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.

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.

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.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

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.

Diagnosis of West Nile virus infections:Evaluation of different laboratory methods

BACKGROUND The diagnosis of West Nile virus(WNV)is challenging due to short-term and low-level viremia,flavivirus cross-reactivity,and long immunoglobulin M(IgM)persistence.AIM To evaluate different methods for WNV detection[reverse transcription-polymerase chain reaction(RT-PCR),IgM/IgG antibodies,IgG avidity]in serum,cerebrospinal fluid(CSF),and urine samples of patients with confirmed WNV infection.METHODS The study included patients with confirmed WNV neuroinvasive infection(n=62),asymptomatic WNV seropositive individuals(n=22),and individuals with false-positive WNV IgM antibodies(n=30).WNV RNA was detected using RT-PCR.A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test(VNT).IgG-positive samples were tested for IgG avidity.RESULTS The WNV-RNA detection rates were significantly higher in the urine(54.5%)/serum(46.4%)than in CSF(32.2%).According to the sampling time,the WNV-RNA detection rates in urine collected within 7 days/8-14/≥15 days were 29.4/66.6/62.5%(P=0.042).However,these differences were not observed in the CSF.The median RT-PCR cycle threshold values were significantly lower in urine(32.5,IQR=28-34)than in CSF(34.5,IQR=33-36).The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF.Positive IgM/IgG antibodies were detected in 84.3/9.3%of serum samples collected within 7 days,100/71.1%of samples collected 8-14,and 100%samples collected after≥15 days.Recent WNV infection was confirmed by low/borderline avidity index(AI)in 13.6%of asymptomatic individuals.A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG.No significant correlation between ELISA IgG and VNT was found.CONCLUSION The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples.IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Field evaluation of COVID-19 rapid antigen test:Are rapid antigen tests less reliable among the elderly?

BACKGROUND The global outbreak of coronavirus disease 2019(COVID-19)leads to the development of accessible and cost-effective rapid antigen-detection tests(RATs),as quick and accurate diagnosis is crucial to curb the pandemic.AIM To evaluate the Humasis COVID-19 Ag Test(Humasis Co.,Ltd.,Gyeonggi-do,Republic of Korea)in the diagnosis of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2).METHODS This retrospective study was carried out at the Croatian Institute of Public Health and included patients with clinical symptoms of COVID-19 lasting no longer than 5 d prior to testing,whose nasopharyngeal swabs were primarily tested with RAT.Negative RAT samples underwent confirmatory real-time reverse transcription-polymerase chain reaction(RT-PCR).Diagnostic efficacy was determined compared to RT-PCR.The patients were divided into three age groups(<18,19-65,>65 years).Statistical analysis was performed with the significance level set at P<0.05.RESULTSIn total,2490 symptomatic patients were tested;953 samples were positive on RAT,and 1537 werenegative.All negative RAT samples were subjected to RT-PCR;266 samples were positive andmarked as false-negative results on RAT.The calculated negative predictive value as a measure ofRAT efficacy was 82.69%.The χ^(2) test and Kruskal-Wallis test showed a significant difference in theproportion of false negatives(P<0.001)and RT-PCR cycle(Ct)values for false-negative RATs(P=0.012)among the age groups.The young age group was significantly less likely to be falsenegative,whereas the false negatives from the elderly group experienced significantly lower Ctvalues than the other two age groups.CONCLUSIONEvaluated RAT demonstrated satisfactory performance with more reliable results in youngerpatients.Humasis COVID-19 Ag RAT is potentially a valuable tool in areas where access tomolecular methods is limited;however,RT-PCR remains a gold standard for SARS-CoV-2detection.

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。

Effect of gender on the reliability of COVID-19 rapid antigen test among elderly

Defining con-founders that affect the reliability of diagnostic tests for coronavirus disease 2019 is vital to breaking the chain of infection.The elderly population is a higher risk group for the emerging virus.However,gender seems to exert a critical role in modifying the infection risk among women owing to hormonal changes.The menopause transition is an exceptional period for older women where the protective and immunomodulatory effects of the estrogen hormone are lost.Accordingly,attention should be given to postmenopausal women since they will have an increased risk compared to their pre-menopausal peers.

Role of microRNAs in gastric cancer

Although gastric cancer(GC)is one of the leading causes of cancer-related death,major therapeutic advances have not been made,and patients with GC still face poor outcomes.The prognosis of GC also remains poor because the molecular mechanisms of GC progression are incompletely understood.MicroRNAs(miRNAs)are noncoding RNAs that are associated with gastric carcinogenesis.Studies investigating the regulation of gene expression by miRNAs have made considerable progress in recent years,and abnormalities in miRNA expression have been shown to be associated with the occurrence and progression of GC.miRNAs contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors,affecting cell proliferation,apoptosis,motility,and invasion.Moreover,a number of miRNAs have been shown to be associated with tumor type,tumor stage,and patient survival and therefore may be developed as novel diagnostic or prognostic markers.In this review,we discuss the involvement of miRNAs in GC and the mechanisms through which they regulate gene expression and biological functions.Then,we review recent research on the involvement of miRNAs in GC prognosis,their potential use in chemotherapy,and their effects on Helicobacter pylori infections in GC.A greater understanding of the roles of miRNAs in gastric carcinogenesis could provide insights into the mechanisms of tumor development and could help to identify novel therapeutic targets.

Increased CD163 expression is associated with acute-on-chronic hepatitis B liver failure

AIM: To assess CD163 expression in plasma and peripheral blood and analyze its association with disease in acute-on-chronic hepatitis B liver failure (ACHBLF) patients. METHODS: A retrospective study was conducted from January 1, 2011 to January 1, 2012. Forty patients with ACHBLF (mean age 44.48 ± 12.28 years, range 18-69 years), 40 patients with chronic hepatitis B (CHB) (mean age 39.45 ± 12.22 years, range 21-57 years) and 20 ageand sex-matched healthy controls (mean age 38.35 ± 11.97 years, range 28-60 years) were included in this study. Flow cytometry was used to analyze the frequency of CD163+ peripheral blood mononuclear cells (PBMCs) and surface protein expression of CD163. Real-time transcription-polymerase chain re-action was performed to assess relative CD163 mRNA levels in PBMCs. Plasma soluble CD163 (sCD163) levels were measured by enzyme-linked immunosorbent assay. Clinical variables were also recorded. Comparisons between groups were analyzed by Kruskal-Wallis H test and Mann-Whitney U test. Statistical analyses were performed using SPSS 15.0 software and a P value < 0.05 was considered statistically significant. RESULTS: Flow cytometry showed that the population of CD163+ PBMCs was significantly greater in ACHBLF patients than in CHB patients and healthy controls (47.9645% ± 17.1542%, 32.0975% ± 11.0215% vs 17.9460% ± 6.3618%, P < 0.0001). However, there were no significant differences in mean fluorescence intensity of CD163+ PBMCs within the three groups (27.4975 ± 11.3731, 25.8140 ± 10.0649 vs 20.5050 ± 6.2437, P = 0.0514). CD163 mRNA expression in ACHBLF patients was significantly increased compared with CHB patients and healthy controls (1.41 × 10 -2 ± 2.18 × 10 -2 , 5.10 × 10 -3 ± 3.61 × 10 -3 vs 37.0 × 10 -4 ± 3.55 × 10 -4 , P = 0.02). Plasma sCD163 levels in patients with ACHBLF were significantly increased compared with CHB patients and healthy controls (4706.2175 ± 1681.1096 ng/mL, 1089.7160 ± 736.8395 ng/mL vs 435.9562 ± 440.8329 ng/mL, P < 0.0001). In ACHBLF patients, plasma sCD163 levels were significantly positively associated with model for end-stage liver disease scores (r = 0.5075, P = 0.008), hepatitis B virus-DNA (r = 0.6827, P < 0.0001), and negatively associated with prothrombin activity (r = -0.3348, P = 0.0347), but had no correlation with total bilirubin (r = 0.2551, P = 0.1122). Furthermore, sCD163 was obviously elevated in non-surviving patients compared with surviving patients with ACHBLF (5344.9080 ± 1589.5199 ng/mL vs 3641.7333 ± 1264.5228 ng/mL, P = 0.0321). CONCLUSION: CD163 and sCD163 may be related to disease severity and prognosis in ACHBLF patients.

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.

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Circulating hTERT mRNA as a tumor marker in cholangiocarcinoma patients

AIM： To investigate human telomerase reverse transcriptase （hTERT） mRNA in the serum of cholangiocarcinoma patients. METHODS： The serum of thirty three cholangiocarcinoma patients, forty one benign biliary tract disease patients and ten healthy volunteers were collected and analyzed for the expression of hTERT mRNA by real-time reverse transcriptase-polymerase chain reaction （RT-PCR）. We then examined the correlation between values of serum hTERT mRNA and the pathological staging of cholangiocarcinoma. RESULTS： hTERT mRNA was detected in 28 of 33 （84.85%） of serum obtained from cholangiocarcinoma patients and 9 of 41 （21.9%） of serum obtained from benign biliary tract disease patients, hTERT mRNA was not detected in any serum obtained from healthy volunteers, on the other hand the common tumor marker, CA19-9 was detected in 20 of 33 （60.6%） of serum obtained from cholangiocarcinoma patients and 8 of 41 （19.5%） of serum obtained from benign biliary tract disease patients. However, no correlation was found between the present of serum hTERT mRNA and tumor staging. CONCLUSION： These results indicate that the detection of circulating hTERT mRNA was identified in almost all cholangiocarcinoma patients. It offers a novel tumor marker, which can be used as a complementary study for diagnosis of cholangiocarcinoma .

Analysis of Survivin Expression in the Subtypes of Lymphoma

Objective： To detect the expression of survivin in subtypes of lymphoma and its value in classifying subtypes of lymphoma. Methods： Paraffin-embedded samples collected from 219 cases of lymphoma and 13 cases of lymph node reactive proliferation from affiliated hospitals of Sun Yat-sen University during 2001-2003 were examined for the expression of survivin by using immunohistochemical staining. Reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the mRNA expression of survivin in K562, HL60, Raji, and Jurkat cell lines. Semi-quantitative assay was used to evaluate the quantity of survivin protein and mRNA expression in subtypes of lymphoma. Results： Protein expression of survivin was high and strong in diffuse large B-cell lymphoma （DLBL） （88.6%, 70/79）, Burkitt lymphoma （BL） （100%, 2/2）, and lymphoblastic lymphoma （LBL） （92.3%, 12/13）, while their expression was always lower and weaker in follicular lymphoma （FL） （18.2%）, extranodal marginal zone B-cell lymphoma of mucosaassociated lymphoid tissue （MALT lymphoma） （40.9%） and marginal zone lymphoma （MZL） （33.3%）. There was a significant difference between the higher expression group （DLBL, BL and LBL） and lower one （FL, MZL, and MALT） in the expression of survivin （Chi-square test, X^2=24.77, P〈0.01）. Almost all of Reed-Sternberg cells （R-S cells） in Hodgkin lymphoma （HL） strongly expressed survivin. The protein expression of survivin was positively correlated with mRNA （r=0.6270, P〈0.01）. Conclusion： The expression level of survivin mRNA and protein shows significant difference in subtypes of lymphoma. The expression of survivin mRNA might act as a biomarker to classify the subtypes of lymphoma.

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.

Identification of Sheep Endogenous Beta-Retroviruses with Uterus-Specific Expression in the Pregnant Mongolian Ewe

The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses （enJSRVs）, and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morphogenesis. This study was aimed to assess the expression of mRNAs of an enJSRV and its receptor, HYAL2, in the uterus and conceptuses of Mongolian ewes throughout gestation, using real-time reverse transcription polymerase chain reaction and in situ hybridization analysis. The results showed that enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium, chorion, placenta, and conceptus. The enJSRV mRNA was most abundant in the placenta on day 90 of pregnancy, in the endometrium on day 30 and 50, and in the chorion on day 70 and 110. However, HYAL2 mRNA was most abundant in the endometrium on day 30. These differences were all significantly different from each other （P〈0.01）. In situ hybridization showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, trophoblastic giant binucleated cells （BNCs）, endometrial caruncles, placental cotyledons, stroma, trophectoderm, as well as multinucleated syncytia of the placenta and blood vessel endothelial cells. Collectively, little is known about the molecular mechanisms by which trophoblastic differentiation and multinucleated syncytia formation are regulated by enJSRVs. However, the temporal and spatial distributions of enJSRV expression in the uterus and conceptus indicate that differentiation of BNCs and the formation of a multinucleated syncytiotrophoblast involve enJSRV and possibly its cellular receptor, HYAL2. Therefore, enJSRV and HYAL2 appear to play important roles in the female reproductive physiology in this breed of sheep.

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.

Insulin-like growth factor receptor-1 overexpression is associated with poor response of rectal cancers to radiotherapy

AIM: To explore the potential correlation between insulin-like growth factor receptor-1 (IGF-1R) expression and rectal cancer radiosensitivity.