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Objective:To investigate the effect of S-phase kinase-associated protein 2 (SKP2) expression level on radiosensitivity of human hepatocellular carcinoma (HCC) cells and the correlation of SKP2 expression with clinical prognosis of patients with HCC.Methods:The expression levels of SKP2 gene in liver cancer tissues and normal tissues were validated and its correlation with clinical prognosis of HCC patients was analyzed based on the TCGA database. Western blot was used to determine the SKP2 protein levels in HCC cell lines before and after radiation. CRISPR/Cas9 technology was employed to delete the promoter and first exon of SKP2 gene in PLC/PRF/5 (PLC) and Hep3B HCC cells for generating the SKP2 knockout cell lines. The difference of radiosensitivity and cell survival rate between normal (SKP2 +/ +) and SKP2 knockout (SKP2 -/ -) HCC cells was determined by using cell clonogenic assay and CCK8 kit. Results:Compared with normal tissues, the expression levels of SKP2 gene in HCC were increased based on the results of TCGA database analysis. K-M analysis showed that the HCC patients with high SKP2 expression had relatively poor prognosis. The 5-year overall survival (OS) was 34.6% in high SKP2 expression HCC patients and 50.6% in low SKP2 expression HCC patients, respectively ( HR=2.18, 95% CI=1.46-3.27, P<0.001). In vitro experiment showed that the expression levels of SKP2 were significantly increased after radiation in HCC cells. Simultaneously, deletion of SKP2 significantly increased the radiosensitivity of HCC cells. Conclusion:The expression level of SKP2 gene is increased in HCC patients, and patients with high SKP2 expression have worse prognosis than those with low expression. Radiation can upregulate the SKP2 expression levels in HCC cells, while the radiosensitivity of the cells is significantly increased after SKP2 deletion.
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ObjectiveTo explore the effect of Scutellariae Barbatae Herba extract on on the cycle arrest of nasopharyngeal carcinoma cells and the possible mechanism by adding different concentration of Scutellariae Barbatae Herba extract （0.25， 0.5， 1 g·L-1） in the culture medium， taking CNE1 （nasopharyngeal carcinoma cells） as the research object. MethodAfter the treatment of CNE1 by Scutellariae Barbatae Herba extract， cell counting kit-8 （CCK-8） was used to detect cell proliferation， and Giemsa staining was used to detect the clone formation rate. Flow cytometry was used to detect cell cycle distribution， and reverse transcription-polymerase chain reaction （ RT-PCR） assay and Western blot assay were used to detect the relative expression of messenger ribonucleic acid （mRNA） by small interfering RNA （siRNA） or overexpression. ResultAs compared with the blank group， the proliferation and colony formation rate of CNE1 in the Scutellariae Barbatae Herba extract group significantly decreased （P<0.05， P<0.01） in a dose and time-dependent manner， whereas the percentage of cells in the presynthetic phase （G0/G1） increased （P<0.05， P<0.01）. The expression level of S-phase kinase associated protein 2 （SKP2） in the Scutellariae Barbatae Herba extract group significantly decreased （P<0.01） as compared with the blank group. As compared with the Scutellariae Barbatae Herba extract group， the protein levels of p21 and p27 significantly decreased in the overexpressed SKP2+ Scutellariae Barbatae Herba extract group （P<0.01）. As compared with the blank group， the signal activation and the phosphorylation level of signal transducer and activator of transcription 3 （STAT3） of CNE1 in the S. barbata extract group significantly decreased （P<0.05， P<0.01）. ConclusionScutellariae Barbatae Herba extract effectively inhibits the proliferation of CNE1， and the mechanism may be related to its action on the STAT3/SKP2 pathway.
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ObjectiveTo explore the effect of Scutellariae Barbatae Herba extract on on the cycle arrest of nasopharyngeal carcinoma cells and the possible mechanism by adding different concentration of Scutellariae Barbatae Herba extract （0.25， 0.5， 1 g·L-1） in the culture medium， taking CNE1 （nasopharyngeal carcinoma cells） as the research object. MethodAfter the treatment of CNE1 by Scutellariae Barbatae Herba extract， cell counting kit-8 （CCK-8） was used to detect cell proliferation， and Giemsa staining was used to detect the clone formation rate. Flow cytometry was used to detect cell cycle distribution， and reverse transcription-polymerase chain reaction （ RT-PCR） assay and Western blot assay were used to detect the relative expression of messenger ribonucleic acid （mRNA） by small interfering RNA （siRNA） or overexpression. ResultAs compared with the blank group， the proliferation and colony formation rate of CNE1 in the Scutellariae Barbatae Herba extract group significantly decreased （P<0.05， P<0.01） in a dose and time-dependent manner， whereas the percentage of cells in the presynthetic phase （G0/G1） increased （P<0.05， P<0.01）. The expression level of S-phase kinase associated protein 2 （SKP2） in the Scutellariae Barbatae Herba extract group significantly decreased （P<0.01） as compared with the blank group. As compared with the Scutellariae Barbatae Herba extract group， the protein levels of p21 and p27 significantly decreased in the overexpressed SKP2+ Scutellariae Barbatae Herba extract group （P<0.01）. As compared with the blank group， the signal activation and the phosphorylation level of signal transducer and activator of transcription 3 （STAT3） of CNE1 in the S. barbata extract group significantly decreased （P<0.05， P<0.01）. ConclusionScutellariae Barbatae Herba extract effectively inhibits the proliferation of CNE1， and the mechanism may be related to its action on the STAT3/SKP2 pathway.
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@#[Abstract] Objective： ：The co-immunoprecipitation and mass spectrometric analysis was carried out to obtain the S-phase kinase-associated protein 2 (SKP2)-binding proteins in HeLa cells, and the biological functions of these binding proteins were forecast. Methods: The co-immunoprecipitation system was established by co-immunoprecipitation and Western blotting assay; the specific protein gel of SKP2-binding proteins was obtained by SDS-PAGE and silver staining assay; the potential SKP2-binding proteins was identified by mass spectrometric analysis; and the GO (Gene ontology) analysis and KEGG analysis was carried out by bioinformatics technique. Results: The expression level of SKP2 protein in HeLa cells was high enough for co-immunoprecipitation assay; the co-immunoprecipitation system was established successfully, and SKP2-binding proteins was obtained; a total of 563 proteins were identified by mass spectrometric analysis, and 270 proteins with high credibility were obtained after screening. The GO analysis and KEGG analysis was carried out for the 270 proteins to forecast their functions and pathways. Conclusion: The SKP2-binding proteins were screened successfully, and it was the foundation for the subsequent screening of target-binding proteins and the search for targeting drugs.
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Objective To investigate the expression of microRNA-340 (miR-340) in hepatocellular carcinoma (HCC) and its effect on cell biological behavior. Methods We collected 40 frozen HCC tissues and adjacent non-tumor tissues from patients undergoing hepatectomy of HCC at The First Affiliated Hospital of Chongqing Medical University from Mar. 2015 to Sep. 2016. The expression of miR-340 in all tissues was detected by qPCR and the relationship between miR-340 expression and clinicopathological parameters was analyzed. Simultaneously, the expression of miR-340 in normal hepatocyte (HL-7702) and four hepatoma cells lines (Hep3B, Bel-7402, HepG2, SMMC-7721) was detected by qPCR after incubation for 48 h. The eukaryotic expression vector with miR-340 or control reagent was transfected into SMMC-7721 cells using EndoFection™-Max to increase or inhibit the expression of miR-340, and then the cells were cultured for 24 h, 48 h and 72 h. The proliferation of SMMC-7721 cells was detected by CCK-8 assay, and the apoptosis was detected by flow cytometry. The target gene of miR-340 was predicted by bioinformatics software, and the effect of miR-340 on target gene was further verified by qPCR and Western blotting. Results The expression of miR-340 in HCC tissues was significantly lower than that in the adjacent non-tumor tissues (P〈0. 01), and was correlated with hepatitis B surface antigen, HBV DNA, tumor size and TNM stage (all P〈0. 01). Besides, the expression of miR-340 in HL-7702 cells was significantly higher than that in Hep3B, Bel-7402, HepG2 and SMMC-7721 cells (P〈0. 05, P〈0. 01). CCK-8 assay results showed that overexpression of miR-340 inhibited proliferation of SMMC-7721 cells, while inhibition of miR-340 promoted cell proliferation (P〈0. 05, P〈0. 01). Overexpression of miR-340 significantly promoted SMMC-7721 cells apoptosis, while suppression of miR-340 significantly inhibited cells apoptosis (all P〈0. 01). S-phase kinase-associated protein 2 (SKP2) was a target gene of miR-340 as indicated by bioinformatics software. Further, qPCR and Western blotting results showed that overexpression of miR-340 inhibited the mRNAand protein expression of SKP2, while inhibition of miR-340 increased the mRNA and protein expression of SKP2. Conclusion The abnormal expression of miR-340 may be associated with the HBV infection, and miR-340 may be an indicator to evaluate the progression and prognosis of HCC. MiR-340 can inhibk proliferation and promote apoptosis of SMMC-7721 cells, which may be effected by inhibiting the SKP2 expression.
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Objective To investigate the effects of liver X receptor (LXR) agonist on the proliferation of mouse pancreatic β cell line MIN6 cells.Methods The viability,changes of cell cycle,mRNA levels of S phase kinase associated protein 2 (Skp2) and p27,and protein levels of Skp2 and p27 in MIN6 cells treated with LXR agonist T0901317 were determined by the CCK-8 method,flow cytometry,real-time RT-PCR and western blot,respectively.Results The viability of MIN6 cells treated with 1 μmol/L,5 μmol/L and 10 μnol/L of T0901317 were (98.54 ±0.94)％,(87.03 ±0.93)％ and (75.57 ± 1.85)％ of the controls,respectively,and there was significant difference among them (F =301.90,P ＜ 0.01).The percentages of G1 phase cells in the MIN6 cells treated with 0 μmol/L,1 μmol/L,5 μmol/L and 10 μmol/L of T0901317 were (35.93 ±2.25)％,(38.45 ±0.91)％,(45.46±1.34)％ and (53.28 ± 1.14) ％,respectively,and there was significant difference among them (F =80.83,P ＜ 0.01).Similarly,the percentages of S phase cells in the MIN6 cells treated with 0 μmol/L,1 μmol/L,5 μmol/L and 10 μmoi/L of T0901317 were (52.87 ± 1.19) ％,(48.65 ± 0.85) ％,(36.31 ± 1.37) ％ and (31.45 ± 1.22) ％,respectively,and there was also significant difference among them (F =221.30,P ＜ 0.01).The protein levels of p27 in the MIN6 cells treated with 10 μmol/L of T0901317 (2.84 ± 0.14) were significantly higher than that in the controls (2.28 ± 0.10) (t =4.54,P ＜ 0.05),while there was no significant difference in the mRNA levels of p27 between them (t =0.28,P ＞ 0.05).However,10 μmol/L of T0901317 significantly decreased mRNA (0.52 ± 0.02,t =29.22,P ＜ 0.01) and protein levels (0.98 ± 0.12 vs 1.89 ± 0.01,t =10.98,P ＜ 0.01) of Skp2 in MIN6 cells.Based on the control siRNA transfection group as a reference (100％),the cell survival rates of the p27 siRNA transfection group,10 μmol/L of T0901317 treatment group and the intervention group (p27 siRNA transfection + T0901317 treatment) were (100.97 ± 1.08) ％,(75.03 ± 1.83) ％ and (86.67 ± 2.45) ％,respectively.There was no significant difference between the control siRNA and p27 siR-NA transfection groups (t =1.542,P ＞ 0.05).Compared with the control siRNA transfection group,the cell survival rates of the T0901317 treatment group decreased (t =23.58,P ＜ 0.01).There was also significant difference in the cell survival rates between the T0901317 treatment group and the intervention group (t =7.77,P ＜ 0.01).Conclusion The activation of LXR may induce pancreatic β cell cycle arrest by up-regulating the expression of p27 and down-regulating the expression of Skp2.
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OBJECTIVE: Emerging evidence has shown that the F‑box protein S‑phase kinase‑associated protein 2 (Skp2) plays an important role in the pathogenesis of breast cancer (BC). Our study aimed to evaluate the prognostic value of Skp2 in BC patients using meta‑analysis based on the published studies. MATERIALS AND METHODS: Eligible studies were identified by searching the online databases such as PubMed, EMBASE, and Web of Science up to October 2015. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to clarify the correlation between Skp2 expression and indicators of BC clinical outcomes, including overall survival (OS), disease‑free survival (DFS), and BC‑specific survival. RESULTS: In total, nine studies with 1820 BC patients were included for final analysis. The meta‑analysis suggested that Skp2 overexpression was associated with poor OS (HR = 2.58, 95% CI: 1.83–3.63, P = 0.000) and poor DFS (HR = 2.12, 95% CI: 1.48–3.05, P = 0.000) in BC patients. CONCLUSIONS: This meta‑analysis indicates that enhanced Skp2 is an independent prognostic factor for poor cancer survival.
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Objective To study the expression and functional role of S phase kinase associated protein 2 (Skp2) in development of glioma. Methods Sixty surgically removed specimens of primary glioma and 4 normal brain specimens were obtained from the Department of Neurosurgery, Navy General Hospital of PLA, from June 2001 to June 2006. All the specimens were graded according to WHO Criteria as astrocytoma (grade II, n=20), anaplastic astrocytoma (grade III, n=20) and glioblastoma (grade IV, n=20). Western blotting and immunohistochemistry were used to detect the expression of Skp2 in specimens of glioma of different grades and normal brain tissue. Results Western blotting demonstrated that the expression of Skp2 in normal brain tissue was significantly lower than that in glioma specimens, and the expression increased in degree along with the elevation of malignant grade. Immunohistochemical staining showed that the Skp2 positively expressed in both the normal brain tissues and gliomas. However, Skp2 was weakly positive and mainly found in the cytoplasm in normal brain tissue and low grade glioma (grade II), while it was strongly positive and mainly observed in the nuclei in high grade glioma (grade III and IV). Furthermore, the positive expression of Skp2 was also observed in vascular endothelial cells of glioma tissue, and the glioma cells with positive Skp2 were found to gather around the vessels in glioma specimens. Statistically analysis showed that the expression of Skp2 was significantly higher in high grade glioma tissues (grade III and IV) than in normal brain tissues and low grade glioma (grade II) with significant difference (P<0.01). Conclusions Along with the increase in malignancy of glioma, the expression of Skp2 was found increasing gradually, and it transferred from cytoplasm to nuclei. Skp2 positively expressed in both vascular endothelial cells of glioma tissue and the glioma cells around vessels in glioma specimens. The change in Skp2 expression might be closely related to an increase in malignancy of glioma, the formation of new vessels, and metastasis of tumor cells.
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Objective To study the expression and functional role of S phase kinase associated protein 2 (Skp2) in development of glioma. Methods Sixty surgically removed specimens of primary glioma and 4 normal brain specimens were obtained from the Department of Neurosurgery, Navy General Hospital of PLA, from June 2001 to June 2006. All the specimens were graded according to WHO Criteria as astrocytoma (grade II, n=20), anaplastic astrocytoma (grade III, n=20) and glioblastoma (grade IV, n=20). Western blotting and immunohistochemistry were used to detect the expression of Skp2 in specimens of glioma of different grades and normal brain tissue. Results Western blotting demonstrated that the expression of Skp2 in normal brain tissue was significantly lower than that in glioma specimens, and the expression increased in degree along with the elevation of malignant grade. Immunohistochemical staining showed that the Skp2 positively expressed in both the normal brain tissues and gliomas. However, Skp2 was weakly positive and mainly found in the cytoplasm in normal brain tissue and low grade glioma (grade II), while it was strongly positive and mainly observed in the nuclei in high grade glioma (grade III and IV). Furthermore, the positive expression of Skp2 was also observed in vascular endothelial cells of glioma tissue, and the glioma cells with positive Skp2 were found to gather around the vessels in glioma specimens. Statistically analysis showed that the expression of Skp2 was significantly higher in high grade glioma tissues (grade III and IV) than in normal brain tissues and low grade glioma (grade II) with significant difference (P<0.01). Conclusions Along with the increase in malignancy of glioma, the expression of Skp2 was found increasing gradually, and it transferred from cytoplasm to nuclei. Skp2 positively expressed in both vascular endothelial cells of glioma tissue and the glioma cells around vessels in glioma specimens. The change in Skp2 expression might be closely related to an increase in malignancy of glioma, the formation of new vessels, and metastasis of tumor cells.
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ObjectiveTo investigate the correlation of Skp2,p27kiP1 and p21WAF1 expression with the clinicopathological features of ovarian serous cystadenocarcinomas.Methods Expressions of Skp2 ,p27kiP1 and p21WAF1 were examined by immunohistochemical staining in 124 epithelial ovarian tumors (25 serous cystadenomas, 19 borderline serous cystadenomas, and 80 serous cystadenocarcinomas) Results(1) The expression of Skp2 in serous cystadenocarcinomas (47.5％)was significantly higher than that in borderline serous cystadenomas (0％)and serous cystadenomas (0％)(P ＜ 0.001) .The p27kiP1 expression in serous cystadenocarcinomas (35.0％) was significantly lower than that in borderline serous cystadenomas(73.7％)and serous cystadenomas (80.0％) .The p21WAF1 staining frequency in serous cystadenocarcinomas (38.8％)was significantly lower than in borderline serous cystadenomas (73.7％)and serous cystadenomas (80.0％) .(2) The Skp2 protein expression in serous cystadenocarcinomas was positively correlated with clinicopathological stage,histological differentiation degree and lymph node metastasis of the tumors.The p27kiP1, p21WAF1 protein expression in serous cystadenocarcinomas was reversely correlated with clinicopathological stage and histological differentiation degree of the tumors(Ps ＜ 0.05) .(3) The Skp2 protein expression in serous cystadenocarcinomas was reversely correlated with that of p27kiP1 , p21WAF1.Conclusion The Skp2 protein expression in serous cystadenocarcinomas was increased and positively correlated with the clinicopathological features of ovarian serous cystadenocarcinomas.Skp2 protein expression was reversely correlated with p27kip1 ,p21WAF1.Skp2 protein expression may play an important role in the development and progression of serous cystadenocarcinomas.
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Objective To investigate whether there is any functional link between p27~(Kip1) function and all-trans retinoic acid (RA) in the control of neuronal differentiation of immortalized human neural progenitor cells (hSN12W-TERT cells). To investigate the mechanism by which p27~(Kip1) regulates the differentiation of immortalized human neural progenitor cells. Methods hSN12W-TERT cells were derived from the striatums of human embryos at 12 weeks gestation and cultured with serum-free medium in presence of EGF and bFGF. At the appropriated time, hSN12W-TERT cells were exposed to 1μmol/L RA for 3, 5, 7 days respectively. The experiment was repeated there times. Cell cycle analysis was performed by flow cytometry analysis (FACS). The expression of p27~(Kip1), p21~(cip1), cyclin-dependent kinase 2 (cdk2), p-cdk2 and S-phase kinase-associated protein 2 (skp2) in hSN12W-TERT cells before and after RA treatment cells were determined by using Western blotting analysis. Results FACS result showed that 77.25% of proliferating hSN12W-TERT cells were in the G1/G0-phase while 9.38% of cells in the S-phase. Following RA treatment, cell growth was arrested, and 85.68% of cells accumulated in G1/G0-phase while 8.57% of cells in the S-phase. Western blotting analysis demonstrated that the levels of p27~(Kip1) in the hSN12W-TERT cells increased following 3 days' treatment with RA compared with those of normal untreated cells, with a peak at 5 days (P<0.05). The similar results were acquired both in nuclear proteins and in cytoplasm proteins of hSN12W-TERT cells. The expression level of p21~(cip1) decreased in response to RA treatment. RA did not affect the expression of cdk2, but the expression of p-cdk2, which represented the activity of cdk2, was markedly decreased in response to RA treatment. Skp2, which was required for the ubiquitin-mediated degradation of p27~(Kip1), was detected in proliferating hSN12W-TERT cells. The expression of skp2 reduced dramatically in response to RA treatment in a time-dependent manner.Conclusion There is a functional link between RA and p27~(Kip1) function in the control of neuronal differentiation in hSN12W-TERT cells. P27~(Kip1) plays a key role during neuronal differentiation. Moreover, high levels of p27~(Kip1) are associated with its degradation inhibiting through reducing proteasome-dependent proteolysis.
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Objective To study the expressions of SKP2 and p27 in gastric carcinoma and pericancerous tissues and to detect the relationship between their expressions and clinicopathological features. Methods Forty-nine cases of gastric carcinoma spicemen and 20 cases of tissue adjacent to the carcinoma were cut and made into paraffin-embedded slices. The expressions of SKP2 and p27 were then detected by SP immunohistochemical method. Results The positive expression rate and score of SKP2 were both significantly higher in the gastric carcinoma tissues than those in pericancerous tissues (P
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AIM:To investigate the effect of trichostatin A(TSA)on p27kip1 gene expression in vascular smooth muscle cells.METHODS:Reverse transcription-polymerase chain reaction(RT-PCR)was used to measure the level of p27kip1 mRNA.The protein levels of p27kip1 and S-phase kinase-associated protein-2(skp2)were determined by Western blotting.20S proteasome activity was quantified by using a fluorogenic proteasome-specific substrate.RESULTS:TSA did not affect mRNA level of p27kip1 in VSMCs,but attenuated serum-induced downregulation of p27kip1 through stabilizing p27kip1 turnover.In addition,TSA decreased the expression of skp2,an F-box protein that targets p27kip1 for degradation,but had no effect on proteasome activity.CONCLUSION:TSA regulates p27kip1 expression at the post-translational level in VSMCs.