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Objective To investigate the activation effect of microRNA-1280 (miR-1280) on the expression of p21 gene in bladder cancer cell line BIU-87 and its effect on cell cycle and proliferation of bladder cancer cell line.Methods Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expressions of miR-1280 in bladder cancer cell lines T24,5637,J82,BIU-87 and normal bladder epithelial cells SV-HUC-1.miR-1280 mimics (experimental group) and miR-NC (control group) were transfected into the bladder cancer cells with the lowest expression of miR-1280.The expressions of miR-1280 and p21 mRNA were detected by qRT-PCR.Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR-1280 and p21 gene promoter.Western blotting was used to detect the expressions of p21,cell cycle-dependent kinase 1 (CDK1),Cyclin A2 mRNA and protein in the two groups.Cell cycle was detected by flow cytometry,and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay.Results The results of qRT-PCR indicated that the expression levels of miR-1280 in bladder cancer cell lines T24,5637,J82 and BIU-87 and normal urothelium cell line SV-HUC-1 were 0.503 ±0.094,0.611 ±0.054,0.567 ± 0.077,0.257 ± 0.032 and 1.014 ± 0.090 respectively,with a significant difference (F =1.880,P ＜0.001).Compared with bladder cancer cell lines T24,5637 and J82 cells,the expression of miR-1280 in BIU-87 cell was the lowest (P =0.026,P =0.003,P =0.008).Compared with the control group,the expression of miR-1280 in BIU-87 cell was significantly increased (1 041.000 ± 157.500 vs.1.023 ± 0.118,t =6.606,P ＜0.001),and the expression of p21 mRNA was also significantly increased (5.280 ± 0.660 vs.1.007 ± 0.070,t =6.440,P ＜ 0.001).Western blotting showed that p21 protein expression was up-regulated,CDK1 and Cyclin A2 protein expressions were down-regulated.ChIP experiments showed that compared with the miR-NC transfection group,the concentration of biotin modified miR-1280 in the p21 gene promoter region was significantly increased (1.246 ±0.171 vs.0.519 ± 0.087,t =3.787,P =0.009).The proportion of G0-G1 cells in the experimental group BIU-87 cells was significantly higher than that in the control group (68.360％ ±3.064％ vs.46.970％ ±3.971％,t =4.263,P =0.005).The results of MTT showed that compared with the control group,the cell proliferation ability of BIU-87 cells after being transfected miR-1280 was significantly decreased starting from day 3 (0.826 ± 0.099 vs.1.224 ± 0.057,t =3.505,P =0.013).Conclusion miR-1280 can activate the expression of p21 gene in bladder cancer cell line BIU-87 by binding the promoter region of p21 gene,blocking the progression of cell cycle and inhibiting cell proliferation,which provides a new direction for bladder cancer targeted therapy theory.
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Objective To investigate the mechanisms of radiotherapy sensitization role of curcumin in cervical carcinoma Hela cells.Methods Western blot was used to detect expressions of p53, p21, Bax,and Bcl-2 proteins after cervical cancer Hela cells were treated.Results The expressions of p53 and p21 proteins were increased.The ratio of Bcl-2/Bax was decreased when curcumin and irradiation were applied to cells alone (P ＜ 0.05).This change were much more obvious when irradiation and curcumin were combined than either of them was used alone(P ＜0.01).Conclusions The mechanisms of curcumin enhancing radiosensitivity on Hela cells might be due to upregulation of p53, p21, and Bax proteins and downregulation of Bcl-2 protein.
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Background: In colorectal cancer, BRAF and KRAS mutation are mutually exclusive, but both are independent prognostic factors for the disease. Aim: To determine the frequency of BRAF V600E mutation in colorectal cancer. Material and Methods: A KRAS mutation study was carried out in 100 tissue samples of primary and metastatic adenocarcinomas of colon and rectum from patients aged 61.1 ± 62 years (56 women). Negative KRAS mutation cases underwent study of BRAF V600E mutation by restriction fragment length polymorphism (RFLP) and direct sequencing. Results: Primary tumors were located in the colon and rectum in 88 and six cases respectively. Five were liver metastases and in one case, the sample location was undetermined. Forty two samples were KRAS positive (mutated). In 12 of the 58 KRAS negative (wild type) samples, the V600E mutation in codon 15 of the BRAF gene was demonstrated. No differences in the frequency and distribution of mutations, stratified by gender, age, primary tumor versus metastasis, or tumor location were observed. Conclusions: Twelve percent of KRAS negative colorectal cancer samples showed BRAF gene mutation. Considering that 42% of samples have a KRAS mutation, 54% of patients should not respond to therapies with monoclonal antibodies directed against epidermic growth factor (EGFR) pathway.
Subject(s)
Female , Humans , Male , Middle Aged , Adenocarcinoma/genetics , Colorectal Neoplasms/genetics , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Genotype , Neoplasm Staging , Polymorphism, Restriction Fragment LengthABSTRACT
A incidência do melanoma cutâneo em pacientes adultos jovens tem aumentado consideravelmente nos últimos anos. Há, contudo, carência de conhecimentos clinicopatológicos e moleculares sobre os melanomas que ocorrem nessa faixa etária. O presente estudo teve por objetivo avaliar 132 casos de melanoma cutâneo primário em pacientes com idade entre 18 e 30 anos, com ênfase no estudo das características clínicas, histopatológicas e avaliação molecular das mutações nos genes BRAF, NRAS e KIT. Em relação aos achados clínicos e histopatológicos, houve predomínio de indivíduos do sexo feminino (61,4%), sendo o tronco o sítio anatômico mais comumente envolvido (44,3%) e o melanoma extensivo superficial o tipo histológico predominante (79,5%). A mutação V600E no gene BRAF (BRAFV600E) foi analisada em 93 casos, utilizando-se a técnica de RT-PCR. Essa mutação foi identificada em 38,7% (36/93) e, estatisticamente, associada à fase vertical de crescimento (p = 0,01), infiltrado inflamatório discreto (p = 0,02) e presença de mitose intradérmica (p = 0,004). Houve, ainda, forte indício de associação com a presença de ulceração (p = 0,05). Todas essas variáveis apresentaram associação com pior prognóstico do melanoma cutâneo. Observou-se predomínio da mutação BRAFV600E em regiões anatômicas relacionadas à exposição solar intermitente. Nenhum caso de melanoma com fenômeno de regressão apresentou mutação BRAFV600E (p < 0,05). Não houve associação significativa entre BRAFV600E e sexo, tipo histológico, nível de Clark, índice de Breslow, elastose solar, invasão angiolinfática e perineural, satelitose, nevo melanocítico coexistente e sobrevida. A pesquisa de mutações NRAS, pela técnica de RT-PCR, detectou frequência de 3,95% (3/76). As três mutações encontradas foram do tipo 61K e ocorreram em pacientes do sexo masculino e em região de cabeça e pescoço. As mutações BRAFV600E e NRAS, quando presentes, eram mutuamente exclusivas. A frequência de mutações KIT, analisadas por...
The incidence of cutaneous melanoma in young adults has dramatically increased in recent years. However, there is scarce data about the clinicopathological and molecular characteristics on the melanomas occurring at this age group. The present study aimed to evaluate 132 patients aged between 18 and 30 years with primary cutaneous melanoma with emphasis on the study of clinical, histopathological characteristics and molecular evaluation of mutations in BRAF, NRAS and KIT genes. Regarding the clinical and histopathological findings, the following results were found: female predominance (61.4%), trunk was the most commonly anatomical site involved (44.3%) and superficial spreading melanoma, was the most common histological type (79.5 %). The V600E mutation in BRAF (BRAFV600E) gene was analyzed in 93 cases, using RT-PCR. It was present in 38.7% (36/93) and statistically related to the vertical growth phase (p = 0.01), mild inflammatory infiltration (p = 0.02) and the presence of intradermal mitosis (p = 0.004). There was, also, strongly evidence of an association with the presence of ulceration (p = 0.05). Worse prognosis was associated with these variables. There was a predominance of BRAFV600E mutation in anatomical regions related to intermittent sun exposure. No cases of melanoma with BRAFV600E mutation showed regression phenomenon (p < 0.05). There was no significant association between BRAFV600E and gender, histological type, Clark level, Breslow thickness, solar elastosis, angiolymphatic and perineural invasion, sattelitosis, coexisting melanocytic nevus and survival. The presence of a mutation in NRAS, by RT-PCR was seen in 3.95% (3/76) of the cases. All these three mutations were of type 61K, occurred in male patients and the head and neck region. BRAFV600E and NRAS mutations, when present, were mutually exclusive. The frequency of KIT mutations, analyzed by sequencing, was 11.1% (3/27). The three mutations identified in this gene were located in...
Subject(s)
Humans , Male , Female , Adolescent , Young Adult , Adult , Melanoma , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins c-kit , Skin Neoplasms , Young AdultABSTRACT
Objective To investigate the expressions of PTEN and p21 genes in Hazak patients with esophageal can-cer. Methods The expressions of PTEN and p21 genes were detected by RT-PCR in 48 samples (cancer tissues and nor-mal tissues) of patients with esophageal cancer. The relationship between the expressions of PTEN and p21 genes, tumor dif-ferentiation, TNM stage, clinical phase and lymph node metastasis were analyzed. Results The positive rates of PTEN gene were 75%and 45.8%in cancer and distant normal tissues. The expression of PTEN was significantly higher in cancer tis-sues than that of distant normal tissues (χ2=8.537,P＜0.05). The positive rates of p21 gene were 95.8%and 97.9%in cancer and distant normal tissues, and no significant difference between them (χ2=0.344,P>0.05). There was no correlation be-tween expressions of PTEN and p21and the tumor differentiation, the depth of invasion and lymph node metastasis in esopha-geal cancer. Conclusion PTEN and p21 genes are not the primary genes for the carcinogenesis of esophageal cancer in Hazak.
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Objective To research the expression change and the sense of histone acetylation and p21WAF1 protein in breast cancer. Methods Pathological morphologic changes of breast cancer were identified by H. E. staining. Immunohistochemical study for p21WAF1 protein expression was performed on 80 breast cancers and 80 normal breast tissues. The expressions of acetylated histone H3, H4 and p21WAF1 protein in 80 breast cancers and 80 normal breast tissues were analyzed by Western blot. Results H.E. staining discovered that the tissue structure and cell morphous of breast cancer had visible atypia, compared with normal breast tissues. The immunohistochemical results displayed that the positive expression of p21 WAF1 in 80 breast cancers and 80 normal breast tissues was 49 cases(61.25 %) or 3 cases (3.75%), respectively. There was significantly difference in the expression level between the 80 breast cancers and 80 normal breast tissues (P ＜ 0. 05). The expression levels of p21WAF1 protein in breast cancer tissues were higher than that in normal breast tissues, 0. 78 ± 0. 095 or 0. 65 ± 0. 055, respectively. The expression levels of acetylated histone H3, H4 protein in normal breast tissues were higher than those in breast cancer tissues. H3 was 2. 35 ± 0. 340 or 1.07 ± 0. 067, respectively, H4 was 3.44 ± 0. 202 or 1.11 ± 0. 086, respectively. There was significantly difference in the expression levels between the 80 breast cancers and 80 normal breast tissues (P ＜0. 01). Conclusions The occurrence and development of breast cancers may be related to the expression change of histone acetylation and p21WAF1 protein.
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Objective To investigate the photo-protective mechanisms of hydroxychloroquine and epigallocatechin gallate (EGCG) on HaCaT cells damaged from UVB irradiation. Methods Subconfluent HaCaT cells were irradiated with different doses of UVB irradiation and treated with the above listed agents. The mRNA expression levels of p53, p21, c-fos and GADPH genes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Results UVB irradiation induced mRNA expression of p53, p21 and c-fos in cultured HaCaT cells, which were alleviated by hydroxychloroquine and EGCG treatment in UVB irradiation group. Conclusions The photo-protective effects of hydroxychloroquine and EGCG on HaCaT cells by UVB irradiation might be related to inhibition of the expression of p53,p21 and c-fos genes.