Establishment and evaluation of a LAMP-CRISPR-based nucleic acid detection method for Schistosoma japonicum / 中国热带医学

@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

Epidemiology of Sports-Related Sudden Death in Guangdong Province / 法医学杂志

OBJECTIVES@#By retrospective study of the epidemiological characteristics of sports-related sudden death (SrSD), the risk factors associated with SrSD were analyzed and explored to provide a scientific basis for comprehensive prevention and treatment of SrSD.@*METHODS@#The personal information (sex, age, occupation, etc.), case information (time, place, type of sports, relative time between SrSD occurrence and exercise, etc.), death related information (sign or prodrome, medical history and surgical history, etc.), rescue situation (witnesses, on-site assistance, the availability of paramedics, etc.) of 374 SrSD cases in Guangdong Province from 2017 to 2021 were collected. Statistical analysis was conducted aiming at the key factors.@*RESULTS@#In the 374 cases, there were significantly more males than females (19.78:1); the number of people aged between >39 and 59 was the largest (151, 40.37%); non-manual workers (68.98%) were more than manual workers; the top three sports with the highest number cases were basketball (34.49%), running (19.52%) and badminton (12.03%); from 3 pm to 9 pm (63.10%) was the time period with the highest incidence of events; sudden death mainly occurred during exercise (75.27%) and within 1 h after exercise (20.05%); the on-site rescue rate was very low (6.15%); the rate of autopsies was extremely low (1.07%); sudden cardiac death was the most common cause (67.11%).@*CONCLUSIONS@#SrSD is most common in males aged >39 to 59 years old, mostly in non-manual workers, and usually occurs in basketball and running. Sudden death is more likely to occur during exercise and within 1 h after exercise. Therefore, the above potential risk factors should be focused on and studied in daily comprehensive prevention and treatment to provide scientific basis for accurate prevention and first aid of such sudden death.

Risk of obesity-related gene polymorphism on the incidence and durative of childhood obesity / 中华流行病学杂志

Objective To examine the impact of single nucleotide polymorphisms (SNPs) in obesity-related genes on the incidence and durative of obesity in childhood and adolescence.Methods Based on the Beijing Child and Adolescent Metabolic Syndrome (BCAMS) Study,780 school children aged 6 to 16 years were followed-up in 2010,and assessed for body size parameters.Venipuncture blood samples were collected after a 12-hour overnight fast.Genomic DNA was isolated from peripheral blood white cells under the salt fractionation method.SNPs were genotyped by ABI 5700 real time PCR (FTO rs9939609) and TaqMan Allelic Discrimination Assays with the GeneAmp 7900 Sequence Detection System (Applied Biosystems,Foster City,CA,USA) (FTO rs6499640,FAIM2 rs7138803,NPC1 rs1805081,MC4R rs17782313,BDNF rs6265,GNPDA2 rs10938397).Both overweight and obesity were diagnosed by the Chinese age-and sex-specific body mass index (BMI)cutoffs.Two independent sample t-test,Chi-square test and multiple logistic regression analysis were performed.Results During the 6 years follow-up period,the incidence of obesity in the total sample 8.5％,and 65.1％ individuals had persisted their obese status.The genotypes of the SNPs except BDNF rs6265 were in Hardy-Weinberg equilibrium in each group (P＞0.05).The incidence rates of obesity increased with FTO rs9939609 TT,TA and AA genotypes (x2 for trend=8.030,P＜0.05).In the non-obese sub-cohort,after adjusted for sex,age at the initial time of follow up and residential area,when compared with children carrying FTO rs9939609 T-allele,a significantly relative risk of obesity was observed for children carrying the rs9939609 A-allele (OR=2.42,95％CI:1.31-4.47,P=0.005).In the obese sub-cohort,FTO rs9939609 A-allele was significantly associated with durative of obesity (OR=1.72,95％CI:1.07-2.77,P=0.026).However,no statistical significant associations were seen between other SNPs (FTO rs6499640,FAIM2 rs7138803,NPCI rs1805081,MC4R rs17782313,GNPDA2 rs10938397) and the incidence or durative of obesity (all P＞0.05).The genetic risk scorewas associated with the risk of occurrence of obesity (OR=16.42,95％ CI:3.59-75.10,P＜0.001)after adjusted for residential area,sex,age at the initial time of follow up and baseline BMI.Conclusion We confirmed the association of FTO rs9939609 with incidence and durative of obesityin children.Early intervention was recommended on the high risk individuals who carrying more riskalleles in obesity-related genes.

Assessment of right ventricular function for patients with rheumatic mitral stenosis by 64-slice multi-detector row computed tomography: comparison with magnetic resonance imaging / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Right ventricular (RV) dysfunction ensues due to rheumatic mitral stenosis (RMS). The evaluation of RV function is clinically important for the diagnosis, treatment, and follow-up for patients with different degrees of RMS. The purpose of this study was to determine whether the 64-slice multi-detector row computed tomography (64-slice MDCT) can assess the RV function in RMS with high accuracy and reproducibility when compared to MR imaging (MRI).</p><p><b>METHODS</b>Right ventricular end-diastolic and end-systolic volumes (RV-EDV and RV-ESV), stroke volume (RV-SV), ejection fraction (RV-EF), cardiac output (RV-CO), and wall mass (RV-Mass) were measured with dedicated cardiac analysis software on 64-slice MDCT and compared with values measured with MRI in 43 consecutive patients with RMS. Agreement between MRI and 64-MDCT results were compared with Bland and Altman analysis and linear regression analysis. Repeated measurements were performed to determine intraobserver and interobserver variability.</p><p><b>RESULTS</b>No significant differences were revealed in calculated RV function parameters between the two methods. RV-EDV, RV-ESV, RV-SV, RV-EF, RV-CO, and RV-Mass by 64-slice MDCT were similar to those by MRI (P > 0.05). There were good correlations (r = 0.98, 0.97, 0.96, 0.96, 0.95 and 0.77, respectively) and close agreement (bias = -0.2 ml, -1.0 ml, 0.8 ml, 0.5%, 26.1 ml, and 0.5 g, respectively, P > 0.05). The variability in 64-slice MDCT measurements was similar to that in MRI values.</p><p><b>CONCLUSION</b>ECG-gated 64-slice MDCT could assess the RV function in RMS with high accuracy and reproducibility when compared to MRI.</p>

Applicability of the multiplex quantitative antibody array system for early diagnosis of hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To develop an early and accurate detection method for hepatocellular carcinoma (HCC) based on detection of tumor-associated serum markers using a multiplex quantitative antibody array.</p><p><b>METHODS</b>The double-antibody sandwich principle was used to establish an antibody array composed of eight cancer-related serum markers, including alpha-fetoprotein (AFP), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-b1), and vascular endothelial growth factor (VEGF). Serum samples from 160 cases of clinically diagnosed HCC and from 58 cases of liver cirrhosis (LC; controls) were obtained to test the array. Sixty percent of the samples were randomly selected for use as the training set (HCC, n = 96; LC, n = 36), and the remaining 40% was used as the test set (HCC, n = 64; LC, n = 22). The SPSS statistical software was used to perform logistic regression analysis and to create a diagnostic model.</p><p><b>RESULTS</b>When used with the training set, the model had sensitivity of 93.3%, specificity of 83.3%, and accuracy of 90.9%. When used with the test set, the model had sensitivity of 89.0%, specificity of 77.3%, and accuracy of 86.0%. The traditional serum AFP value (cut-off value of 20 ng/mL) showed 70.0% diagnostic sensitivity, 59.0% specificity, and 64.0% accuracy.</p><p><b>CONCLUSION</b>The newly developed multiplex quantitative antibody detection system has high sensitivity and specificity. The diagnostic model with AFP and seven other cancer-related factors was superior to the traditional AFP only approach for early diagnosis of liver cancer, indicating its potential clinical value.</p>

The effects of stathmin on cell proliferation and tumor-related genes expressions in HCCLM3 cells / 中华肝脏病杂志

To explore the biological function and possible underlying mechanism of stathmin gene during hepatocarcinogenesis. Three pairs of chemically synthesized small interfering RNA (siRNA) targeting on stathmin were transfected into HCCLM3 by LipofectamineTM 2000. After confirming the interfering effects of stathmin siRNAs through reverse transcription PCR and Western blotting, the HCCLM3 cells proliferation and apoptosis were detected by cell count kit-8 (CCK-8) and flow cytometry analysis, and the expressions of tumor-related genes (c-myc, c-fos, p53, etc) were observed by real-time PCR. Stathmin expression was effectively inhibited up to 90% by stathmin silencing in HCCLM3 cells (P is less than to 0.05) . By using CCK8 assay, it was shown that HCCLM3 cells proliferation were obviously depressed by 13.04%+/-0.10%, 28.10%+/-0.41% and 37.36%+/-2.15% at the time point of 24 h, 48 h and 72 h with the comparison to Mock group (F = 4.21, P is less than to 0.05). The results of flow cytometry demonstrated that the percentage of apoptotic cells was increased to 25.11%+/-1.62% in RNAi group, compared with 9.20 %+/-0.64 % in Mock group (F = 44.67, P is less than to 0.01). The results of real-time PCR showed that oncogenes c-myc and c-fos expressions were repressed, proliferation-associated gene ki-67 was down-regulated, and apoptosis-promoting gene caspase-3, bax and p53 were induced (P is less than to 0.05). Stathmin may promote cell proliferation, inhibit cell apoptosis and induce malignant transformation of hepatocytes by regulating some tumor-related genes expressions.

Association between obesity in childhood and hypertension incidence: a prospective cohort study / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the impact of obesity level and the level change in childhood on hypertension incidence.</p><p><b>METHODS</b>A perspective cohort study was conducted. As part of Beijing Child and Adolescent Metabolic Syndrome Study, 2189 aged 6 - 16 year non-hypertensive children was followed up in December, 2010. In this study, height, weight, waist circumference (WC) and blood pressure was measured at follow-up, and body mass index (BMI) and WC was respectively used to assess overweight, obesity and abdominal obesity. Non-conditional logistic regression was used to evaluate the association between baseline obesity status, change of obesity status and hypertension incidence. OR and 95%CI were computed in the model using obese status as dummy variable and hypertension at follow up visit as dependent variable.</p><p><b>RESULTS</b>The total hypertension incidence of 1184 subjects during 6 years follow-up was 19.9% (236/1184). The hypertension incidence in male (23.2%, 149/643) was higher than that in female (16.1%, 87/541) (χ(2) = 9.257, P = 0.002). The hypertension incidence of non-overweight, overweight and obese children at baseline was 8.7% (45/519), 19.3% (35/181) and 32.4% (156/484) respectively (χ(2) = 9.332, P < 0.001), and the incidence of non-obese and abdominal obese children at baseline was respectively 10.3% (63/613) and 30.7% (173/567) (χ(2) = 77.753, P < 0.001). Hypertension incidence in the baseline obesity group was higher than the non-overweight (BMI: OR = 4.9, 95%CI: 3.4 - 7.0) and non-obese group (WC: OR = 3.9, 95%CI: 2.8 - 5.3). The hypertension incidence increased with the follow-up BMI/WC level, based on the same baseline level of BMI and WC. The hypertension risk increased to 0.21 and 0.07 times respectively with elevation of baseline BMI level by 1 kg/m(2) and WC level by 1 cm, and OR (95%CI) were 1.21 (1.16 - 1.26) and 1.07 (1.05 - 1.09), respectively. Similarly, the hypertension risk increased 0.16 and 0.05 times respectively with the elevation of BMI level change by 1 kg/m(2) and 1 cm, and OR (95%CI) were 1.16 (1.11 - 1.22) and 1.05 (1.03 - 1.07), respectively.</p><p><b>CONCLUSION</b>Obesity and increased obesity level change in childhood can increase the risk of incident hypertension.</p>

Relationship between CD44 expression or glycosylation and hepatocellular carcinoma metastasis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the relationship of CD44 expression or glycosylation and hepatocellular carcinoma (HCC) metastasis.</p><p><b>METHODS</b>IHC, Quantum dots detection, RT-PCR, Western blot, Cellular immune fluorescence and MS-PCR were used to identify CD44 expression in HCC samples and a series of human HCC cell lines with different metastatic potentials. Lectin array was used to reveal the relationship of CD44v6 glycosylation and human HCC metastasis.</p><p><b>RESULTS</b>Immunohistochemistry analysis showed that CD44v6 was mainly distributed on the cell membrane, while CD44S immunoreactivity was prominently in the cytoplasm, CD44v3 and CD44v4/5 were in cytoplasm on membrane. Among CD44S and those CD44 variants, only the expression of CD44v6 was higher in metastasis HCC samples as compared to that in non-metastasis group (x²=8.828, P less than 0.05). This result was also re-confirmed by the result of Quantum dots (t = 2.392, P < 0.05) and serum detection (t = 2.56, P < 0.05). We found completely methylation of CD44v6 gene in Hep3B and incomplete methylation in MHCC97H and MHCC97L cell lines with metastatic potentials. The lectin affinity assay indicated that lectin MAL, SNA and WGA showed more affinity to MHCC97H and MHCC97Lcell lines than that of the non-metastatic Hep3B cell lines.</p><p><b>CONCLUSIONS</b>CD44v6 over-expression presents a positive correlation with HCC metastatic potential, which may be associated with DNA methylation level in promoter sequence. The increasing sialic acid modified glycan of CD44v6 might be related to HCC metastatic ability.</p>

Identification of metastasis-related osteopontin expression and glycosylation in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To test expression level and glycosylation level of OPN in HCC cell lines with different metastatic potential and HCC tissues, and investigate the correlation between the glycosylation change and the liver cancer transporting as well as its significance.</p><p><b>METHODS</b>The level of OPN expression in liver cancer tissue(6 cases of non-metastasis and 7 cases of metastasis)as well as HCC cell lines with different metastatic potential (L02, Hep3B, MHCC97L, MHCC97H, HCCLM3, HCCLM6)was identified by immunohistochemistry and Western Blot, and then OPN was purified from HCC tissues by immunoprecipitation, followed by glycosylation detection of OPN from non-metastatic and metastatic HCC tissues by multiple lectin blot. Data were analyzed by t-test and variance analysis.</p><p><b>RESULTS</b>Different levels of OPN expression were observed in HCC cell lines with different metastatic potential (F = 5.04, P = 0.008). Additionally, OPN expression level in HCC tissues with metastasis was higher than that in non-metastasis group (t = 2.447, P < 0.05). Relative optical density value was 0.69 ± 0.21 and 0.45 ± 0.14 respectively. OPN in liver cancer tissue was successfully purified using immunoprecipitation. Followed lectin blotting result showed that OPN protein in metastasis group showed lower affinity to MAL, PHAE, DSA, ConA as compared with that in non-metastasis group (P < 0.05).</p><p><b>CONCLUSIONS</b>The expression of OPN was positively correlated with the enhanced metastasis potential of HCC. OPN from metastasis HCC tissues presented lower level of some specific glycan structures such as a2, 3- sialic acid, bisecting GlcNAc, biantennary, muti-antennary and high mannose type N-glycan structure. This study not only indicates the role of OPN in HCC metastasis for the first time, but also provide experimental support for the mechanism of the function of OPN in the transportation of liver cancer cells as well as offer potential target for clinical treatment.</p>

Effects of AKR1B10 gene silence on the growth and gene expression of HCC cell line MHCC97H / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the biological function and possible underlying mechanism of aldo-keto reductase family 1 member B10 (AKR1B10) gene during hepatocarcinogenesis.</p><p><b>METHODS</b>A pair of chemically synthesized small interfering RNA (siRNA) targeting on AKR1B10 was transfected into liver cancer cell line MHCC97H by LipofectamineTM 2000. After confirming the interfering effects of AKR1B10-siRNAs through Quant SYBR Green polymerase chain reaction (Real-time PCR), Western blot and enzymatic activity assay, the capabilities of proliferation and apoptosis of the transfected cells were observed by CCK-8 assay and flow cytometry analysis, and the expressions of a group of tumor-related gene such as c-myc, c-fos, N-ras were observed through Real-time PCR.</p><p><b>RESULTS</b>The expressions of AKR1B10 and the enzymatic activity were down-regulated significantly in AKR1B10-siRNA-transfected cells. Compared with mock and blank control groups, cell growth in AKR1B10-siRNA-transfected group was inhibited by 26.6%+/-3.1% at 72h after transfection. The ratio of apoptotic cells was 37.3%+/-1.0% in AKR1B10-siRNA-transfected group, which was significantly higher than that in mock and blank control groups (P < 0.01). Real-time PCR showed that the expressions of oncogene c-myc, c-fos and N-ras, and the proliferation-associated gene ki-67 were down-regulated in AKR1B10-siRNA-transfected cells, while the expressions of apoptosis-promoting gene caspas-3 and bax were up-regulated.</p><p><b>CONCLUSIONS</b>AKR1B10 might promote proliferation, inhibit apoptosis and then induce malignant transformation of hepatocytes by regulating the expression level of some tumor-related genes.</p>

Protein profiles of multinodular hepatocellular carcinoma with multicentric occurrence or with intrahepatic metastasis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the protein expression profiles of multinodular hepatocellular carcinoma (HCC) with multicentric occurrence (MO) or with intrahepatic metastasis (IM).</p><p><b>METHODS</b>5 IM and 6 MO patients were divided into groups of IM1, IM2, MO1 and MO2 according to the size of node of HCC. Two dimensional gel electrophoresis (2-DE) and mass spectrum were used to analyze the protein expression profiles. Western blot was used to confirm the results obtained by mass spectrum.</p><p><b>RESULTS</b>2-DE of IM1, IM2, MO1 and MO2 indicated that 30 protein dots were differentially expressed in these tumors. By mass spectrum, 25 proteins were identified. Gene ontology classification indicated that these proteins are associated to cell movement, signal transduction, oxidoreduction, lipid metabolism, and amino acid metabolism.</p><p><b>CONCLUSION</b>The protein expression profiles of IM is different from that of MO, 2-DE and mass spectrum can be used to identify the molecular markers of IM and MO of HCC.</p>

Differentially expressed proteins in the precancerous stage of rat hepatocarcinogenesis induced by diethylnitrosamine / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the differentially expressed proteins especially at the precancerous stage of diethylnitrosamine (DEN) induced hepatocarcinogenesis by comparative proteome research.</p><p><b>METHODS</b>Rats were divided into normal and DEN groups and sacrificed periodically. The liver samples were stained with gamma-glutamyl transpeptidase (GGT) and HE to distinguish the preneoplastic lesion (pre-HCC) from the normal and HCC tissues. The two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) were then applied to analyze the differentially expressed protein between pre-HCC and normal tissues, pre-HCC and HCC, as well as HCC and normal tissues. A few of the candidate proteins such as laminin receptor 1 (67LR) and agmatinase were validated by Western blot and RT-PCR.</p><p><b>RESULTS</b>Totally, there were 82 proteins that differentially expressed two fold or more in one kind of tissues sample than the other, 47 of which occurred in the pre-HCC tissues. Eight proteins including 67LR were consistently up-regulated from normal tissue to pre-HCC and then to HCC tissues, while 22 proteins including agmatinase showed progressively down-regulated in these tissues samples.</p><p><b>CONCLUSION</b>The protein expression profiles are different during the process of hepatocarcinogenesis. Further study on the differentially expressed protein, especially these upregulated in the precancerous stage such as 67LR and agmatinase, might contribute to prevention and early diagnosis of human HCC.</p>

Comparison of the serum proteomes of pathological stages during hepatocarcinogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To compare the 2-DE profiles for serum proteins of different pathological stages during hepatocarcinogenesis.</p><p><b>METHODS</b>Sera from hepatocellular carcinoma patients, cirrhosis patients, chronic hepatitis patients and healthy controls were collected. After sonication, albumin and immunoglobulin (IgG) depletion, and desalination, sera were subjected to 2-DE, the differential protein spots were identified by MALDI-TOF-MS. Western blot was used to validate these differentially expressed proteins.</p><p><b>RESULTS</b>2-DE sera protein profiles were obtained from the patient suffering from HCC, liver cirrhosis, chronic hepatitis, healthy controls in each group. From optimized 2-DE gel images of the above groups, 96 protein spots with more than 2-fold difference in intensity between the two groups were selected by image master 6.0 software, differential proteins including haptoglobin, SAA1 and SP40 were identified by MALDI-TOF-MS/MS. 7 different spots within more than 30 protein spots belonged to the same haptoglobin family. The differential expression of haptoglobin was confirmed by western blot.</p><p><b>CONCLUSIONS</b>Four protein expression patterns have been identified during the pathological stages of hepatocarcinogenesis. Haptoglobin is significantly increased from liver cirrhosis to HCC. It implies that haptoglobin might be a potential biomarker in the early diagnosis of liver cancer.</p>

Analysis of the expression of Slit/Robo genes and the methylation status of their promoters in the hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the expression of genes in the Slit/Robo signaling pathway, and the methylation status of their promoters in hepatocellular carcinoma (HCC) cell lines.</p><p><b>METHODS</b>Genomic DNA and total RNA were isolated from 9 HCC cell lines of different metastatic ability (Hep3B, HepG2, PLC/PRF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, LM3, LM6) and a control cell line L-02. The expression profiles of Slit1, Slit2, Slit3, Robo1, and Robo3 were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the promoters was detected by methylation specific polymerase chain reaction (MSP).</p><p><b>RESULTS</b>The promoters of Slit1, Slit2 and Slit3 genes were almost methylated in all the HCC cell lines. The Slit1 and Slit3 RNAs were not detected in most of the cell lines. Furthermore, the mRNA Slit2 was decreased gradually as the metastatic potential of the cell lines increased. As the candidate ligand of the Slit2 gene, Robo1 was frequently methylated in HCC cell lines whereas its mRNA was detected in all of these cells except SMMC-7721, BEL-7402 and L-02. Robo3 was unmethylated in HCC cell lines while its mRNA was not detected in these HCC cell lines.</p><p><b>CONCLUSION</b>The hypermethylation status of Slit/Robo signaling pathway related genes is a universal event in the HCC. The hypermethylation status of Slit1, Slit2, Slit3 genes associated with the loss of expression or reduced expression. Those data suggest that Slit/Robo pathway may play a significant role in the progress or metastasis of HCC.</p>

Interferon-alpha upregulates thymidine phosphorylase expression via JAK-STAT transcriptional activation and mRNA stabilization in human hepatocellular carcinoma SMMC-7721 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To examine how the thymidine phosphorylase (TP) gene expression is upregulated by interferon-alpha (IFN-alpha) in human hepatocellular carcinoma SMMC-7721 cells.</p><p><b>METHODS</b>TP mRNA levels were determined by RT-PCR. Whether the JAK-STAT cascade mediates IFN-alpha-induced TP mRNA expression was studied by pretreatment with Janus Kinase (JAK) inhibitor, AG-490. Effects of IFN-alpha on TP mRNA stability were detected with additional actinomycin D.</p><p><b>RESULTS</b>The expression of TP mRNA was induced by IFN-alpha in a dose- and time-dependent manner in SMMC-7721 (human hepatocellular carcinoma) cells. TP mRNA levels rose at 8 h, reached the peak value at 12 h, and remained at a high level up to 72 h in SMMC-7721 cells treated with IFN-alpha 10000 U/ml. IFN-alpha at a dose of 5000 or 10000 U/ml up-regulated TP expression about 3 fold compared with that of non-treated cells (P < 0.05). Induction of TP mRNA expression by IFN-alpha was significantly inhibited in SMMC-7721 cells by pretreatment with AG-490, in comparison with that treated with IFN-alpha alone. Pretreatment of SMMC-7721 cells with IFN-alpha 10000 U/ml for 24 h caused a substantial stabilization of TP mRNA, with a half-live of 35.8 h, compared with 8.5 hr in the control SMMC-7721 cells.</p><p><b>CONCLUSION</b>IFN-alpha at certain doses upregulates TP mRNA expression via both JAK-STAT transcriptional activation and post-transcriptional mRNA stabilization in human hepatocellular carcinoma SMMC-7721 cells.</p>

The biological function of peroxiredoxin II on Hep3B cells and its underlying mechanism / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B.</p><p><b>METHODS</b>Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell.</p><p><b>RESULTS</b>The two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05).</p><p><b>CONCLUSION</b>PrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.</p>

Analysis of gene expression profile during malignant transformation of rat liver oval-like cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the changes of gene expression profile during malignant transformation of rat liver oval-like cells and to analyze the significances of these changes.</p><p><b>METHODS</b>MNNG initiated WB-F344 cells were exposed to H2O2 once a week (repeated 21 times) to induce their malignant transformation. The characteristics of the transformed cells were confirmed by morphology, genetics and soft agar assay. Then, gene expression profiles of the transformed cells at different time points were evaluated using rat Cancer PathwayFinder Oligo Microarray.</p><p><b>RESULTS</b>The transformed cells possessed heteroploid karyotypes with anchoring-independent growth characteristics. Transmission electron microscopy showed that the transformed cells had more cellular organelles, gap junctions and microvilli than the controls. The 21 differential expression genes were mainly involved in regulating cell proliferation, apoptosis, adhesion and motility. The expression of PTEN was gradually up-regulated and cdkn1a was gradually down-regulated in three groups of cells. Myc, fos, and casp-8 were first up-regulated in WB-5 cells and then down-regulated in WB-21 cells.</p><p><b>CONCLUSION</b>The disorder of proliferation and apoptosis may play an important role in malignant transformation of WB cells. Cdkn1a, PTEN, myc and fos may be crucial factors involved in the process.</p>

Screening for differential methylation status by CpG island microarray in the hepatocellular carcinoma cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To profile the methylation alterations of CpG islands in hetpatocellular carcinoma cell lines.</p><p><b>METHODS</b>A global analysis of DNA methylation using the Human CpG-island 12K Array (HCGI12K) from Canada University Health Network was performed on nine human hepatocellular carcinoma (HCC) cell lines (Hep3B, HepG2, PLC/RPF/5/RPF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, HCCLM3, HCCLM6) and a control cell line Chang's liver. Metastatic potential related alterations were also screened in MHCC97 series cell lines (MHCC97-H, MHCC97-L, HCCLM3, HCCLM6), using MHCC97-L, a cell line with low metastatic potential, as control. To screen the key genes which are hypermethylation or hypomethylation in the HCC cell lines compared with the normal liver cell line by normalization processing and cluster analysis of microarray data. Two randomly selected genes was analyzed by methylation specific PCR to verify the chip results.</p><p><b>RESULTS</b>By a standard of methylation alteration ratio > or = 2 or < or = 0.5, fifty-eight CpG island cloning sites and sixty-six upstream or downstream tumor-related genes were identified. The genes were oncogenes, tumour suppressor genes and their ligand genes, apoptosis-related genes, cell proliferation and differentiation genes, cell cycle-related gene and cell signaling pathway key genes such as Wnt, ras, and FGF pathway-related genes. The methylation specific PCR results were consistent with those obtained by chips.</p><p><b>CONCLUSION</b>The results of this study demonstrate that there are a series of CpG island methylation alterations in HCC cell lines. The expression of many oncogenes, tumor suppressor genes and other key genes may be up- or down-regulated, respectively, because of their CpG island hypomethylation or hypermethylation accordingly. It may provide a basis for screening HCC biological markers by CpG island methylation profilling.</p>

Application of SELDI-TOF-MS in detection of liver metastasis from colorectal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To establish a serum protein fingerprint model for prediction of liver metastasis from colorectal cancer by SELDI-TOF-MS analysis, and to determine the differentiatial proteins associated with the metastatic liver cancers.</p><p><b>METHODS</b>Data were collected from the Department of General Surgery in Zhongshan Hospital. A group of patients with colorectal cancer (CRC) without liver metastasis (n = 36) and another group with liver metastasis (n = 36) were included in this study. Serum samples were collected from peripheral venous blood before operation. Special serum protein or peptide fingerprint was determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The obtained data were analyzed by Biomarker Wizard software to screen the serum protein markers discriminating colorectal cancer patients with and without liver metastasis. A serum protein fingerprint model was established. This model was blindly verified in of CRC patients with and 44 cases without liver metastasis.</p><p><b>RESULTS</b>Comparing the characteristic proteins in those two groups of patients, 10 specific protein peaks were identified with statistical significance (P < 0.05). According to m/z growing from small to large, they were: 2398, 2814, 4084, 4289, 4465, 6422, 6619, 11 482, 11 649 and 13 714. The predictive model had a sensitivity of 91.7% and a specificity of 97.2%. The validation showed a sensitivity of 75.0% and a specificity of 81.8%.</p><p><b>CONCLUSION</b>A predictive model based on differentiatial serum protein fingerprint with high sensitivity and specificity has been successfully established. It should be a very useful tool in detection and diagnosis of liver metastasis in colorectal cancer patients.</p>

Establish predictive model of colorectal cancer by using surface enhanced laser desorption/ionization-time of flight-mass spectrometry / 中华外科杂志

<p><b>OBJECTIVE</b>To establish serum proteome fingerprinting predictive models and search for proteins associated with colorectal cancer.</p><p><b>METHODS</b>Thirty-six randomly selected colorectal cancer patients and 36 cases with hernia or gall bladder diseases scheduled for elective operation were enrolled as cancer group and control group respectively. Peripheral venous blood samples were collected before the operations. Special serum protein or peptide fingerprint was investigated by using surface enhanced laser desorption/ ionization-time of flight-mass spectrometry (SELDI-TOF-MS) measurement after blood sample had been treated with weak cation exchange protein chip (CM10) for each case. The obtained data were analyzed by Biomarker Wizard software to screen serum proteome tumor markers and set up diagnosis predictive model for colorectal cancer. Blind validation of the model with 44 healthy controls and 88 colorectal cancer patients were carried out by using Biomarker Patterns Software.</p><p><b>RESULTS</b>In comparing colorectal cancer group with control group, 5 specific protein peaks (P < 0.05) were found. The predictive model had a sensitivity of 100% and a specificity of 97.2%. A sensitivity of 71.6% and a specificity of 72.7% was got with the blind validation. The specific protein peaks with a mass-to-charge ratio (m/z) of 8908 and 13,707 showed in all the results and it showed their strong relationship with colorectal cancer.</p><p><b>CONCLUSIONS</b>The predictive models built by the differences of serum proteome fingerprint could be a very useful diagnostic tool in colorectal cancer. Proteins with m/z of 8908 and 13,707 would possibly be the tumor markers of colorectal cancer.</p>