Correlation between mutation of p53 gene 2-4 exons from peripheral blood and HPV16 positive cervical cancer susceptibility and clinical significance / 中华妇产科杂志

Objective To detect mutations of p53 gene 2-4 exons from peripheral blood and to explore their relevance in HPV16-positive cervical cancer susceptibility and clinical significance. Methods Collected firstly cases from the Third Affiliated Hospital of Kunming Medical University from October 2012 to April 2014, included 167 cases HPV16-postive cervical cancer and 160 cases HPV-negative healthy women. Genomic DNA from the host peripheral venous blood was taken, mutations of p53 gene 2-4 exons were analyzed with software DNAstar after PCR and bidirectional sequencing. Meanwhile,mutations of p53 gene 2-4 exons among different clinicopathological characteristics in HPV16-postive cervical cancer were distinguished. Results (1)Three mutations and an 16-bp insertion/deletion sequences were found in p53 gene exons 2-4, included C/G mutation of single nucleotide polymorphism(SNP)11827 in intron2, A/C mutation of SNP11992 in intron3, C/G mutation in codon 72 (rs1042522) of exon4 and 16-bp(acctggagggctgggg) repeat insertion or deletion in intron3 (rs17878362), while deletion recorded as A1, insertion recorded as A2. No significant differences were found in each point allele and genotype frequency(P＞0.05). (2) Stratified analysis for cervical cancer group resulted with some differences. Compared group of non-squamous carcinoma with squamous carcinoma group, there were obviously decreased in allete A2 [11.8%(4/34) vs 3.5%(10/284); χ2=4.90,P=0.027], genotype A1A2 [4/17 vs 7.0%(10/142); χ2=5.14,P=0.023], and haplotype C-A2 [11.8%(4/34)vs 3.5%(10/284);χ2=4.91,P=0.027]. Compared with poorly differentiated group,allele C of SNP11827 and rs1042522 were obviously decreased in medium high differentiation group [50.8%(61/120)vs 38.8%(62/160);χ2=4.07,P=0.044], while haplotype G-A1 were apparently higher [49.2%(59/120)vs 61.2%(98/160);χ2=4.07,P=0.044], genotype GG of SNP11827 and rs1042522 were obviously decreased in superficial myometrial invasion depth group than that in deep myometrial invasion depth group [46.3%(25/54) vs 21.1%(8/38); χ2=7.06,P=0.029]. No significant differences were found between stage Ⅰ and Ⅱ, pelvic lymph node metastasis or not (all P＞0.05). Conclusions No obvious correlation is found between polymorphisms in exons 2-4 of p53 gene and susceptibility of HPV16-postive cervical cancer. But the patient with allete C and A2, genotype GG and A1A2, haplotype C-A2 and G-A1 may be increase risk of poorly differentiation, deep muscular invasion and bad pathological type. Analysis of p53 gene polymorphism may be provide a basis for the prognosis evaluation and individualized treatment of cervical cancer.

Diagnostic value of combined detection of autoantibodies against p53 and Bmi-1 in lung cancer / 中国医师杂志

Objective To evaluate the diagnostic value of combined detection of serum autoantibodies against against p53 and Bmi-1 in lung cancer (LC).Methods Serum levels of autoantibodies against p53 and Bmi-1 were measured by enzyme-linked immunosorbent assay (ELISA) in 92 patients with LC and 80 normal controls.The combined diagnostic value was evaluated with the receiver operating characteristic (ROC) curve.Results The serum levels of autoantibodies against p53 and Bmi-1 were significantly higher in LC than in normal controls (0.438 ±0.705 vs 0.045 ±0.035,Z =-7.667,P ＜0.01;0.117±0.061 vs 0.068 ±0.031,Z =-7.179,P ＜0.01).The levels of autoantibodies against p53 and Bmi-1 were not related to age,gender,pathological classification,lymph node status or tumor-node-metastasis (TNM) stage (P ＞ 0.05).The combined detection of two autoantibodies provided an enhanced sensitivity of 63.0％,a specificity of 91.2％ and an area under curve (AUC) of 0.881,which showed better diagnostic efficiency compared to individual autoantibodies.Conclusions Combined detection of autoantibodies against p53 and Bmi-1 shows good diagnostic value,which may aid diagnosis of LC.

Expression of Blimp1 mRNA in the PBMCs of patients with RA and the corresponding regulation of IL-21 / 中华检验医学杂志

Objective To Investigate the expression of IL-21 and Blimp1 mRNA in Rheumatoid arthritis ( RA) patients and the influence on the expression of Blimp 1 in peripheral blood mononuclear cells (PBMCs) of RA patients after IL-21 stimulated; To further explore the mechanism of IL-21 and blimp1 in the pathogenesis of RA.Methods Case control study.The samples of peripheral venous blood from 50 RA patients of department of rheumatology of The First Affiliated Hospital of Zhengzhou University and 50 healthy people were collected respectively , then the plasma and PBMCs was separated.IL-21 in plasma was measured by ELISA; the correlation between patients clinical index DAS 28, anti-CCP antibody and IL-21 was analyzed.Blimp1 mRNA of patients′PBMCs was detected by qPCR; PMBCs were isolated from RA patient and then cultured in vitro.Blimp1 mRNA level was measured by qPCR and the ratio of CD 20 positive B cell and the ratio of CD138 positive cells in all groups were detected by flow cytometry after 72 h stimulated by IL-21 and CD40L.Results IL-21 content in RA patient blood plasma (130.51 ±11.35)ng/L was significantly higher than that in healthy control (25.46 ±6.05)ng/L, t=5.39,P<0.05.Besides, IL-21 level also had a close relativity with patients DAS28(r=0.658) and anti-CCP antibody (r=0.674, P=0.039 and 0.035).In addition, the expression level of Blimp1 mRNA in RA patient PMBCs (1.321 ± 0.11)was higher than that in healthy control group (1.000 ±0.000), Z=-2.48, P<0.05.While after IL-21 and/or CD40L stimulation, Blimp1 mRNA of IL-21 group and CD40L+IL-21 group(1.084 ±0.029, 1.157 ±0.028)were higher than those of control (1.000 ±0.000)(P=0.002,P=0.001), moreover the expressive level of Blimp1mRNA of CD40L+IL-21 group was higher than that of control group (t=4.862, P=0.02).Compared to negative control group , the ratio of CD20 positive B cells [2.42 ±0.35, 2.63 ± 0.33, 6.35(4.85,6.57),F=278.363,P<0.001] and the ratio of CD138 positive cells(0.474 ±0.110, 0.668 ±0.120, 0.955 ±0.170,F=49.01, P<0.001) in CD40L group, IL-21 group and CD40L+IL-21 group were much higher and the differences among CD 40L+IL-21 group with CD40L group and IL-21 group were statistically significant.Conclusion IL-21 could promote the level of Blimp 1 mRNA in peripheral blood mononuclear cells in RA patient; IL-21 and CD40L could co-promote B cell maturation though regulating Blimp1 mRNA expression and eventually participate in RA pathogenesis.

Correlation between TRIB3 gene rs2295490 polymorphism and coronary heart disease with dyslipidemia / 中华检验医学杂志

Objective To explore the correlation between the polymorphism of TRIB3 gene rs2295490 and coronary heart disease (CHD) with dyslipidemia.Methods In the case control study,we enrolled 220 CHD patients as observation group (60.15 years mean age,150 males and 70 females) and 151 age and gender matched healthy individuals as control group (59.22 years mean age,102 males and 49 fe males).Genotypes of TRIB3 gene rs2295490 polymorphisms in the two groups were detected by PCR and sequence-based testing.At the same time,blood glucose (GLU),total cholesterol (TC),triglyceride (TG),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C) were measured.The correlations between the polymorphism of TRIB3 gene rs2295490 and CHD,biochemical indicators of two groups were analyzed,respectively.The comparison of genotype and allele frequency between two groups used x2 test,while the comparison of biochemical indexes between two groups used t test.Results In the control group,genotypic frequencies of AA,AG and GG of TRIB3 gene rs2295490 were 57％,35％ and 8％,and the frequencies of A and G alleles were 75％ and 25％,respectively.In the CHD group,genotypic frequencies of AA,AG and GG of TRIB3 gene rs2295490 were 50％,41％ and 9％,and the frequencies of A and G alleles were 71％ and 39％,respectively.There was no significant difference of genotype and allele frequencies of polymorphism of TRIB3 gene rs2295490 between CHD and healthy controls(x2 =1.74,P =0.42;x2 =1.26,P =0.243).In the CHD group,the HDL-C levels of the AA genotype were higher than those of AG + GG genotype (t =-9.78,P =0.00);The TG,LDL-C levels of the AA genotype were lower than those of AG + GG genotype (t =2.59,P =0.01;t =6.15,P =0.00).All the differences were statistically significant.Conclusions TRIB3 gene rs2295490 polymorphism has no correlation with CHD,but the G allele may be correlated with dyslipidemia in CHD patients.

The expression and significance of MTA1, MMP-9, and VEGF-C in the gastric adenocarcinoma / 中国医师杂志

Objective To investigate the expression and clinicopathological significance of metastasis-associated 1 (MTA1), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-C (VEGF-C) in a gastric adenocarcinoma.Methods The expressions of MTA1, MMP-9, and VEGF-C were detected with immunohistochemical method in 120 cases of gastric adenocarcinoma tissues and 30 cases of normal gastric tissues.Results The positive percentage of MTA1, MMP-9, and VEGF-C proteins in gastric adenocarcinomas was 76.7％ , 71.7％ , and 64.2％ , respectively, which was significantly higher than that (33.3％, 36.7％, and 30.0％) in normal gastric mucosa tissues (P ＜0.05).The expressions of MTA1, MMP-9, and VEGF-C were significantly higher in gastric carcinoma invasion serous membrane, lymph node metastasis, clinical Ⅲ ～ than that in non-invasion serous membrane, non-lymph node metastasis, and clinical Ⅰ ～ Ⅱ (P ＜ 0.05).The expression of MTA1 had significantly positive correlation with that of MMP-9 (P ＜ 0.05) and VEGF-C (P ＜ 0.05) in gastric adenocarcinomas.Conclusions The expressions of MTA1, MMP-9, and VEGF-C were higher in gastric adenocarcinomas than normal gastric mucosa tissues.They may play an important role in the invasion and metastasis of gastric carcinomas.MTA1 may promote infiltration, invasion, and lymph node metastasis of gastric carcinoma through regulation of the expressions of MMP-9 and VEGF-C.The combined detection of MTA1, MMP-9, and VEGF-C expressions has important value to judge the grade malignance of gastric adenocarcinomas.

Role of neuron-restrictive silencer factor in spinal cord in remifentanil-induced hyperalgesia in a mouse model of incisional pain / 中华麻醉学杂志

Objective To evaluate the role of neuron-restrictive silencer factor (NRSF) in the spinal cord in remifentanil-induced hyperalgesia in a mouse model of incisional pain (IP) .Methods Fifty-six male Kunming mice were randomly divided into 7 groups (n=8 each):control group (group C) ,IP group (group I) ,IP +remifentanil group (group IR ) , NRSF antisense oligonucleotide group (NAS group ) , IP + NRSF antisense oligonucleotide group (I+NAS group ) ,IP + remifentanil + NRSF mismatch oligonucleotide group (IR+NMS group) , and IP + remifentanil + NRSF antisense oligonucleotide group (IR + NAS group ) . Artificial cerebrospinal fluid 5 μl was injected intrathecally once a day for 3 consecutive days in C ,I and IR groups .NRSF antisense oligonucleotide NAS 10μg was injected intrathecally once a day for 3 consecutive days in NAS ,I+NAS and IR + NAS groups . NRSF mismatch oligonucleotide 10 μg was injected intrathecally once a day for 3 consecutive days in IR+NMS group .A 1-cm longitudinal incision was made through skin ,fascia and muscle of the plantar aspect of the right hindpaw to establish the model of incisional pain in sevoflurane-anesthetized rats .At 30 min after the last injection ,normal saline 0.4 ml was infused subcutaneously in C and NAS groups ,the model was established and normal saline 0.4 ml was subcutaneously infused simultaneously in I and I+NAS groups ,and the model was established and remifentanil 0.04 mg/kg was subcutaneously infused simultaneously in IR ,IR+NMS and IR+NAS groups .At 3 days before operation (T0 ) ,4 h before operation (T1 ) and 4 ,12 ,24 and 48 h after operation (T1-5 ) ,mechanical paw withdrawal threshold to von Frey stimuli (PMWT ) and paw withdrawal latency to thermal nociceptive stimulus (PTWL ) were measured .Results Compared with C group ,the PWMT and PWTL were significantly decreased at T2-5 in I ,IR ,I+NAS ,IR+NMS and IR+NAS groups ( P0.05 ) .Compared with I group ,the PWMT and PWTL were significantly decreased at T2-5 in IR and IR+NMS groups ( P0.05) . Compared with IR group ,no significant change was found in the PWMT and PWTL at each time point in IR+NMS group ( P>0.05) ,and the PWMT and PWTL were significantly increased at T2-5 in IR+NAS group ( P<0.05) . Conclusion NRSF in the spinal cord is involved in the development and maintenance of hyperalgesia induced by remifentanil in a mouse model of IP .

Expression of KLF-8 and MMP-9 in placentas and their relationship with the pathogenesis of preeclampsia / 中华妇产科杂志

Objective To evaluate the expression of Krüppel-like factor 8 (KLF-8) and matrix metalloproteinase 9 (MMP-9) in placentas and their relationship with the pathogenesis of preeclampsia (PE).Methods Twenty-two women with PE (mild PE:4 cases; severe PE:18 cases) who received cesarean sections in the First Affiliated Hospital of Chongqing Medical University from September 2011 to March 2012 were recruited as the PE group (n =22).And twenty women who received elective term cesarean section without perinatal complications were chosen as the control group (n =20).Placentas were collected and immunohistochemical SP method were employed to detect the localization of KLF-8 protein.KLF-8 mRNA level was determined by quantitative real-time PCR technique and western blot analysis was used to quantify KLF-8 and MMP-9 protein levels.Results (1) There was no difference of KLF-8 protein distribution in placentas of the PE group and the control group.It was mainly located in the nuclear and cytoplasm of syncytiotrophoblasts.KLF-8 immunostaining was apparently decreased in the placentas of preeclamptic women when compared with the control group.(2)The KLF-8 mRNA levels were significantly decreased in placentas of the PE group (0.69 ±0.08) compared to those of the control group (1.14 ±0.09,P ＜0.01).(3) KLF-8 and MMP-9 protein levels significantly decreased in the PE placentas (0.68 ±0.05 and 0.21 ± 0.03) when compared to the control group (0.94 ± 0.06 and 0.34 ± 0.03,respectively,P ＜ 0.01).(4) There was a positive correlation between the expression of KLF-8 and MMP-9 protein in the placentas from PE and normal pregnancies (r =0.64,P ＜ 0.01).Conclusions KLF-8 mRNA and protein levels were decreased in placentas of PE patients compared to those of normotensive women.KLF-8 protein was primarily located in the invasion-related trophoblast cells and its expression had a positive correlation with MMP-9 levels.KLF-8 might have an important role in the pathogenesis of PE by regulation of trophoblast invasion.

Relationship between MTA1 expression and invasive and metastatic ability of cervical cancer cell / 中华妇产科杂志

ObjectiveTo investigate the relationship between metastasis-associated gene 1 ( MTA1 )expression and invasive and metastatic ability of cervical cancer cell. MethodsThree kinds of plasmids pcDNA3( control group), pcDNA3-MTA1 ( MTA1 group) and pSilencer3. 1-MTA1-siRNA ( MTA1-siRNAgroup) were transfected into human cervical cancer cell line CaSki cells. Reverse transcription (RT)-PCR and western blot were used to detected MTA1 mRNA and protein expressions. The effects of MTA1 expression on CaSki cell growth and proliferation, cell migration, adhesion and invasion, and cell cycles were tested by methyl thiazolyl tetrazolium (MTT), clone formation experiment, wound-healing assay, transwell assay, adhesion assay and flow cytometry, respectively. In animal experiment, three groups of cells were inoculated to BALB/c nude mouse subcutaneously to observe tumor formation ability. ResultsCompared with control group, MTA1 mRNA and protein were significantly overexpressed in MTA1 group, while MTA1-siRNA group showed lower MTA1 expression. Compared with control group, MTA1 group showed significantly accelerated cell growth; while MTA1-siRNA group showed decreased cell growth since the second day (P＜0. 05). Clone formation number in control, MTA1 and MTA1-siRNA group were 133 ±6, 169 ± 10 and 57 ±5,respectively. MTA1 group showed accelerated cell formation, while MTA1-siRNA group showed the reverse effect compared with that in control group(P ＜ 0. 05 ). At 24, 48 and 72 hours after wounding, the healing ability of MTA1-siRNA group significantly lagged behind that in the control group, while MTA1 group showed accelerated cell healing ability. The adhesion rate of control, MTA1 and MTA1-siRNA group were (69. 3 ± 3. 6) ％, ( 80. 4 ± 5. 6 ) ％ and ( 39. 2 ± 7.4 ) ％ separately at 90 minutes after cell seeding. In contrast with control group, MTA1 group promoted the adhesion of CaSki cell to matrigel matrix, while MTA1-siRNA group inhibited the adhesion process (P ＜0. 05 ). In the migration assay, the number of cells migrated to the bottom side of the membrane in control,MTA1 and MTA1-siRNA group were 153 ± 17,247 ± 38 and 82 ± 10, respectively. The number of cells in the invasion assay were 231 ± 19,354 ± 36 and 76 ± 7, respectively. Compared with the control group, MTA1 group significantly increased the migration and invasion ability, while MTA 1-siRNA group showed lower cell migration and invasion ability (P ＜ 0. 05 ). In cell cycle experiment, no significant differences of cell proportions including G1, S and G2 stage were found among three groups (P ＞ 0.05).In animal experiment, compared with control group,MTA1 group showed accelersted tumor formation and growth,whilethe MTA1-siRNA group showed the reverse effect ( P ＜ 0. 05 ). ConclusionsMTA1 may play its roles to promote cervical cancer cell invasion, migration, adhesion, as well as cell growth and colony formation, while RNA interference against MTA1 may decrease the malignant phenotypes. This study shows that it will be an effective beginning to explore metastasis mechanisms and cancer gene therapy strategy targeting MTA1 in cervical cancer.

Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues / 中华妇产科杂志

Objective To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2)in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. Methods Spontaneous abortion embryo tissues were collected,including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos(35 cases)from the Department of Gynaecology and Obstetrics of the Affilisted Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007.FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein;primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis.Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAIY2 genes in embryonic cells which have normal karyotypes;the groups were defined as the first experimental group(transfeeted with pshRNA-hsMAD2-1),the second experimental group(transfected with pshRNA-hsMAD2-2),the third experimental group(transfected with pshRNA-hsMAD2-3),the first control group(transfected with nothing),the second control group(transfected with pTZU6+1)and the independent group(transfected with pshRNA-N1).Interference efficiency was demonstrated by FQ-PCR and western blot:cell prolireration was meagured by methyl thiazolyl tetrazolium(MTT)assay;cell-cycle was assessed by flow cytometry (FCM):the chromosome numbers were calculated to analyze the variation of chromosomes.Results(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue,twice or more spontaneous abortion tissue and indueed abortion tissue were 0.00879±0.00035.0.00901±0.00033 and 0.00941±0.00026 respectively,and there Wag no significant ditierence(P＞0.05)compared with each other;however,the protein levels of hsMAD2 in three groups were 0.2791±0.0311.0.0431±0.0020 and 0.5790±0.0331 respectively,and there were significant difierences(P＜0.05)compared with each other.(2)Recombinant shRNA plagmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells.Compared with the first control group(4%)and the second control group(3%),the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection(P＜0.05):compared with the first control group(8.2%)and the second control group(8.0%),the ratios of G2/M phase cells in the experimental group(17.9%)was significantly increased(P＜0.05);compared with the first control group (4.8%),the ratios of abnormal chromosomes in the experimental group was increased to 30.0%(P＜0.05).Conclusions Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration,abnormal embryo development and the occurrence of spontaneous abortion.

PIAS1 interacts with the KRAB zinc finger protein, ZNF133, via zinc finger motifs and regulates its transcriptional activity

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.

Retrovirus-mediated cellular repressor of E1A-stimulated genes inhibits neointima formation in the rat carotid artery after balloon injury / 中国病理生理杂志

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes(CREG) mediated by retrovirus on neointima formation in injured rat carotid.METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM ?-actin and Ki-67 were detected.RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM ?-actin expression.CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.