Combined identification of long non-coding RNA XIST and HIF1A-AS1 in serum as an effective screening for non-small cell lung cancer

Objective: Long non-coding RNAs (lncRNAs) XIST and HIF1A-AS1 have been shown to play important regulatory roles in cancer biology, and lncRNA-XIST and HIF1A-AS1 are upregulated in several cancers such as glioblastoma, breast cancer and thoracoabdominal aorta aneurysm, however, its value in the diagnosis of non-small cell lung cancer (NSCLC) is unclear. The aim of this study is to evaluate the clinical significance of serum XIST and HIF1A-AS1 as a biomarker in the screening of NSCLC. Methods: Expression levels of lncRNA-XIST and HIF1A-AS1 in tumor tissues and serum from NSCLC patients were evaluated by quantitative real-time PCR, and its association with overall survival of patients was analyzed by statistical analysis. Moreover, the XIST and lncRNA-XIST expression correlation between tumor tissues and plasma was demonstrated by linear regression analysis. Results: The levels of XIST (P < 0.05) and HIF1A-AS1 (P < 0.05) were significantly increased in tumor tissues or serum from NSCLC patients as compared to those of control group. Correlation of lncRNA-XIST or HIF1A-AS1 expression between tumor tissues and serum from the same individuals was confirmed in NSCLC patients. Moreover, serum levels of XIST and HIF1A-AS1 were significantly decreased after surgical treatment as compared to pre-operative. The ROC curves illustrated strong separation between the NSCLC patients and control group, with an AUC of 0.834 (95% CI: 0.726-0.935; P < 0.001) for XIST and 0.876 (95% CI: 0.793-0.965; P < 0.001) for HIF1A-AS1, however, the combination of XIST and HIF1A-AS1 yielded an AUC of 0.931 (95% CI: 0.869-0.990; P < 0.001), which was significantly improved as compared to XIST or HIF1A-AS1 alone. Conclusion: Our results demonstrated that increased serum XIST and HIF1A-AS1 could be used as a predictive biomarker for NSCLC screening, and that combination of XIST and HIF1A-AS1 had a higher positive diagnostic efficiency of NSCLC than XIST or HIF1A-AS1 alone.     

长链非编码RNA调控肿瘤细胞凋亡的研究进展

长链非编码RNA(long non-coding RNA,lncRNA)是一类长度>200个核苷酸、通常不编码蛋白质的RNA。近年研究表明,lncRNA在肿瘤的的发展过程中发挥抑癌或促癌作用,参与细胞增殖、凋亡等过程。本综述简要介绍lncRNA的生物学功能及其调控细胞凋亡的研究进展。     

Paeonol alleviates CCl4-induced liver fibrosis through suppression of hepatic stellate cells activation via inhibiting the TGF-β/Smad3 signaling

Objective: Paeonol is a natural phenolic component isolated from the root bark of peony with multiple pharmacological activities. We investigated the anti-fibrotic effect and underlying mechanism of paeonol.

Methods: Twenty-four male C57BL/6J mice were divided into 4 groups (n?=?6 in each group), injected with CCl₄ to induce liver fibrosis and administrated with paeonol according to the regimen. The serum activity of ALT and AST, and H&E staining were to assess liver injury. Sirius and Masson staining, and hydroxyproline content were to evaluate the degree of liver fibrosis. TNF-α, IL-6, TGF-β, MDA, GSH-PX, SOD, and CAT were detected to reflect inflammation and oxidative stress. RT-qPCR and Western blot analysis to assess the activation of HSCs and TGF-β/Smad3 signaling.

Results: Paeonol ameliorated liver injury and liver fibrosis, reflected by the decrease of ALT, AST, less lesion in H&E staining, mitigated fibrosis in Sirius and Masson staining, lessened content of hydroxyproline. Paeonol attenuated the level of IL-6 and TNF-α, and elevated the activity of GSH-PX, SOD, and CAT with reducing the level of MDA. The expression of col 1a, α-SMA, vimentin, and desmin were down-regulated and TGF-β/Smad3 signaling pathway was inhibited.

Conclusion: These data demonstrated that paeonol could alleviate CCl₄-induced liver fibrosis through suppression of hepatic stellate cells activation via inhibiting the TGF-β/Smad3 signaling.     

A novel long non-coding RNA NFIA-AS1 is down-regulated in gastric cancer and inhibits proliferation of gastric cancer cells

Gastric cancer is one of the most common malignant gastrointestinal tumors whose morbidity and mortality account for the second and third place respectively in malignant tumors in China. As an important participant in tumor biology, the abnormal expression of long non-coding RNA (lncRNAs) in cancer cells is closely related to the occurrence and development of tumors and plays the role of oncogenes or tumor suppressor genes. In this study, we identified a novel lncRNA NFIA antisense RNA 1 (NFIA-AS1) and explored its role and clinical significance in gastric cancer. Real-time quantitative PCR was performed to detect the expression of NFIA-AS1 in tumor tissues and corresponding normal tissues from 42 pairs of gastric cancer samples. The lower expression of NFIA-AS1 was significantly associated with larger tumor size, lower histological grade, and advanced TNM stage. Kaplan-meier analysis showed that NFIA-AS1 expression could be used as an independent predictor of overall survival. We also demonstrated that overexpression of NFIA-AS1 significantly inhibited the proliferation of gastric cancer cells through affecting p16 levels. In conclusion, our results suggest that the lncRNA NFIA-AS1 may play the role of tumor suppressor gene, and serve as a biomarker for prognosis or progression of gastric cancer.     

长链非编码RNA HOTAIR在宫颈癌患者组织及HeLa细胞中的表达和影响

目的探讨长链非编码RNA HOTAIR在宫颈癌患者组织中的表达及对宫颈癌细胞系He La细胞增殖和凋亡的影响。方法 66例宫颈癌手术切除标本(包括癌组织和癌旁组织),实时荧光定量PCR检测HOTAIR的表达,分析其与临床病理特征的关系;用慢病毒介导shRNA干扰人宫颈癌细胞He La HOTAIR表达后,分别用CCK-8法、流式细胞术检测细胞增殖、周期和凋亡。结果 HOTAIR在宫颈癌组织中的相对表达量为8.25±0.46,高于癌旁组织的3.60±0.26(P0.001);HOTAIR的表达水平与临床病理分期(P0.05)、肿瘤大小(P0.05)、淋巴结转移(P0.05)密切相关;HOTAIR的表达被干扰后,He La细胞增殖明显减慢,48、72和96 h的抑制率分别为20%、24.6%和33.1%;细胞周期被阻滞于G0/G1期;HOTAIR干扰组细胞凋亡比例为12.37%±2.74%,高于对照组的5.94%±1.27%(P0.01)。结论 HOTAIR在宫颈癌组织中显著高表达,可以作为宫颈癌预测、判断预后的分子标志物和潜在的治疗靶点。     

长链非编码RNA BCYRN1与相关疾病的研究进展

BCYRN1是一种长链非编码RNA,在大脑中高水平表达,并在多种肿瘤类型和其他系统疾病中表达升高。随着对BCYRN1研究的深入,发现BCYRN1与肿瘤、神经系统疾病、呼吸系统疾病以及其他系统疾病密切相关并扮演着重要角色。本文总结了BCYRN1在不同疾病的作用及相关机制的研究进展,为深入了解BCYRN1是如何影响和促进疾病的发生、发展过程提供一点参考或启示。     

下调长链非编码RNA PVT1表达对胰腺癌BxPC-3细胞凋亡、侵袭及转移的影响

目的研究下调长链非编码RNA PVT1的表达对胰腺癌细胞增殖、凋亡及侵袭转移能力的影响。方法体外转染PVT1干扰序列si PVT1-1和si PVT1-2降低胰腺癌细胞系Bx PC-3 PVT1表达,同时转染阴性对照si PVT1-NC作为对照组。CCK-8实验检测细胞增殖,流式细胞术检测细胞凋亡,细胞划痕实验和Transwell实验检测细胞侵袭转移;qRT-PCR检测E-cadherin、N-cadherin、β-catenin、vimentin和PVT1 mRNA表达;Western blot检测凋亡相关蛋白(Bax、Bcl-2、caspase-3)和上皮间质转化相关蛋白(E-cadherin、N-cadherin、β-catenin、vimentin)表达。结果下调胰腺癌细胞Bx PC-3 PVT1表达能明显抑制细胞增殖、促进细胞凋亡和减少细胞侵袭转移数目(P0.05);E-cadherin蛋白和分子水平升高,N-cadherin、β-catenin、vimentin蛋白和分子水平降低(P0.05);Bax和caspase-3蛋白水平升高,而Bcl-2蛋白水平降低(P0.05)。结论下调长链非编码RNA PVT1表达能抑制胰腺癌细胞增殖和侵袭转移能力,促进胰腺癌细胞凋亡,且能够逆转胰腺癌细胞上皮间质转化。     

Combined identification of long non-coding RNA CCAT1 and HOTAIR in serum as an effective screening for colorectal carcinoma

Long non-coding RNAs (lncRNAs) CCAT1 and HOTAIR have been shown to play an important regulatory role in cancer biology, and CCAT1 and HOTAIR are upregulated in several cancers, however, its value in the diagnosis of colorectal cancer (CRC) is unclear. Therefore, the aim of this study is to evaluate the clinical significance of plasma CCAT1 and HOTAIR as a biomarker in the screening of CRC. In our study, we found that the levels of HOTAIR (P < 0.05) and CCAT1 (P < 0.05) were significantly higher in plasma of CRC patients than that of the healthy control. Moreover, the levels of lincRNA-p21 (P < 0.05) were obviously decreased in plasma of CRC patients as compared to those of healthy control. There was highly correlated for CCAT1 (R = 0.752, mean differences = -0.06 ± 1.20), HOTAIR (R = 0.739, mean differences = -0.26 ± 0.76) and lincRNA-p21 (R = 0.848, mean differences = -0.41 ± 0.89) in plasma and serum. By receiver operating characteristic curve (ROC) analysis, plasma CCAT1 provided the higher diagnostic performance for detection of CRC (the area under the ROC curve (AUC), 0.836; P < 0.001; sensitivity, 75.7%; specificity, 85.3%). Moreover, CCAT1 combining with HOTAIR could provide a more effective diagnosis performance (AUC, 0.954, P < 0.001, sensitivity, 84.3%; specificity, 80.2%). Most importantly, this combination was effective to detect CRC at an early stage (85%). In conclusion, our results demonstrated that increased plasma HOTAIR and CCAT1 could be used as a predictive biomarker for CRC screening, and that combination of HOTAIR and CCAT1 had a higher positive diagnostic rate of CRC than HOTAIR or CCAT1 alone.     

长链非编码RNA MEG3对肿瘤细胞调控作用的研究进展

长链非编码RNA(long non-coding RNA,lncRNA)是一类长度超过200 nt的不编码蛋白的RNA分子。lncRNA最初被认为是转录噪音,在近年的研究中发现越来越多功能性的lncRNA,其重要性才渐渐被阐述。母系表达基因3(materally expressed gene 3,MEG3)是一种由母系印记基因编码的lncRNA,在对其功能的研究中发现MEG3几乎参与了所有的生理和病理过程,其在多种肿瘤中表现出抑制作用。本文就lnc RNA MEG3对肿瘤细胞调控的作用机制作一综述。     

长链非编码RNA母系表达基因3对结直肠癌细胞侵袭和迁移能力的影响

目的:检测长链非编码RNA母系表达基因3(maternally expressed gene 3,MEG3)在结直肠癌细胞中的表达,并观察过表达MEG3对结直肠癌细胞侵袭和迁移能力的影响。方法:检测人正常结肠细胞NCM460及结直肠癌细胞SW48、Lo Vo中MEG3的水平,在SW48细胞和Lo Vo细胞中转染MEG3过表达质粒,利用Transwell小室及划痕实验观察过表达MEG3对细胞侵袭和迁移能力的影响,通过Western blotting检测基质金属蛋白酶(matrix metalloproteinase,MMP)家族相关蛋白的变化。结果:结直肠癌细胞SW48和Lo Vo中MEG3的水平明显低于人正常结肠细胞NCM460;在SW48和Lo Vo细胞中过表达MEG3后能够明显抑制细胞的侵袭和迁移能力;Transwell侵袭实验和迁移实验显示MEG3表达组SW48细胞的穿膜数及Lo Vo细胞的穿膜数与对照组比较明显减少。划痕实验中过表达MEG3后细胞间距较对照组明显增大,提示细胞运动能力减弱。同时过表达MEG3可明显降低细胞中MMP-2及MMP-9的表达,增高金属蛋白酶组织抑制物2(tissue inhibitor of metalloproteinase-2,TIMP-2)的表达。结论:结直肠癌细胞中MEG3水平较正常结直肠细胞明显降低;在结直肠癌中过表达MEG3可抑制细胞的侵袭、迁移运动能力,并且可能通过影响TIMP-2、MMP-2及MMP-9的表达发挥上述功能。     

长链非编码RNA AK046999在小鼠大脑皮质发育中的表达及功能

目的检测长链非编码RNA AK046999在小鼠大脑皮质发育过程中的表达及作用。方法 1)通过UCSC基因组浏览器获取其基因组座位,利用Coding Potential Calculator工具对其编码能力进行预测;2)原位杂交及免疫荧光技术确定其在大脑皮质发育过程中的表达谱;3)TALEN技术构建AK046999完全敲除小鼠,并且在DNA及RNA水平对其进行敲除鉴定;4)免疫荧光及TUNEL技术对敲除小鼠大脑皮质在发育过程的功能进行检测。结果 1)AK046999可认为是非编码RNA(Coding Potential Score-1);2)AK046999在小鼠大脑皮质发育过程中,前体区相对高表达;3)成功构建了AK046999敲除小鼠;4)与对照相比,AK046999敲除小鼠中表达PAX6、TBR2和NEUROD2的细胞数量无明显改变,并且在这两组小鼠中凋亡细胞的数量无明显改变。结论 AK046999在发育期的小鼠大脑皮质表达,但敲除后不影响小鼠大脑皮质中神经祖细胞的增殖、分化和细胞凋亡过程。     

Expression of long non-coding RNA SFTA1P and its function in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a major type of lung cancer with high morbidity and mortality. Long non-coding RNAs (lncRNAs) have been reported to be important in development and progression of NSCLC. However, the role of lncRNA SFTA1P remains unclear. This study aims to explore the clinical roles, biological function, and mechanism of SFTA1P in NSCLC. SFTA1P expression was estimated by the quantitative real-time polymerase chain reaction (qRT-PCR) of 90 pairs of tissue samples, the Cancer Genome Atlas (TCGA) database and microarray. After overexpressing SFTA1P, NSCLC cell proliferation, cycle, and apoptosis were detected. We found that the expression of SFTA1P was significantly downregulated in NSCLC tissues with high diagnostic value (AUC = 0.87), which was consistent with the results of TCGA and microarray data. For the analysis of clinical features, the results revealed that SFTA1P expression was closely related to the pathological type (P < 0.01). Furthermore, the cell function results suggested that the overexpression of SFTA1P triggered cell cycle arrest in the S-phase (P < 0.05). From a mechanistic perspective, the results showed that the PI3K-AKT signaling pathway was inhibited after overexpression of SFTA1P in NSCLC. Taken together, this work supported that SFTA1P may play a suppressing role in the tumorigenesis of NSCLC by modulating PI3K-AKT signaling pathway to influence cell cycle, which provides a potential and prospective biomarker for NSCLC.     

长链非编码RNA WIF1-1在食管鳞状细胞癌组织中的表达及其临床意义

目的:探究食管鳞状细胞癌组织及癌旁组织中长链非编码RNA(long noncoding RNA,lncRNA)WIF1-1的表达水平,并评估其对患者预后的价值.方法:使用实时荧光定量PCR检测50例食管鳞状细胞癌组织样本及其对应同源的癌旁组织样本中lncRNA WIF1-1的表达水平,分析临床病理特征及其与患者预后的相关性.结果:食管鳞状细胞癌组织中lncRNA WIF1-1的表达水平明显低于癌旁组织,差异有统计学意义(P<0.001).进一步分析发现:lncRNA WIF1-1的表达水平与食管鳞状细胞癌的淋巴结转移、TNM分期和临床分期之间呈负相关.对预后资料分析发现:lncRNA WIF1-1表达水平较低的食管鳞癌患者的总生存时间(overall survival,OS)以及无进展生存期(progression-free survival,PFS)均较短.单因素及多因素分析结果显示:lncRNA WIF1-1可作为食管鳞状细胞癌患者OS和PFS的独立危险因素.结论:lncRNA WIF1-1可能在食管鳞状细胞癌中发挥抑癌基因的作用,其表达量与患者预后具有相关性,有可能成为食管鳞状细胞癌的治疗靶点.     

shRNA表达载体pWH1的构建及用于HIF1基因的沉默

目的:构建用于RNAi的shRNA(small hairpin RNA)表达载体及检测其对低氧诱导因子-1(Hypoxia-inducible Factor-1,HIF1)基因的沉默效果.方法:从人血基因组中PCR扩增出H1基因启动子,克隆入酶切处理后的pEGFP-C1载体片段中,此载体命名为pWH1.以人HIF1 cDNA基因为靶标设计引物,退火后克隆入pWH1.新的载体转染SGC7901细胞,然后用RT-PCR和Western blot检测HIF1基因的表达改变.结果:构建的pWH1载体能很好地表达针对HIF1基因的shRNA,RT-PCR和Western blot的结果显示HIF1基因的mRNA和蛋白表达水平均明显下降.结论:成功构建了shRNA表达载体pWH1,这对于基因的功能研究具有重要的意义.