ABSTRACT
Objective To investigate the regulatory effect of miRNA-370(miR-370)on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth. Methods RCC cells were transfected with dsRNA known lack homology to human genes (control group) and miR-370 (experimental group) by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry (FCM). Cell viability and proliferation ability were measured by cell viability assay (MTS) and colony culture assay. Results The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04±0.33, 1.04±0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68±0.62 (t=7.535, P<0.001), 3.15±0.29 (t=9.975, P<0.001). Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1phase after transfection of miR-370.MTS results showed that after transfection of miR-370,the number of colonies formed by ACHN and 786-O cells in the control group was 113±30 and 106±27 respectively. The number of colonies formed by experimental group was significantly reduced by 53±17 (t=2.982, P=0.041) and 50±16 (t=3.089, P=0.037). Conclusion miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.
ABSTRACT
Objective To investigate the regulatory effect of miRNA-370(miR-370)on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth. Methods RCC cells were transfected with dsRNA known lack homology to human genes (control group) and miR-370 (experimental group) by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry (FCM). Cell viability and proliferation ability were measured by cell viability assay (MTS) and colony culture assay. Results The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04±0.33, 1.04±0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68±0.62 (t=7.535, P<0.001), 3.15±0.29 (t=9.975, P<0.001). Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1phase after transfection of miR-370.MTS results showed that after transfection of miR-370,the number of colonies formed by ACHN and 786-O cells in the control group was 113±30 and 106±27 respectively. The number of colonies formed by experimental group was significantly reduced by 53±17 (t=2.982, P=0.041) and 50±16 (t=3.089, P=0.037). Conclusion miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.
ABSTRACT
Objective To investigate the effect of dsP21-555 transfection on the expression of tumor suppressor gene p21 in renal clear cell carcinoma cell lines ACHN and 786-O.Methods Renal clear cell carcinoma cells were transfected with dsControl and dsP21-555 with Lipofectamine 3000 respectively.Real-time quantitative PCR (RT-qPCR) and Western blotting were used to detect the expression of p21 mRNA and protein.Cell cycle distribution was detected by flow cytometry (FCM).Cell viability and proliferation were analyzed by cell viability assay (MTS method) and colony culture assay.Results In ACHN and 786-O cells, the expressions of p21 mRNA in dsP21-555 group (2.86±0.33, 1.96±0.35) were significantly higher than those in dsControl group (1.05±0.34, 1.01±0.14), which were increased to 2.72 times (t=7.640, P<0.001) and 1.95 times (t=5.058, P=0.002).Western blotting showed that the expressions of P21 protein were up-regulated in both renal cell lines, which was consistent with p21 mRNA up-regulation.The result of FCM showed that the cell cycle was blocked in G0-G1 phase (57.08%±5.66% vs.46.06%±4.60%, t=3.023, P=0.023;61.58%±6.23% vs.42.25%±6.08%, t=4.444, P=0.004) after transfection of dsP21-555 in renal clear cell carcinoma cells.MTS result showed that the vitality of both cell lines after transfection of dsP21-555 decreased compared with dsControl group, their absorbance values were 0.85±0.20 vs.1.27±0.13, t=3.410, P=0.014;1.04±0.25 vs.1.55±0.10, t=3.758, P=0.009.Colony culture experiments showed that the numbers of colonies formed by ACHN and 786-O in the dsControl group were 110.91±26.21 and 129.99±22.87 respectively, and the numbers of colonies formed in the dsP21-555 group were 59.37±14.23 (t=3.456, P=0.014) and 71.26±21.38 (t=3.745, P=0.010), indicating that the proliferation of cells in the dsP21-555 group was significantly reduced.Conclusion dsP21-555 can up-regulate the expression of p21 gene in renal clear cell carcinoma cells and inhibit the growth of carcinoma cells, suggesting that dsP21-555 may become a new gene therapy tool.
ABSTRACT
Objective To investigate the expressions of p15,p16,and p21 in skin of uygur patients with psoriasis and its significance in the development of psoriasis.Methods The expressions of p15,p16,and p21 were studied with immunohistochemical method in the Xinjiang Uygur psoriatic and normal Uygur skins.Results The positive expression rate of p16 gene was 12.5％ in skin lesions of patients with psoriasis Uygur,and 66.67％ in the normal group.The positive expressions of p15,and p21 genes in skin lesions of patients with psoriasis and healthy Uygur population were higher.Conclusions There is a significant correlation between the development of uygur psoriasis vulgaris and abnormal expression of p16.
ABSTRACT
Objective To determine the expression of HPV16/18,31/33 DNA and p53,p21~(WAF1) and MDM2 proteins in invasive squamous cell carcinoma of cervix (ISCC)and to indicate the significance of them in the occurrence and development of ISCC.Methods Using tissue microarray,in situ hybridization (ISH) and immunohistochemical method,we detected the expression of HPV16/18,31/33 and investigate the expres- sion of p53,p21~(WAF1),MDM2 and proteins in ISCC,CIN and NCE.Results was analysed by SPSS vision 12.5. Results The positive expression rate of HPV16/18,p53,p21~(WAF1),MDM2 in ISCC was markedly higher than in CIN and NCE.We found the difference between HPV31/33 and lymph node transfer.Significant relation- ship was observed between p53 protein expression and histological grade and lymph node metastasis of the cancer.There was positive correlation between the expression of p21~(WAF1) protein and the depth of invasion(P
ABSTRACT
BACKGROUND: Cyclin-dependent kinase inhibitors (CDKI), including p21, p27 and p57 of the KIP family, are negative regulators of cell cycle progression and potentially act as tumor suppressors. The expression of p21 is induced by tumor suppressor gene p53. Mutations of p53 are common and found in various human cancers. Thus, the function of p21 as a tumor suppressor may be not retained after p53 mutation in human cancers. The aim of our study was to evaluate the tumor suppressive activity of p21 and p53 in human gastric cancer. METHODS: One hundred and two patients who underwent surgery for gastric cancer at Chonnam National University Hospital were selected retrospectively for this study. The primary selection criteria were the availability of formalin-fixed and paraffin-embedded blocks and sufficient clinical follow-up for tumor-specific survival analysis. In this study, we examined the expression of p21 and p53 in human gastric cancer tissue by immunohisto-chemistry and the correlation between their expression and clinicopathological variables. RESULTS: p21 and p53 immunoreactivities were localized in the nuclei of carcinoma cells. Positive nuclear expression of p21 and p53 was demonstrated in 63.7 and 33.3% of cancer tissues, respectively. No apparent correlation was noted between p21 and p53 expression. Negative expression of p21 correlated with advanced stage and lymph node metastasis (p=0.028 and 0.017, respectively). Moreover, negative expression of p21 correlated with poor survival (p=0.037). Positive expression of p53 correlated with depth of tumor invasion (p=0.029). However, no significant correlation could be observed between the status of p53 expression and survival. Combined analysis of p21 and p53 status showed that p21 negative and p53 positive tumors had a poorer survival than other group tumors (p=0.026). CONCLUSION: These results suggest that the status of p21 and p53 expression may help in predicting the aggressive behavior of gastric cancer. However, further studies are warranted to clarify the impact of p53 on the function of p21 as a tumor suppressor.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma/genetics , Gene Expression , Genes, p53 , /genetics , Prognosis , Tumor Suppressor Protein p53/genetics , Retrospective Studies , Stomach Neoplasms/genetics , Survival Analysis , Biomarkers, Tumor/geneticsABSTRACT
Objective To investigate the expression of p21ras and microvascular density (MVD) in pancreatic carcinoma and to identify their clinico-pathological significance. Methods Expressions of p21ras and MVD were immunohistochemically assessed in 48 cases of pancreatic carcinomas. Results The expression rate of p21ras in pancreatic carcinomas was 60.40%, the MVD was (22.207?5.815) and (18.053?5.502) respectively in the p21ras-positive group and p21ras-negative group (P
ABSTRACT
Objective To investigate the expression of p21ras and vascular endothelial growth factor (VEGF) in pancreatic carcinoma, and to elucidate its clinicopathological implication. Methods Expressions of p21ras and VEGF were immunohistochemically examined in 48 cases of pancreatic carcinomas. Results The expressions of p21ras and VEGF in pancreatic carcinomas were 60.40% and 64.58%, respectively, and they were significantly higher than those in adjacent tissues (P
ABSTRACT
Objective To study the relationship between the expression of p53 and its downstream effecter p21~(waf1)protein and multi-drug resistance(MDR) of Tca8113/BLM cell line.Methods The cDNA of wildtype p53 gene was introduced into the drug-resistance cell line Tca8113/BLM by Lipofectamine-mediated transfection.The expression of p53 mRNA in Tca8113/BLM and its parental counterpart Tca8113 cell lines were analyzed using molecular beacons.The expression of p53,p21~(waf1),P-gp,and MRP proteins in Tca8113/BLM cell line transfected by wt-p53 cDNA,the untransfected control Tca8113/BLM cell line,its parental counterpart Tca8113 were analyzed by Western blotting.MTT assay was used to determine the drug-sensitivity of different cell lines.The cell cycle distribution and apotosis of different cell lines were also observed by flow cytometery(FCM) and laser confocus microscope(LCM),respectively.Results The expression of p53 protein was significantly lower,and no p21~(waf1) protein was detected in Tca8113/BLM cells.The protein expression of P-gp and MRP were significantly higher in Tca8113/BLM cells than in Tca8113 cells.By Western Blotting,the expression of p53 and p21~(waf1)proteins was significantly increased,while the expression of P-gp and MRP proteins was significantly decreased in Tca8113/BLM/p53 cells.Comparing to Tca8113/BLM cells,the sensitivity of BLM treatment in Tca8113/BLM/p53 cells was 10.1 fold increased.Tca8113/BLM/p53 cells decreased at G1 and S phase,and increased at G2 phase.LCM results showed that the apoptosis of Tca8113/BLM/p53 cells was more obvious than Tca8113/BLM cells when treated with the drug BLM.Conclusion The multi-drug resistance in Tca8113/BLM cell line is involved in down-regulation of p53 and p21~(waf1) protein levels,and up-regulation of P-gp and MRP protein levels.
ABSTRACT
AIM: To explore the effects of cholestoral and mevalonate synthesis inhibitor lovastatin (LOV) on the proliferation of NB4 cells and elucidate the mechanisms. METHODS: Cell proliferation was analyzed by MTT assay;the expression of H, K, N- ras oncogenes in NB4 cells at different time point after LOV treatment were determined by semi-quantitative RT-PCR. Both total p21 Ras protein and p21 Ras protein on the cellular membrane were examined by flow cytometry. RESULTS:①LOV inhibited the proliferation of NB4 cells. ②All three kinds of ras were expressed in NB4 cells. ③LOV caused no increase in H, K, N- ras mRNA level. Amount of total p21 Ras protein did not change as the time varied. Concomitantly,p21 Ras protein localized on the cellular membrane decreased. CONCLUSION:LOV inhibits the proliferation of NB4 cells. Targeting HMG-CoA reductase, LOV blocks the isoprenylation of p21 Ras protein which affects its anchorage on the cellular membrane. No change in the H, K, N- ras mRNA and total p21 Ras protein expression is detected.