Effect of miR-3619-5p on proliferation of human bladder cancer cell lines EJ and T24 / 中华泌尿外科杂志

Objective To study the effects of a synthetic miR3619-5p mimics on bladder carcinoma cell lines of EJ and T24 in vitro.Methods EJ and T24 cells were cultured in vitro and treated with three different processing:negative control group(tinfection with dsControl),positive control group(infection with dsP21-322) and the experimental group(infection with miR-3619-5p)during October 2015 to March 2016.Real-time fluorescent quantitative PCR (qPCR) was performed to detect the expression of p21 mRNA,cell cycle protein D1 (CyclinD1) and cell cycle-dependent kinase (CDK4 and CDK6) mRNA.Western Blot method was conducted to evaluate the expression of p21,CyclinD1 and CDK4 and CDK6 proteins;the change of cell cycle was displayed by flow cytometric analysis.Colony formation assay was used to test the ability of single cancer cell clone proliferation.Cell proliferation assay(MTS) was implemented to observed the inhibitive effect of cell proliferative potential.Results qPCR results showed that miR-3619-5p upregulated p21 mRNA expression (P ＜ 0.05),while the expression of CyclinD1,CDK4 and CDK6 were a little lower(P ＜ 0.05) in EJ and T24cells,respectively.Western Blot analysis testified that the expressions of p21,CyclinD1,CDK4 and CDK6 were difference among groups.Flow cytometry displayed that,the G0/ G1 phase increased significantly after transfected with miR-3619-5p and dsP21-322,compared with dsControl group(P ＜ 0.05),indicating that the cell cycle block in G0/G1 phase.Cell colony formation assay certified that the colony formation rates were less in the groups of miR-3619-5p and dsP21-322 than in that of dsControl group(P ＜ 0.05).Cell proliferation assay demonstrated that,cell proliferation ability decreased obviously when transfected with miR-3619-5p and dsP21-322 (P ＜0.05),compared with dsControl group.Conclusions miR-3619-5p could up-regulate the expression of p21 by RNA activation pathway and remarkably induced cell cycle arrest in G0/G1 phase,inhibiting the proliferation of bladder cancer cells.

Validation of the silencing site of rhesus monkey P21 gene in COS-7 cells / 中国实验动物学报

Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey . Methods To detect the expression of P21 gene in COS-7 cells ( derived from the kidney of African green monkey , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern blot assay .Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA.Their silencing rate was (91.82 ±3.21)%, (82.47 ±2.48)%, (81.31 ±2.69 )% and ( 87.35 ±4.59 )%, and the protein expression was ( 11.97 ±0.70 )%, ( 20.22 ±0.65 )%, ( 23.21 ± 0.63)%and (14.42 ±0.86)%, respectively.Conclusions Four effective silencing target sequences are screened at cel-lular level , which can be used in gene silencing research of rhesus monkeys .

Polymorphism at P21 codon 31 and dinucleotide polymorphism of P73 gene and susceptibility to bladder cancer in individuals from North India.

BACKGROUND AND AIM: p73, a novel P53 homolog and plays an important role in modulating cell cycle control, apoptosis and cell growth while P21, functions to negatively control the cell cycle. P53 up regulates p21 expression in response to deoxyribonucleic acid damage leading to cell cycle arrest at G1 checkpoint. In the present study, we are targeting p21 codon 31 and p73 gene variants of G4C14‑to‑A4T14 (Exon 2) polymorphism for bladder cancer (BC) risk in North Indians. MATERIALS AND METHODS: The above gene variants of P21 and P73 were assessed in the case‑control study comprising of 200 BC cases and 200 healthy controls of the same age, gender and similar ethnicity. Genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism method and PCR‑based confronting two‑pair primers (PCR with CTPP). RESULTS: The variant genotype of p73Exon 2 polymorphism showed significant risk for BC (p = 0.014). While combining with heterozygous genotype, variant genotype of p73Exon2 showed a significant association with BC risk (p = 0.010). While in case of p21 codon31 showed no significant association for BC risk at genotypic level. Significant association between p73Exon2 polymorphism and smoking was observed for BC risk. Furthermore, gene combination analysis revealed that AT/AT‑Ser/Ser is associated with risk for BC. Variant genotype of P73Exon2 was associated with reduced risk of recurrence (p = 0.039) in superficial BC patients receiving Bacillus Calmette‑Guerin treatment thus showing least survival (log rank = 0.029). CONCLUSION: Our study provided evidence that the p73 G4C14 > A4T14 (Exon2) polymorphisms were associated with higher risk of BC in North Indian population.

Effects of ionizing radiation on expression of P21 protein in Jurkat cell line and p21 gene in thymocytes and splenocytes of mice / 中华放射医学与防护杂志

Objective To investigate the effects of ionizing radiation on the expression of P21 protein in Jurkat cell line and p21 gene in thymocytes and splenocytes of mice.Methods Flow cytometry (FCM)was used to analyze the expression of P21 protein in Jurkat cells at 12 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Real-time PCR was used to detect the expression of p21 gene in thymocytes and splenocytes of mice at4 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Multi-staining was used to analyze the micronucleus rates of Rct in bone marrow.Results The expressions of P21 protein were increased in a dose-dependent manner during 0.5-4.0 Gy(t=-24.23--3.96,P<0.05),but decreased at 6.0 Gy at 12 and 24 h post-irradiation(t=-11.19,-14.50,P<0.05).The expressions of p2 1 gene in both thymocytes and splenocytes of mice were increased in dose-dependent manner in the range of 0-6.0 Gy(including 6.0 Gy)(t=-29.96-8.80,P<0.05),and reached to the peak at 6.0 Gy at 4 and 24 h post-irradiation(t=-11.84--3.42,P<0.05),except thymocytes at 4 h and 1.0 Gy post-irradiation(t=-3.42,P>0.05).Conclusions The expressions of P21 protein and p21 gene could be increased by X-ray irradiation.which shows good dosedependent manners in certain range of dose.

Effects of p21 gene transfection in human arterial smooth muscle cells / 中国临床药理学与治疗学

AIM: To find a new way for the treatment of restenosis after coronary artery inserted stent,the effect of p21 gene transfection in human arterial smooth muscle cells(HASMCs) was investigated and its antagonistic effect of restenosis after coronary artery inserted stent was studied.METHODS:P21 gene was transfected into HASMC via Lipofectamine 2000 liposomes and the expression of its encoded protein in HASMCs was detected by immunohistochemistry.The effects of p21 gene transfection on the growth,proliferation and apoptosis of HASMCs were described by cell growth curves,the WST-1 assay and flow cytometry(FCM) analysis respectively.RESULTS: Immunohistochemical result indicated that the protein encoded by p21 gene was highly expressed in the cytoblast of HASMCs.The expression of p21 gene significantly inhibited the growth and proliferation of HASMC and remarkably promoted its apoptosis.Compared with the control group,the difference was statistically significant.CONCLUSION:These results demonstrate that the encoded protein of the p21 gene transfected into HASMCs may be involved in inhibiting cell growth and inducing apoptosis,suggesting that the technology of p21 gene transfected into HASMCs can be a new strategy to prevent and treat restenosis after coronary artery inserted stent.

The Relationship Between Mutation of p53 Gene, p21 Gene and Bacteria L- Form in Gallbladder Carcinoma / 中国普外基础与临床杂志

0.05). The positive expression rate of p21 and p53 in gallbladder carcinoma was 62.5%(25/40) and 65.0%(26/40) respectively. The expression values of p21 and p53 in chronic cholecystitis was 2.5%(1/40) and 5.0%(2/40) respectively, which was significantly different from that of gallbladder carcinoma ( P

Polymorphisms of p53, p21 and IRF-1 and Cervical Cancer Susceptibility in Korean Women / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The aim of this study was to identify gene- gene and gene-environmental factor on cervical carcinogenesis in Korean women. MATERIALS AND METHODS: We evaluated 185 women patients who had cervical cancer with 345 normal control healthy women. The single nucleotide polymorphisms (SNPs) of the p53 codon 72, the p21 codon 31 and the IRF-1 intron 6 were evaluated from extracted DNA of peripheral blood with an automatic DNA sequencer. The difference of each SNP, gene-gene and gene-environmental interaction between normal controls and patients, were evaluated in an adjusted environmental background. RESULTS: With regard to environmental factors, the cervical cancer increased in the women with a lower level of education, a younger age at first sexual intercourse and with the increased number of children borne. The women who had p53 (Arg/Arg), IRF-1 (T/T) and an education of less than 6 years showed a 14.7 fold increased risk of cervical cancer than those women who had p53 (~Pro), IRF-1 (~C) and an education of more than 15 years. The women who had p53 (Arg/Arg), p21 (Ser/Ser) and more than 3 children showed a 6.4 fold increased risk of cervical cancer than those women who had p53 (~Pro), p21 (~Arg) and had borne no child. The women who had p53 (Arg/Arg), IRF-1 (T/T) and had experience of first sexual intercourse before the age of 22-years showed a 5.5 fold increased risk of cervical cancer than those women who had p53 (~Pro), IRF-1 (~C) and had experience of first sexual intercourse after the age of 26-years. CONCLUSION: We found that the level of education, the age at first intercourse, and the number of children borne, were independent risk factors in cervical carcinogenesis. The specific combination of p53, p21 and IRF-1 gene-gene and gene-environmental interactions were significantly noted in the cervical carcinogenesis of Korean women.

Detection of Human Papillomavirus DNA and p53 , p21 Gene Expression in CIN3 and Uterine Cervical Cancer / 대한암학회지

PURPOSE: Though the etiology of this cancer has not been elucidated, it has been suggested that certain types of human papillomavirus (HPV) and alteration of the p53 gene are closely associated with uterine cervical cancer. The aim of the study was to investigate the role of high risk HPV, p53 and p21 gene in cervical intraepithelial neoplasia III (CIN3) and invasive squamous cell carcinoma. MATERIALS AND METHODS: The presence of high-risk HPV DNA (16, 18, 31, 33, 35, 45, 51, 52, 56) were detected from cervical swab in 240 patients by hybrid capture method. Expression of p53 genes were studied in paraffin-embedded specimens by immunohisto- chemical staining and p53, p21 gene alteration by RT-PCR SSCP using fresh cervical tissues. RESULTS: High risk HPV DNA were detected in 34%, 74.3% and 75.7% in control, CIN3 and invasive squamaus cell carcinoma respectively. In patients with high risk HPV DNA, type 16 were detected of 5.9%, 30.8%, 47.2% respectively. Relative concentration of HPV DNA to control was 16.3+-27.4 in CIN3 and 30.4+-40.8 in invasive squamous cell cancer. Of patients with high risk HPV DNA, p53 expression was found in 42.9% of CIN3 immunohistochemically, while patients with invasive squamous cell carcinoma was de- tected in 50%. In patients without high risk HPV DNA, p53 expression was detected in 17.1% in CIN3, 15.7% in invasive squamous cell carcinoma. But the mutation of p53 and p21 gene by RT-PCR SSCP were not observed in CIN3 and invasive squamous cell carcinoma. CONCLUSION: These observations suggest that carcinogenesis of invasive squamous cell carcinoma from CIN3 may be concerned with high risk HPV concentration and may be occurred via another pathway without HPV and p53 or p21 mutation.

The study of the colony formation of HPP-CFC from bone marrow-derived hematopoietic cells of psoriatic patients and the methylation of p21 gene promotor in HPP-CFC / 中国免疫学杂志

Objective:To investigate the colony formation of high-proliferative potential colony-forming cells(HPP-CFC) from bone marrow-derived hematopoietic cells of psoriatic patients and p21 gene promotor methylation in HPP-CFC,and probe into the relationship between the colony formation and the methylation status of p21 gene promoter.[WT5"HZ] Methods:[WT5"BZ]Bone marrow-derived mononuclear cells were separated by density gradient centrifugation.The cells were cultured in methycellulose semi-solid culture medium with SCF,GM-CSF,IL-3 and IL-6 for 14 days, and then high-proliferative potential colony-forming cells(HPP-CFC) were counted.The HPP-CFC were collected and their genomic DNA was isolated . DNA was subjected to bisulfite treatment,and the modified DNA was studied by using the methylation-specific polymerase chain reaction (MSP).[WT5"HZ]Results:[WT5"BZ]In methycellulose semi-solid culture system, the number of HPP-CFC in bone marrow of psoriatic patients was significantly less than that of normal control. The positive frequency of methylation of p21 gene promoter in HPP-CFC of normal contrasts was higher than that of psoriatic patients. [WT5"HZ]Conclusion:[WT5"BZ]The activity of methylation status of p21 gene promoter of bone marrow derived hematopoietic cells of psoriatic patients is abnomal. The lower positive frequency of methyllation of p21 gene promotor in HPP-CFC perhaps play a role in lower colony-forming capability of HPP-CFC of psoriatic patients.