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.

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.

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。

香蕉MaPRMT1基因的分离及表达分析(英文)

[Objective] The aim of experiment was to lay molecular foundation for studying maturity mechanism of banana after harvest. [Method] The combined method of suppressing subtractive hybridization and cDNA micro-array were used to obtain cDNA segment of one PRMT gene in banana and the whole cDNA sequence of the gene was cloned.The bioinformatics analysis was operated on it,in addition, the expression profile analysis was conducted in different organs and different mature periods of banana.[Result] The whole length of cDNA in MaPRMT1 was 1 158 bp and possessed a complete open reading frame,which could encode 385 amino acids.It had high homology with PRMT in plant,containing one Methyltransf_1 domain.The MaPRMT1 gene was expressed in root,stem,leaf and fruit of banana and the expression levels in stem and leaf were relatively high.As the increase of days after harvest,the expression level declined gradually,however it reached maximum when ethylene release was biggest,then it declined.[Conclusion] MaPRMT1 belonged to the first kind of arginine methyltransferase and it was expressed differently in different organs and fruits at different mature periods.

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.

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.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

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.

Combination of Cytogenetic Analysis and Molecular Screening in Patients with de novo Acute Myeloid Leukemia

Nowadays the role of genetic findings in determining the diagnosis,therapy and prognosis of acute myeloid leukemia(AML) has become more valuable.To improve and validate the detection of clonal chromosomal aberrations in leukemia,we designed a combined application of karyotyping with multiplex reverse transcription-polymerase chain reaction(RT-PCR) and fluorescence in situ hybridization(FISH),and addressed the expression and distribution of fusion genes among the subtypes of Chinese adult patients with de novo AML.Multiplex RT-PCR assays were performed on 477 samples from newly diagnosed AML patients,and cytogenetic data were obtained from 373 of them by R or G banding techniques and those in some cases were confirmed by FISH.The PCR products in some suspected cases were tested by two-directional sequencing.The results showed that except unqualified samples,fusion genes were detected by multiplex RT-PCR in 211 of 474 patients(44.51%),including AML1-ETO,CBFβ-MYH11,PML-RARα,PLZF-RARα,NPM-RARα,MLL rearrangements,BCR-ABL,DEK-CAN,SET-CAN,TEL-PDGFR,TLS-ERG,AML1-MDS1(EVI-1).In 373 patients,who took both multiplex RT-PCR and karyotype analysis,the detection rate of chromosomal aberrations by using multiplex RT-PCR and karyotyping was 160/373(42.89%) and 179/373(47.98%) respectively,and the combination could optimize the detection rate of clonal genetic abnormalities to 216/373(57.90%).The PCR results from 11 cases 'normal' in karyotyping but abnormal in RT-PCR for MLL rearrangements were confirmed by two-directional sequencing.It is concluded that karyotype studies remain the cornerstone for genetic testing;conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in AML,especially for the cryptic or submicroscopic aberrations.Once a genetic marker has been identified by combined analysis,it could be used to monitor residual disease during/after chemotherapy,by quantitative RT-PCR and/or FISH.

Silencing the enhancer of zeste homologue 2,Ezh2,represses axon regeneration of dorsal root ganglion neurons

Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes.This eventually leads to axonal regeneration of injured neurons.Although some regeneration-related genes have been identified,the regulatory network underlying axon regeneration remains largely unknown.To explore the regulator of axon regeneration,we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion(DRG)neurons at different time points(0,3,6,12 hours,1,3 and 7 days)after rat sciatic nerve crush.The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining.We found 1228 differentially expressed genes in the injured sciatic nerve tissue.The hub genes within these differentially expressed genes include Atf3,Jun,Myc,Ngf,Fgf2,Ezh2,Gfap and Il6.We verified that the expression of the enhancer of zeste homologue 2 gene(Ezh2)was up-regulated in DRG neurons after injury,and this up-regulation differed between large-and small-sized dorsal root ganglion neurons.To investigate whether the up-regulation of Ezh2 impacts axonal regeneration,we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed.In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis,we found some regulators of Ezh2(including Erk,Il6 and Hif1a)and targets(including Atf3,Cdkn1a and Smad1).Our findings suggest that Ezh2,as a nerve regeneration-related gene,participates in the repair of the injured DRG neurons,and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons.All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China(approval No.S20191201-201)on March 21,2019.

Polypeptide Daintain as a New Biomarker for Detecting Breast Tumor

Daintain, a novel bioactive peptide produced and secreted by macrophages, was expressed in breast tumor tissues. The spatial distributions of daintain in 66 breast tumor specimens were investigated with immuno-histochemistry method. Reverse transcription polymerase chain reaction （RT-PCR） and in situ hybridization inspection system were also used to detect daintain in 45 cases of malignant breast tumors. The final results show that 93% high positive responses to daintain on breast cancer tumors. RT-PCR demonstrated that, no smear of daintain transcripted in benign tissues was found, and light smear in peri-cancer tissue was observed. Distribution of daintain was distinguishable among benign tissues, hyperplasia tissues, immature hyperplasia and invasive breast cancer, which can be used to mark the progression of the malignant lesion development. We conclude that the expression of daintain is up-regulated in breast cancers, which indicates that the peptide is closely associated with the disease progression. So daintain could be used as the biomarker for detecting breast cancer.