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30 November 2021, Volume 33 Issue 6
甲基丙烯酸环氧丙酯诱导16HBE细胞恶性转化相关m6A修饰异常mRNA的分析
WANG Miao, WANG Quankai, LI Xinwei, MA Shunpeng, WUHAN Baolier, XU Jianning
2021, 33(6):  405-409.  doi:10.3969/j.issn.1004-616x.2021.06.001
Abstract ( 435 )   PDF (1762KB) ( 210 )  
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OBJECTIVE: To investigate m6A methylation levels of mRNAs which are related to glycidyl methacrylate (GMA)-induced malignant transformation of 16HBE cells and their functional roles. METHODS: After 16HBE cells were repeatedly exposed to GMA(8 μg/mL),the 30th generation cells of GMA group and DMSO control group were collected. m6A methylation levels of mRNAs which were related to 16HBE malignant transformation were detected by high-throughput human apparent transcriptome chip. The differentially m6A-methylated mRNAs and differentially expressed mRNAs were screened by Visual Studio Code. The Omicshare tools were used for GO enrichment and KEGG pathway analyses of these mRNAs. RESULTS: A total of 454 lncRNAs with differential m6A methylation were identified in GMA-induced 16HBE malignant transformed cells,including 334 which were hypermethylated mRNAs and 120 which were hypomethylated mRNAs (|FC|>2.0,P<0.05). There was a total of 434 differentially expressed mRNAs,among which 236 were up-regulated and 198 were down-regulated (|FC|>2.0,P<0.05). There were 45 m6A-methylated mRNAs. The GO analyses showed that SNAP receptor activity,SNARE binding and SNARE complex were the main biological processes. KEGG pathway analyses showed that m6A-methylated mRNAs were enriched in SNARE interactions in vesicular transport,non-homologous end-joining,and phenylalanine metabolism. CONCLUSION: Our data indicate that m6A methylation played an important role in the process of GMA-induced 16HBE malignant transformation.
胶原蛋白作为胃癌潜在生物标志物的生物信息学分析
YANG Chanjun, CHEN Wentian, LIU Jing
2021, 33(6):  410-419.  doi:10.3969/j.issn.1004-616x.2021.06.002
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OBJECTIVE: To identify potential biomarkers for gastric cancers (GC) through bioinformatics methods. METHODS: Gene Expression Omnibus (GEO) were retrieved from GC-related datasets,followed by R software and Venn analyses for differentially expressed genes (DEGs) in GC. DAVID software was used to distinguish DEGs' functional annotation. Expression patterns of DEGs were analyzed by Oncomine and their prognostic values were examined by Kaplan-Meier plotter. RESULTS: Three GC-related GEO datasets were found and downloaded from NCBI. After integrating them,110 DEGs were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that these DEGs were significantly enriched in ECM-related functions and pathways. A group of collagen genes was significantly upregulated in the GC tissues and constituted a protein-protein interaction network as important nodes,including COL1A1,COL1A2,COL2A1,COL12A1,COL6A3,and COL10A1. Based on the miRNA-mRNA analysis,hsa-miR-29a-3p,hsa-miR-29b-3p,and hsa-miR-29c-3p which belonged to the hsa-miR-29 family,has the potential for regulation of COL1A1,COL1A2,COL6A3 and COL2A1. Compared with normal gastric tissues,the expression of COL1A1,COL1A2,COL12A1,COL6A3 and COL10A1 were significantly up-regulated in GC (P<0.05). From survival analyses,the GC patients with high expression of COL1A1,COL1A2,COL12A1,COL6A3,COL2A1 and COL10A1 had poorer prognosis than the other GC patients (P<0.05). CONCLUSION: This study identified that expression of collagen genes was significantly up-regulated in GC tissues and associated with GC patients' survival. Their oncogenic roles and prognostic values in GC indicate that collagens could serve as potential therapeutic targets of GC.
Orai1对鼻咽癌细胞侵袭和上皮-间充质转化的影响及其机制
GUO Zhaomeng, ZHANG Hua, ZHANG Peng
2021, 33(6):  420-425.  doi:10.3969/j.issn.1004-616x.2021.06.003
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OBJECTIVE: The study was aimed to investigate mechanisms of Orai1 on nasopharyngeal carcinoma cell invasion and epithelial-mesenchymal transformation (EMT) in vitro. METHODS: A nasopharyngeal epithelial cell line (NP69) and nasopharyngeal carcinoma cell lines (CNE1,CNE2,HONE1 and 5-8F) were cultured. Real-time quantitative PCR and Western blot were used to detect expressions of Orai1 mRNA and protein. The short hairpin RNA (shRNA) plasmid targeting Orai1 was used to down-regulate expressions of Orai1 in CNE2 cells. Store-operated Ca2+ entry (SOCE) was detected by calcium imaging,cell migration and invasion were detected by Transwell chamber,and E cadherin,N cadherin and Vimentin were detected by real-time quantitative PCR and Western blot. RESULTS: Orai1 was expressed in NP69,CNE1,CNE2,HONE1 and 5-8F cells,and the expression of Orai1 was higher in CNE2 cells than that in NP69 cells (P<0.05). The mRNA and protein levels of ORAI1 were significantly decreased in CNE2 cells transfected with Orai1 shRNA (P<0.05). SOCE,migration/invasion and EMT were significantly inhibited in Orai1 shRNA transfected CNE2 cells (P<0.05). CONCLUSION: Blockage of ORAI1 expression inhibited SOCE,migration/invasion and EMT process of nasopharyngeal carcinoma cells. Therefore, Orai1 might be a new target and a new direction for the treatment of nasopharyngeal carcinoma metastasis.
氧化苦参碱对小鼠肉瘤和肝癌的体内外抗肿瘤作用
LIU Ning, LIANG Lanlan, LI Shufang, SHEN Xiangchun, LIANG Bing
2021, 33(6):  426-429,434.  doi:10.3969/j.issn.1004-616x.2021.06.004
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OBJECTIVE: To investigate anti-tumor effects of oxymatrine on S180 and H22 tumor bearing mice in vivo and in vitro,and to provide experimental basis for application of oxymatrine in treatment of cancers. METHODS: In vitro cell culture:S180 and H22 cell suspensions at logarithmic growth stage were precultured for 24 h. Mice S180 and H22 cells were divided into blank control group (RPMI-1640 medium),negative control group (un-supplemented S180 and H22 cell suspension),5-fluorouracil (5-FU,5×10-5 g/L) positive control group and 5 different concentrations of oxymatrine (1×10-4,1×10-5,1×10-6,1×10-7,1×10-8 g/L). After 48 h of continuous culture,proliferation inhibition rates of oxymatrine on S180 and H22 cells were detected by tetramethylthiazolyl blue (MTT) method. In vitro animal experiments using our S180 and H22 tumor-bearing mouse models were:normal control group,model group,positive control group,and oxymatrine high-dose,medium-dose and low-dose groups were set up respectively,with 10 mice in each group. Normal control group and model group were intra-gastrically fed with distilled water,high,medium and low dose mice were intra-gastrically fed with oxymatrine at different concentrations (100,50 and 25 mg/kg),and positive control group was intra-gastrically fed with cyclophosphamide at 30 mg/kg. After 8 days of continuous administration,the inhibitory rate,thymus coefficient and spleen coefficient of oxymatrine on S180 and H22 tumor bearing mice and the survival of S180 and H22 ascites tumor mice were detected. RESULTS: Our results showed that oxymatrine inhibited proliferation of S180 and H22 cells in vitro as detected with the MTT method. Results from our in vivo studies showed that oxymatrine high,medium and low dose group significantly inhibited growth of S180 sarcoma in mice,and the tumor inhibition rate were 48.48%,41.67% and 32.58% respectively;oxymatrine high and medium dose group significantly inhibited the growth of H22 sarcoma in mice,and the tumor inhibition rate were 33.95%,31.63% respectively,and the difference was statistically significant. Oxymatrine medium dose group significantly prolonged the survival time of S180 mice. The life extension rate was 31.18%,and the difference was statistically significant. Compared with the normal group,each dose group of oxymatrine had no effect on the thymus index and spleen index in S180 and H22 mice. CONCLUSION: Oxymatrine demonstrated anti-tumor effects on S180 and H22 in vitro and in vivo.
miR-93-5p和miR-106b-5p在酒精相关性肝癌中的表达及其临床意义
JIAO Wenpeng, HOU Lin, CUI Meijuan, JIAO Wenjing, MA Ming, ZHANG Jinyan
2021, 33(6):  430-434.  doi:10.3969/j.issn.1004-616x.2021.06.005
Abstract ( 493 )   PDF (1305KB) ( 168 )  
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OBJECTIVE: Alcohol-related liver cancer has become a common type of liver cancer. Our aim was to identify tumor markers which could be used to screen patients with early liver cancer from patients with alcoholic cirrhosis and to improve the prognosis of patients,especially in expression levels of miR-93-5p and miR-106b-5p. METHODS: Randomly selected 10 patients with alcoholic liver cirrhosis in the pathology department of our hospital,and alcoholic cirrhosis/liver cancer tissue wax blocks from 10 patients according to gender and age matching. Expression levels of miR-93-5p and miR-106b-5p in the tissue wax blocks were determined. In addition,71 patients with alcoholic liver cirrhosis and liver cancer who were hospitalized in our hospital from January 1,2014 to December 31,2016 were recruited. Serum samples were collected from these patients before treatment and expression levels of miR-93-5p and miR-106b-5p were detected. Relationships between expression levels of miR-93-5p and miR-106b-5p and clinicopathological characteristics and prognosis of patients were analyzed. RESULTS: Expression levels of miR-93-5p and miR-106b-5p in cancer tissues were significantly higher than those from patients with alcoholic liver cirrhosis(all P<0.01). The maximum diameters for tumors in the high miR-93-5p group were lower and the miR-93-5p group was significantly enlarged (P<0.01),and the clinical stage was later (P<0.01). The high miR-106b-5p group had significantly greater tumor maximum diameter than miR-106b-5p group (P<0.01),and the clinical stage of the high miR-106b-5p group was later (P<0.01). In the entire group of patients,the survival rates of patients in the age <60 years were significantly better than that in the ≥ 60 years group (P=0.03). Survival rates of patients in the <5cm tumor diameter group were significantly better than that in the ≥ 5 cm group (P=0.01). Survival rates of patients in the group Ⅰ+Ⅱ were higher than that in group Ⅲ (P=0.02). Survival rates of patients in the high miR-93-5p group was significantly lower than that of the patients in the miR-93-5p group (P=0.01). Survival rates of patients in the high miR-106b-5p group were significantly lower than that in the low miR-106b-5p group (P<0.01). Age and miR-106b-5p expression levels were independent factors affecting prognosis of the patients (P=0.02;P=0.03). CONCLUSION: miR-93-5p and miR-106b-5p showed potential to be tumor markers for alcohol-related liver cancers. Detection of their expression levels before treatment was predictive of pathological characteristics and prognosis of the patients.
青藤碱对HPV阳性宫颈癌SiHa细胞增殖和侵袭的影响
WANG Xiaozhou, LI Hongshi, YU Xi, WEI Ning, WANG Ziying, HE Lijie
2021, 33(6):  442-445.  doi:10.3969/j.issn.1004-616x.2021.06.007
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OBJECTIVE: To investigate effects of sinomenine on proliferation and invasion of HPV-positive cervical cancer cells. METHODS: In the negative control group,0 mmol/L sinomenine was added to the SiHa cell culture medium. In the experimental group,0.2,0.4 and 0.8 mmol/L sinomenine were added. CCK-8 was used to detect cell viability after 24 and 48 h. In the sinomenine group,0.2 mmol/L sinomenine was added to SiHa cell culture medium,and in the negative control group,0 mmol/L sinomenine was added to SiHa cell culture medium. After 24 hours,the number of Ki67-positive cells was detected by immunofluorescence. Transwell experiment was used to detect the number of invaded cells. Western blot was used to detect protein expression levels of invasion-related proteins MMP-2 and MMP-9. RESULTS: After 0.2,0.4 and 0.8 mmol/L of sinomenine treatment for 24 and 48 h,proliferation rates of the SiHa cells were significantly lower than that of the control group (P<0.01). Compared with the negative control group,the numbers of Ki67-positive cells in the sinomenine group were significantly reduced (P<0.01). Compared with the negative control group,invasions in the sinomenine group were significantly reduced (P<0.05). Compared with the control group,expressions of the invasion-related proteins MMP-2 and MMP-9 in the sinomenine group were significantly reduced (P<0.01). CONCLUSION: Sinomenine inhibited proliferation and invasion of HPV-positive cervical cancer cells.
TRIM59在结直肠癌中的表达及其与临床病理指标的关系
GAO Xiaobin, WU Xueliang, WANG Shengjie, SUN Guangyuan, WANG Wenjing, LIANG Feng, ZHAO Yifeng, LIU Zhenxian, ZHANG Yingchun
2021, 33(6):  446-450.  doi:10.3969/j.issn.1004-616x.2021.06.008
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OBJECTIVE: To investigate expression of tripartite motif-containing 59 (TRIM59) in colorectal cancer and its correlation with clinicopathological features. METHODS: Colorectal cancers and adjacent tissues were collected; and expressions of TRIM59 were analyzed using real-time quantitative PCR(qPCR) and immunohistochemistry. Relationships between TRIM59 expressions and clinicopathological features and survival rates were analyzed. RESULTS: Results from the qPCR analyses on 86 cases of pair tissues showed that expression levels of TRIM59 in 20 cases were significantly higher than that in the paired adjacent tissues (t=12.86,P<0.01). Immunohistochemical results showed that positive expression rate of TRIM59 was 62.79% (54/86),which was significantly higher than 32% (37/86) in the adjacent tissues (χ2=6.74,P=0.009)。The high expression of TRIM59 was significantly correlated with TNM stages (χ2=8.515,P=0.003),tumor invasion depths (χ2=7.167,P=0.007) and lymph node metastases (χ2=5.511,P=0.018). Kaplan-Meier survival analyses showed that the overall survival (OS) and disease-free survival (DFS) of patients with high TRIM59 expression were significantly shorter than those of patients with low TRIM59 expression (P<0.05 both). CONCLUSION: TRIM59 was highly expressed in colorectal cancers and the high expressions were closely related to TNM stages,depth of invasions,lymph node metastases and prognosis.
全自动DNA图像分析系统在胰腺恶性肿瘤诊断中的价值
WANG Rui, WANG Heng, WU Juan, JI Xiaokun, GUO Xiao, MA Yang, DU Yun
2021, 33(6):  451-454.  doi:10.3969/j.issn.1004-616x.2021.06.009
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OBJECTIVE: To evaluate values of automatic DNA image analyses (DNA-ICM) in diagnosis of pancreatic malignant tumors. METHODS: Patients with suspected pancreatic malignant tumor who underwent endoscopic ultrasonics guided fine needle puncture were recruited for our study. Collected samples were tested by conventional cytology and DNA-ICM. Diagnostic values of cytology,DNA-ICM and their combination were analyzed using histopathological diagnosis as the standard reference to clinical follow-up data. RESULTS: Among the 108 patients,sensitivity,specificity,positive predictive value,negative predictive value and accuracy of conventional cytological diagnosis were higher than those of DNA-ICM (93.1% vs 90.8%,95.2% vs 90.5%,98.8% vs 97.5%,76.9% vs 70.4%,93.5% vs 90.7%). However,there was no statistical significance between them (P>0.05). The combined diagnosis indexes for the two methods were 95.4%,90.5%,97.6%,82.6%,94.4%,respectively. Sensitivity,negative predictive value and accuracy of the combined diagnosis were higher than those of single diagnosis,but the differences were not statistically significant (P>0.05). CONCLUSION: DNA-ICM combined cytological diagnosis has great application value,and DNA-ICM showed good auxiliary diagnostic values for pancreatic cytology diagnosis.
KeratinoSens试验在医疗器械致敏性检测中的应用研究
CHEN Hong, HUANG Yuanli, WANG Han, LIAN Huan, HAN Qianqian, WANG Chunren
2021, 33(6):  455-460.  doi:10.3969/j.issn.1004-616x.2021.06.010
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OBJECTIVE: This article introduces a new in vitro human-derived cell line as an alternative method (KeratinoSens assay) of evaluation for delayed-type hypersensitivity to,KeratinoSens assay,and discusses its application in the detection of two medical devices. METHODS: Plasmid pGL4.17-AKR1C2-ARE-SV40 was constructed to transfect the human immortalized keratinocytes HaCaT,and the stablye transfected cell line KeratinoSens was obtained by screening with G418. TheUsing KeratinoSens assaywas used to evaluateprocess sensitizers with different sensitization ranks and non-sensitizers. By calculating the Variabilities of gene activity induction variability ofin the negative control were used to identify cell stability. The data were,compared toing the test results with the sensitization properties of the compounds in the literature,and were used to indicateget the predictability of the sensitization of the cell line. After the successful cell transfection is identified,the KeratinoSens assay wasis used to test the medical devices with more clinical sensitization reports,medical natural latex gloves and nickel-containing metals. RESULTS: The negative control hads a variation of 18.6% which meets the stability requirements,and the synthesized cell hadve good sensitization predictability. KeratinoSens assay rResults fromof the two medical devices were are negative. CONCLUSION: The KeratinoSens was successfully constructed and used to initially applied to medical device testing to provide empirical data for itsthe method to be transformationed into a medical device testing method.
深圳市某区低学龄儿童血脂异常与血清中铅和镉浓度的关系
LIU Ruiguo, CHEN Xiaoli, YOU Yingbin, LI Shufan, ZHANG Qingying
2021, 33(6):  461-465.  doi:10.3969/j.issn.1004-616x.2021.06.011
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OBJECTIVE: The aims of this study were to explore correlations between dyslipidemia and serum concentrations of lead (Pb) and cadmium (Cd) in low school-aged children in a district of Shenzhen,and to provide evidence for prevention and treatment of children with dyslipidemia. METHODS: Cluster samplings were used to select all first-grade primary school children in Shenzhen,and blood biochemical tests and lifestyle questionnaires were conducted among them. Children with dyslipidemia were selected as the experimental group (n=663),and healthy ones (n=663) as the control group using 1:1 matching according to age ±0.5 years old and gender. Serum concentrations of Pb and Cd among the children were tested using the inductively coupled plasma mass spectrometry (ICP-MS) method. RESULTS: Serum concentrations of Pb and Cd in children with dyslipidemia (133.08 μg/L and 0.45 μg/L,respectively) were higher than those in controls (84.19 μg/L and 0.29 μg/L,respectively,P<0.05). Conditional Logistic regression analyses show that,after adjustment for covariates,serum Pb[OR=2.52,95%CI (1.66,3.83)] and Cd[OR=4.31,95%CI (2.73,6.82)] were associated with an increased risk of dyslipidemia in children in a dose-response relationship (Ptrend<0.01). CONCLUSION: Dyslipidemia was associated with increased serum Pb and Cd concentrations in low school-aged children in a district of Shenzhen. The results indicate that we should pay more attention to concentrations of heavy metals in their low school-aged children in order to prevent chronic diseases and to promote their health.
抗HIV药物齐夫多定、拉夫米定和克力芝联合用药的急性经口毒性和致突变作用
GE Xianmin, LI Bin, HUANG Chaopei, GAO Yuqiu, LUO Hailan, YANG Hui, WANG Yanwu, WEN Pingjing, LAN Guanghua, CHEN Huanhuan, MENG Qin, LUO Liuhong, DENG Yueqin, LIU Shuaifeng, WU Xiuling
2021, 33(6):  466-469,474.  doi:10.3969/j.issn.1004-616x.2021.06.012
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OBJECTIVE: Combined-therapy intervention with highly effective antiretroviral drugs can significantly reduce vertical transmission of HIV from mothers to children (PMTCT). In order to evaluate their safety,acute oral toxicity and potential mutagenicity of a combined drug consisting of zivdodine,lamivudine and aletra for PMTCT were evaluated. METHODS: Tablets of zivdodine,lamivudine and aletra were mixed in a ratio of 2:1:2 and crushed into powder,and then mixed with pure water to prepare a suspension with an effective component concentration of 250 mg/mL. The suspension was given orally to mice once according to the volume of 0.02 mL/g body mass (5 000 mg/kg dose) to observe its acute oral toxicity. Mutagenicity of the combined drugs was studied by reverse mutation test of bacteria,with 5 dose groups of 50,158,500, 1 581 and 5 000 μg/dish (active ingredient),according to the flat mixing method. The mutagenicity of the drugs was detected also by micronucleus test and chromosome aberration test in mammals with dose of 500, 1 000 and 2 000 mg/kg (by active ingredient,the same below). RESULTS: No signs of poisoning and death was found after given the combined drugs to the mice,the LD50 was more than 5 000 mg/kg. The number of revertant colonies of five test strains (TA97a,TA98,TA100,TA102,TA1535) in five dose groups of the combined drugs,with or without S9 metabolic activation system,were close to that of spontaneous revertant colonies,it was a negative result. However,the micronucleus rates of female and male animals in 500,1 000 and 2 000 mg/kg dose group of the combined drugs were between 4.4‰ and 5.2‰,which were significantly different from that of the negative control group (1.2‰-1.4‰),P<0.05,and were higher than the normal background value of our laboratory (0.4‰-3.6‰). Chromosome aberration rates of spermatogonia of animals in 500,1 000 and 2 000 mg/kg dose group of the combined drugs were 0.4%-0.8%,which were not significantly different from that of the negative control group (0.4%),P>0.05. CONCLUSION: Under our experimental conditions,the combined drugs showed no acute oral toxicity,no mutagenic effect in vitro and no chromosome aberration effect of spermatogonia in mice,but the micronucleus test of bone marrow cells was positive. Therefore,mutagenicity of the combined drugs needs to be further explored and verified.