Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Positive reverse transcription-polymerase chain reaction assay results in patients recovered from COVID-19: Report of two cases

BACKGROUND Coronavirus disease 2019(COVID-19)has spread around the globe.On February 28,2020,the World Health Organization adjusted the risk of spread and impact of COVID-19 to“very high”at the global level.Studies have mainly focused on the etiology,epidemiology,and treatment of COVID-19 to limit further spread and the negative impact of the disease,while less attention has been devoted to the follow-up and reexamination of patients who recovered from COVID-19 or were released from quarantine.CASE SUMMARY This study reports two cases where patients who had negative reverse transcription-polymerase chain reaction(RT-PCR)test results and met the criteria for discharge subsequently had positive RT-PCR test results.The clinical manifestations and computed tomography(CT)findings of these patients were examined.The conversion of RT-PCR test results in these two patients may be related to false-negative and false-positive outcomes of the test.CT images helped track improvement of pulmonary lesions.CONCLUSION The timing of discharge of COVID-19 patients should be determined by comprehensive analysis of CT images and RT-PCR test results.

Detection of hepatocellular carcinoma cells in the peripheral blood with reverse--transcription polymerase chain reaction

In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.

Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in Human Spermatozoa

Objective:Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification data.Recently,several strategies have been utilized for validating reference genes in different human tissues.However,no universal reference genes have been described that accurately summarize transcriptional activity in human spermatozoa.Methods:Using quantitative reverse transcription-polymerase chain reaction(RT-qPCR),we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy rates.We assessed the stability of reference genes using three different algorithms,namely geNorm,NormFinder,and BestKeeper.We then identified the most stable reference genes.Results:Male-enhanced antigen 1(MEA1)was identified as the most stably expressed reference gene,followed by testis-enhanced gene transcript(TEGT).Conclusions:We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

Preliminary research on myosin light chain kinase in rabbit liver

AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver.METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction (RT-PCR);the MLCK was obtained from rabbit liver, and its activity was analyzed by γ-32P incorporation technique to detect the phosphorylation of myosin light chain.RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg-L-1, the activity was at the highest level.CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.

荧光定量RT-PCR法在病原微生物检验中的应用价值

目的:分析荧光定量逆转录聚合酶链式反应(RT-PCR)法在病原微生物检验中的应用价值。方法:选取2019年4月-2021年1月在长沙市中心医院进行病原微生物检测的46例疑似微生物感染患者作为研究对象,均实施常规RT-PCR法、荧光定量RT-PCR法检测,统计两种方法的检验结果;以特殊染色镜检结果作为“金标准”,比较两种检验方法检测微生物感染的准确度、灵敏度、特异度以及对各类病原微生物的检出率,统计受试者对两种检验方法的满意度。结果:特殊染色镜检结果显示,46例受试者病原微生物感染阳性43例,阴性3例。两种检验方法检测微生物感染的准确度、特异度比较,差异无统计学意义(P>0.05);荧光定量RT-PCR法检测灵敏度高于RT-PCR法,差异有统计学意义(P<0.05)。特殊染色镜检结果显示,43例病原微生物感染患者中,衣原体感染7例,支原体感染8例,寄生虫感染9例,细菌感染7例,真菌感染5例,病毒感染7例,两种检验方法对各类病原微生物的检出率及总检出率比较,差异无统计学意义(P>0.05)。受试者对荧光定量RT-PCR法检验总满意度高于RT-PCR法,差异有统计学意义(P=0.026)。结论:荧光定量RT-PCR法在病原微生物检验中的应用价值较好,能够提高检测灵敏度,受试者满意度高。

陕西省咸阳地区人乳头瘤病毒的基因分型及亚型分布特征

目的对陕西省咸阳地区妇女人乳头瘤病毒(HPV)感染及亚型分布特征进行研究。方法采用聚合酶链反应(PCR)-反向点杂交技术对7 708例宫颈脱落细胞样本进行HPV的基因分型检测。结果在被研究的7 708例妇女中感染HPV者2 477例,总感染率为32.14%(2 477/7 708),高危型HPV感染率(包括高危多重感染)为23.96%(1 847/7 708),在被检测的18种高危亚型中,常见的类型为16、52、58、51、56和66型。HPV单一基因型的感染率为27.58%(2 126/7 708),单一高危型的感染率为19.94%(1 537/7 708),单一高危型感染常见的亚型为16、52、58、51、33和39型。多重感染率为4.55%(351/7 708),多重高危型的感染率为4.53%(349/7 708)。在不同年龄组妇女中,31~40岁组感染率最高(10.20%)。在31~40岁组妇女HPV高危型感染的亚型主要为16、52、58、51、56和33型,其他各年龄组感染的亚型分布各不相同。结论陕西省咸阳地区妇女的宫颈HPV感染率较高,常见高危型为16、52、58、51、56和66型。各年龄段妇女感染HPV的亚型分布特征各不相同,30~50岁年龄段妇女应作为HPV监测的重点人群。

Msi1基因沉默对人肝癌HepG2细胞的影响

目的:探讨siRNA沉默Msi1基因表达后,人肝癌HepG2细胞的增殖与凋亡的情况。方法:设计合成针对Msi1 mRNA序列的siRNA多条序列,分别转染人肝癌HepG2细胞。使用RT–PCR方法检测各组细胞Msi1基因表达情况;Western blot法检测survivin蛋白、caspase3蛋白表达情况;MTT法、平皿克隆实验检测HepG2细胞增殖情况;流式细胞术检测各组HepG2细胞凋亡情况。结果:(1)RT-PCR结果:转染后24 h后,序列a,b,c转染效率分别为65%,64%及62%。其抑制率分别是Msi1 siRNAa(68%)、Msi1 siRNAb(56%)、Msi1 siRNAc(35%)。(2)流式细胞术检测各组细胞凋亡:空白组、阴性组、脂质体组、Msi1 siRNAa组凋亡率分别为(4.14±0.26)%、(4.51±0.78)%、(4.19±1.21)%,(16.8±0.26)%,Msi1 siRNAa组与空白对照组比较差异有统计学意义(P<0.05)。(3)Msi1 siRNAa最能有效抑制肝癌HepG2细胞中Msi1表达,与空白对照组比较,Msi1 siRNAa组HepG2细胞生长、增殖速度明显减缓(P<0.05);survivin蛋白表达水平显著下调(P<0.05),而caspase3蛋白表达水平上调(P<0.05);Msi1 siRNAa组凋亡率增高(P<0.05)。结论:沉默Msi1可抑制人肝癌HepG2细胞的增殖,诱导其凋亡。

卵巢浆液性癌组织中microRNA表达谱分析

目的寻找与卵巢浆液性癌相关的microRNA,积累以国内患者为数据来源的microRNA表达谱资料,并初步探讨其临床意义。方法利用microRNA芯片,筛查卵巢浆液性癌组织及正常卵巢组织中差异表达的microRNA,用实时荧光定量PCR在58份卵巢组织标本中进行验证,并分析microRNA表达水平与卵巢浆液性癌临床病理特征之间的关系。结果与正常卵巢组织比较,芯片结果显示卵巢浆液性癌有9个microRNA明显上调,分别是hsa-mi R-141-3p、hsa-mi R-200a-3p、hsa-mi R-200b-3p、hsa-mi R-200c-3p、hsa-mi R-224-5p、hsa-mi R-7-5p、hsa-mi R-142-3p、hsa-mi R-21-5p、hsa-mi R-142-5p,6个microRNA明显下调,分别是hsa-mi R-424-5p、hsa-mi R-214-3p、hsa-mi R-125b-5p、hsa-let-7b-5p、hsa-mi R-199a-5p、hsa-mi R-100-5p。实时荧光定量PCR验证结果与芯片结果一致。对差异microRNA进行分层分析发现,与卵巢浆液性癌Ⅰ-Ⅱ期、淋巴结未转移患者比较,Ⅲ-Ⅳ期、淋巴结有转移患者的hsa-mi R-21-5p表达均增高,hsa-mi R-125b-5p表达均降低(P<0.05);而在高级别与低级别卵巢癌患者之间microRNA的表达水平差异均无统计学意义(P均>0.05)。结论卵巢浆液性癌中异常表达的microRNA可能与肿瘤的发生发展相关。

微小RNA差异表达谱在原发性干燥综合征的应用

目的探索原发性干燥综合征(primary Sjgren’s syndrome,p SS)患者血清中相关的差异表达微小RNA(microRNA,miRNA)。方法健康对照组和pSS组各选1例进行芯片分析,筛选出p SS患者与健康对照组表达差异有统计学意义的miRNA;选取6例健康对照和6例pSS患者治疗前后的血清,采用荧光定量法对差异表达的miRNA进行验证;通过Target Scan、miRanda以PicTar预测miRNA作用的靶基因并分析相关信号通路的生物学信息。结果共发现53条miRNA表达差异有统计学意义,选取22条差异倍数>1.5倍的miRNA,p SS治疗前组与对照组比较miR-146a-5p、miR-4484和miR-4687-5p表达均上调(P<0.05),miR-146a-5p和miR-4687-5p验证结果与芯片一致。治疗前组和治疗后组比较,miR-146a-5p和miR-4484表达均下调(P<0.05),而miR-4687-5p表达依然上调。靶基因预测结果显示p SS中miRNA可能通过Notch信号通路和花生四烯酸以及溶酶体等代谢发挥信号转导作用。结论 pSS患者与健康人群存在差异表达的miRNA,这些miRNA表达可能参与pSS疾病的发生和发展,对p SS诊断有一定价值。

人IL-6 mRNA的实时定量检测法

目的:建立TaqMan-MGB技术实时定量检测人IL-6 mRNA的方法,并用于检测人外周血单个核细胞在牛膝多糖诱导下IL-6 mRNA的表达。方法:(1)抽取人外周静脉血,EDTA抗凝,用淋巴细胞分离液分离外周血单个核细胞,用Trizol裂解液提取总RNA,将总RNA逆转录为cDNA,PCR扩增IL-6后纯化PCR产物,与pMD 18-T载体进行连接,转化宿主菌DH-5α。提取重组质粒DNA,作为实时荧光定量检测的标准品。建立TaqMan-MGB技术实时定量检测IL-6 mRNA的方法。(2)终浓度分别为0、100、200、400、800和1600mg/L的牛膝多糖作用于人外周血单个核细胞,定量检测IL-6 mRNA的表达。结果:(1)酶切分析、PCR扩增以及测序结果均证实IL-6 cDNA成功重组到pMD 18-T载体上。(2)TaqMan-MGB技术检测IL-6 mRNA线性范围广;灵敏度高;特异性好;重复性好。(3)牛膝多糖作用于人外周血单个核细胞后IL-6mRNA表达增高。结论:TaqMan-MGB技术能较为精确地定量IL-6 mRNA。

Correlation between MMP-2 and NF-κ B expression of intracranial aneurysm

Objective:To investigate the correlation between expressions of MMP-2 and NF-κB in the intracranial aneurysm wall,and explore their role in the mechanism of the occurrence,growth and rupture of intracranial aneurysms.Methods:RT-PCR was used to detect the expression of MMP-2 and NF-κB mRNA of 30 cases of intracranial aneurysm tissue and 10 cases of normal intracranial arterial tissue:Immunohistochemical method was used to detect the expression of MMP-2 and NF-κB protein.Results:the semi-quantitative analysis of MMP-2 and NF-κB in aneurysms tissues and normal tissues were statistically significant different from each other(P【0 05).Immunohistochemical staining results showed NF-κB was expressed in different layers.The expression of them were positive in intimal and medial,and the expression sites were located in the nucleus.MMP-2 were expressed in different layers of the aneurysm wall,and the expressions were positive in media and extima.The MMP-2 and NF-κB-positive expression of aneurysm wall were significantly higher than in normal cerebral arteries(P【0.05).MMP-2 and NF-κB mRNA expression showed positive correlation in the aneurysm wall tissue(r = 0.689,P = 0.005). Conclusions:The expressions of MMP-2 and NF-κB in the intracranial aneurysm wall tissue were significantly higher than in the normal intracranial arterial tissues.They have a synergistic effect on the formation of intracranial aneurysms.

Diagnosis of nervous necrosis virus in orange-spotted grouper,Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

Expression and functional characterization of platelet-derived growth factor receptor-like gene

AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry in CRC and colorectal normal tissues.PDGFRL prokaryotic expression vector was carried out in Escherichia coli(E.coli),and purified by immobilized metal affinity chromatography.The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT),clone counting,cell cycle,and wound healing assay.RESULTS:Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues.Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E.coli,and the target protein was expressed in the form of inclusion bodies.After purification and refolding,recombinant human PDGFRL(rhPDGFRL)could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT,clone counting and wound healing assay.Moreover,rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase.CONCLUSION:PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.

Expression of huCdc7 in colorectal cancer

AIM:To detect the expression of huCdc7 in colorectal cancer.METHODS:The mRNA and protein expression of huCdc7 in 39 colorectal cancer tissue specimens and matched tumor-adjacent normal colorectal tissue specimens was detected by reverse transcription-polymerase chain reaction and immunohistochemistry,respectively.RESULTS:The relative expression level of huCdc7 mRNA in colorectal cancer was significantly higher than that in tumor-adjacent normal colorectal tissues(0.03675 ± 1.00 vs 0.01199 ± 0.44,P < 0.05).huCdc7-positive cells displayed brown granules in the nucleus.Tumor tissues contained many huCdc7-positive cells,whereas normal colorectal tissues contained very few positive cells.CONCLUSION:huCdc7 may play an important role in the development and progression of colorectal cancer.

In advanced gastric cancer:Prognosis and treatment of patients with positive peritoneal cytology

Positive peritoneal cytology in gastric cancer is classified as M1 disease by the 7thEdition of American Joint Committee on Cancer staging system.With the introduction of laparoscopy and peritoneal washing cytology in the staging of gastric cancer a new category of patients has been identified.These are patients with no macroscopic peritoneal metastases but with peritoneal cytology positive(P0C1).Prognosis and treatment of such patientsrepresent a controversial issue.We evaluate the state of the art of staging system in gastric cancer and discusss tandardisation in staging and treatment procedures.There is still a lack of uniformity in the use of laparoscopy with peritoneal cytology in clinical decision making and in the surgical treatment for gastric cancer.Survival of this patient subset remains poor.Multimodal therapies and new therapeutic strategies are required to improve the survival of these patients.

Significance of combined detection of LunX mRNA and tumor markers in diagnosis of lung carcinoma

Objective：To evaluate the significance of combined detection of LunX mRNA,carcinoembryonic antigen （CEA）,neuron-specific enolase （NSE）,and cytokeratin 21-1 fragment （CYFRA21-1） in clinical diagnosis of lung carcinoma.Methods：Based on the quantitative RT-PCR and chemiluminescence immunoassay,the expression levels of LunX mRNA,CEA,NSE,and CYFRA21-1 in 113 patients with lung carcinoma （case group） and 30 healthy participants （control group） were detected.Meantime,the sensitivity,specificity,and accuracy of the combination detection were also explored.Results：The positive rates of LunX mRNA in peripheral blood and CEA,NSE,and CYFRA21-1 in serum were significandy higher in case group than those in control group （x2=17.295,16.825,19.148,and 17.450; P＜0.05）.There was no statistical significance when positive rate of LunX mRNA was evaluated among different pathological types （x2=0.047,P＞0.05）.The positive rate of LunX mRNA in stage Ⅰ ＋ Ⅱ,Ⅲ,and Ⅳ had a significandy increasing tendency （x2=10.565,32.462,P＜0.05）.The positive rate of CYFRA21-1 was highest in squamous carcinoma （78.5％）,the positive rate of NSE was highest in small cell carcinoma （86.7％）,and the positive rate of CEA wag highest in lung adenocarcinoma （80.4％）.The sensitivity and accuracy of the combination detection were 91.1％ and 88.1％,respectively.Conclusions：The combined detection of LunX mRNA and tumor markers （TMs） including CEA,NSE,and CYFRA21-1 in peripheral blood is helpful to increase the diagnostic accuracy of lmg cancer.Also,it can inform the pathological typing of lung carcinoma.

Methods for the extraction and RNA profiling of exosomes

AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent respectively.Identity and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 marker.Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit.Finally,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)methodology.RESULTS:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these vesicles.The isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher yields.Exosomes were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r RNA.CONCLUSION:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.

Infiltration Related miRNAs in Bladder Urothelial Carcinoma

This study aimed to investigate infiltration related microRNAs(miRNAs) in bladder urothelial carcinoma(BUC).Twenty patients with BUC were enrolled and divided into 2 groups according to infiltration or not:infiltrating BUC group(n=12) and non-infiltrating BUC group(n=8).Gene chip was used to detect infiltration related miRNAs in the BUC samples.In other recruited 17 patients with BUC who were divided into infiltrating BUC samples(n=14) and non-infiltrating BUC samples(n=3),and in 4 BUC cell lines(EJ,5637,T24 and BIU-87),the expression of miRNAs was assayed by using reverse transcription-polymerase chain reaction(RT-PCR).In infiltrating BUC group,as compared with non-infiltrating BUC group,there were 7 differentially expressed miRNAs:hsa-miR-29c,hsa-miR-200a,hsa-miR-378,hsa-miR-429,hsa-miR-200c and hsa-miR-141 were up-regulated,while hsa-miR-451 was down-regulated.In the BUC samples,the results of RT-PCR were consistent with those by the miRNA array.In the cancer cell lines,RT-PCR in T24 only revealed the similar expression pattern of miRNAs to that by the miRNA array.It is suggested that infiltration of BUC is related with different expression of miRNAs,which may provide a novel platform for further study on function and action mechanism of miRNAs.