles in tumor progress is unknown. In recent years, p54nrb has been report in bladder cancer and prostate cancer that different expression of p54nrb between normal tissue and tumor tissue. p54nrb is upregulated in bladder cancer and prostate cancer. However, the mechanism about transcriptional regulation of p54nrb is still unknown. In this study, we characterize p54nrb function in non-small cell lung cancer cell (NSCLC) line and the transcriptional regulation of p54nrb promoter. First we find p54nrb transcription start site and use PCR technology to clone -1671/+1261 ( 2952 bp ) p54nrb promoter. Then we analyzed p54nrb promoter region by deletion of p54nrb promoter 5' end and luciferase report assay. The results of luciferase assay demonstrated that there is an important regulation region in p54nrb promoter +311/+414 fragment. And deletion of p54nrb promoter +109/+203 region can cause a part of luciferase activity decreasing. Furthermore, we used programe to predict what kind of transcription factor binding site on these two p54nrb promoter region. And we found three CCAAT box and AP-1, AP2α, Sp1, PEA3, NF-1, YY1 binding site. To investigate which transcription factors involve in transcriptional regulation of p54nrb. We observed the change of p54nrb after overexpressed AP-1, AP2α and Sp1 in CL1-0, CL1-5 and A549 cell line. But , the protein level and mRNA level of p54nrb don't change after overexpressed AP-1, AP2α and Sp1. On the other hand, our previous data showed that enforced p54nrb expression can suppress CL1-5 migration and invasion. In this study, we silenced p54nrb with siRNA and investigated the effect of migration and invasion. The result showed that there is no effect on migration and invasion after p54nrb silence. And so far, we still try to establish p54nrb silence stable clone.

p54nrb(Non-POU domain containing, octamer-bindin)在前人研究即被指出是一個多功能的核蛋白，參與多種細胞調節作用。其中包含轉錄調節、DNA的解旋、RNA修飾機制、雙股DNA修復與腫瘤新生。細胞內的p54nrb會與PSF形成複合體，並參與 pre-mature RNA的剪接作用，但p54nrb在癌症內所扮演的角色目前還不是很清楚。近幾年有研究指出，其中有研究指出在膀胱癌與前列腺癌的腫瘤組織與正常組織的p54nrb蛋白質表現量有明顯差異，腫瘤組織的p54nrb表現量會高於正常組織。然而目前對於p54nrb基因表現調控的機制還不是很清楚。本篇研究主要目的為定義出p54nrb基因在非小細胞肺癌之上游調控轉錄的區域，並分析可能的轉錄因子參與調控。首先我們找出p54nrb的轉錄起始位置，並利用聚合酶連鎖反應合成p54nrb啟動子-1671/+1261區域。接著利用5’缺失構築與冷光報導基因分析，結果發現p54nrb啟動子+311/+414區域為一段相當重要調控轉錄活性的區域，此外p54nrb啟動子+109/+203區域缺失也會造成部分啟動子的活性下降。進一步利用程式分析這兩個p54nrb啟動子片段，發現此區域具有三個CCAAT box與AP-1、AP2α、Sp1、PEA3、NF-1、YY1等轉錄因子結合位。為了進一步了解哪個轉錄因子可能參與到p54nrb基因的轉錄調控，我們將AP-1、AP2α、Sp1轉錄因子大量表現在CL1-0、CL1-5、A549肺癌細胞株，並觀察其對p54nrb基因表現的調控。根據實驗結果發現p54nrb的蛋白質與mRNA的表現量，在大量表現AP-1、AP2α、Sp1細胞中沒有明顯的差異。另外在實驗室先前的研究中發現大量表現p54nrb於CL1-5細胞株，會抑制其移動能力與侵襲能力。在本篇研究中，我們以siRNA將CL1-0、CL1-5細胞中的p54nrb靜默並觀察對細胞移動能力與侵襲能力的影響，結果發現在靜默p54nrb後對CL1-0、CL1-5細胞的移動能力與侵襲能力沒有顯著的影響。目前我們仍致力於挑選穩定靜默p54nrb的細胞株。