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肺癌是世界范围内致死率较高的恶性肿瘤之一,近年来肺癌在我国的发病率和病死率均高居首位,其主要原因在于肺癌的高侵袭转移能力和高复发率[1]。非小细胞肺癌是肺癌中发病率最高的类型,手术治疗和放化疗是最常用和最基本的治疗手段,但预后依然较差,5年生存率不足17%[2]。因此,研究肺癌的代谢及转移机制在一定程度上能提高肺癌的诊疗效果,进而降低肺癌的病死率。
在RNA中,大部分为非编码RNA,其中长链非编码RNA(long non-coding RNA,lncRNA)长度一般超过200 bp,且缺乏明显的开放阅读框,因此其不编码蛋白质,但是广泛参与人体生理、病理活动,包括参与或介导肿瘤的发生、发展过程[3-3]。早期研究结果发现,lncRNA尿路上皮癌胚抗原(urothelial carcinoma associated,UCA)1在多种肿瘤细胞中表达上调,降低UCA1的表达可以抑制肿瘤细胞的增殖、转移等生物学行为,调节肿瘤细胞的葡萄糖代谢[4],因此UCA1可作为肿瘤早期诊断、预后监测的新型生物标志物[5-6]。
本研究首先通过检测lncRNA UCA1在肺腺癌A549细胞中的表达,然后体外实验进一步验证lncRNA UCA1在肺腺癌中的作用,旨在进一步揭示肺癌的发生、发展机制,并为lncRNA UCA1作为肺癌新的早期诊断指标及治疗靶点提供证据。
长链非编码RNA UCA1对肺腺癌A549细胞糖代谢及侵袭转移的影响
The functional role of long non-coding RNA UCA1 in lung adenocarcinoma A549 cells on glucose metabolism, invasion and metastasis
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摘要:
目的 探讨长链非编码RNA(lncRNA)尿路上皮癌胚抗原1(UCA1)在肺腺癌A549细胞中的表达及对糖代谢关键酶和细胞侵袭、转移的影响。 方法 构建lncRNA UCA1沉默及其对照稳定转染的肺腺癌A549细胞系,将小干扰RNA(siRNA)转染细胞分为NC组(阴性对照组,转染siRNA-UCA1序列)和siRNA-UCA1组(转染siRNA-UCA1敲降序列)。采用实时荧光定量PCR、Western blot检测糖代谢相关指标葡萄糖转运蛋白(GLUT)1、己糖激酶(HK)2和丙酮酸激酶(PKM)2水平的变化。采用Transwell实验和划痕实验检测siRNA-UCA1的侵袭和迁移能力。采用18F-氟脱氧葡萄糖(FDG)摄取实验检测siRNA-UCA1的摄取率。计量资料的比较采用t检验。 结果 UCA1在肺腺癌A549细胞中高表达。与NC组相比,siRNA-UCA1组能够抑制GLUT1、HK2和PKM2基因在肺腺癌A549细胞中的表达(t=19.66、5.81、11.98,均P<0.001),且三者的蛋白表达水平明显下降(t=61.35、145.90、88.19,均P<0.001)。与NC组相比,siRNA-UCA1组肺腺癌A549细胞的侵袭能力、迁移运动能力和对18F-FDG的摄取率均明显降低(t=19.43、7.71、5.79,均P<0.05)。 结论 LncRNA UCA1能够抑制糖代谢的关键酶并促进肺腺癌的转移能力,其可能成为肺癌新的诊断指标和治疗靶点。 Abstract:Objective To explore the expression of long non-coding RNA (lncRNA) urothelial carcinoembryonic antigen 1 (UCA1) in lung adenocarcinoma A549 cells and its influence on key enzymes involved in sugar metabolism, cell invasion, and metastasis. Methods The lncRNA UCA1 silencing and its control stable transfected lung adenocarcinoma A549 cells were constructed, the small interfering RNA (siRNA) transfected cells were divided into NC group (negative control group, transfected with siRNA-UCA1 sequence) and siRNA-UCA1 group (transfected with siRNA-UCA1 knockdown sequence). Quantitative real-time PCR and Western blot analyses were used to detect changes in the levels of glucose transporter protein (GLUT)1, hexokinase (HK)2 and pyruvate kinase isozymes M (PKM)2 related to glucose metabolism. Transwell and scratch tests were conducted to detect the invasion and migration ability of siRNA-UCA1. 18F-Fluorodeoxyglucose (FDG) uptake experiment was used to detect the uptake rate of siRNA-UCA1. T test was used to compare the measurement data. Results UCA1 was highly expressed in lung adenocarcinoma A549 cell line. Compared with the NC group, the siRNA-UCA1 group can inhibit the expression of GLUT1, HK2, and PKM2 in lung adenocarcinoma A549 cells (t=19.66, 5.81, 11.98; all P<0.001), and the protein expression levels of the three significantly decreased (t=61.35, 145.90, 88.19; all P<0.001). Compared with the NC group, the siRNA-UCA1 group's migration ability and 18F-FDG uptake rate of A549 cells were significantly reduced (t=19.43, 7.71, 5.79; all P<0.05). Conclusions LncRNA UCA1 can affected the key enzymes of glucose metabolism and promoted the metastasis ability of lung adenocarcinoma. Thus, lncRNA UCA1 may be a new diagnostic index and therapeutic target for lung cancer. -
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