Scutellariae Barbatae Herba Extract Promotes Cycle Arrest of Nasopharyngeal Carcinoma Cells by Inhibiting STAT3/SKP2 Signaling Pathway / 中国实验方剂学杂志

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.

Scutellariae Barbatae Herba Extract Promotes Cycle Arrest of Nasopharyngeal Carcinoma Cells by Inhibiting STAT3/SKP2 Signaling Pathway / 中国实验方剂学杂志

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.

The expression level of SKP2 gene affects the radiosensitivity in hepatocellular carcinoma cells / 中华放射肿瘤学杂志

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.

Effects of SKP1 gene interference on apoptosis and regulation mechanism of ubiquitin-proteasome system in Parkinson's disease cell model / 中华行为医学与脑科学杂志

Objective:To investigate the effect of interfering S-phase kinase associated protein 1 (SKP1) gene on apoptosis in Parkinson's disease(PD) cell model induced by 1-methyl-4-phenyl-pyridine ion (MPP+ ) and the mechanism of ubiquitin proteasome system degradation of α-synuclein (α-syn) influence.Methods:SH-SY5Y cells were divided into control group, MPP+ group, SKP1 interference group, and SKP1 interference+ MG132(UPS inhibitor) group.The cells in the control group were cultured normally. The cells in the latter three groups were incubated with MPP+ (0.5 mmol/L) for 24 h as PD model cells.The cells in SKP1 interference group were transfected with lentivirus SKP1-siRNA, and the cells in SKP1 interference+ MG132 group were transfected with lentivirus SKP1 siRNA and added with MG132 (0.5 μmol/L) for 24 h. The protein levels and mRNA levels of SKP1, microtubule-associated protein light chain 3 (LC3), lysosome-associated membrane protein (LAMP), α-syn, ubiquitin activating enzyme E1 (UBE1), parkin, and p27 in cells were detected by Western blot and RT-PCR.Flow cytometry was used to detect cell apoptosis and cycle level, and CCK-8 method was used to detect cell proliferation level.Co-immunoprecipitation method was used to explore the interaction between SKP1 and p27. SPSS 23.0 software was used for statistical analysis. One-way ANOVA was used for comparison among groups, and LSD test was used for further pairwise comparison.Results:RT-PCR and Western blot results showed that the mRNA levels and protein levels of autophagy related proteins and ubiquitin related proteins LC3, LAMP2, α-syn, UBE1, parkin and p27 in the four groups were statistically significant(mRNA: F=99.155, 43.028, 138.464, 28.200, 22.009, 28.147, all P<0.05; F=245.517, 157.634, 315.920, 2 336.472, 477.429, 2 350.201, all P<0.05). The mRNA and protein levels of LC3, Lamp2, α-syn and p27 in SKP1 interference group were lower than those in MPP+ group (all P<0.05), while the mRNA and protein levels of UBE1 and parkin were higher than those in MPP+ group (all P<0.05). The mRNA and protein levels of LC3, α-syn and p27 in SKP1 interference+ MG132 group were higher than those in SKP1 interference group (all P<0.05), and the mRNA and protein levels of UBE1 and parkin were lower than those in SKP1 interference group (all P<0.05). The results of flow cytometry and CCK-8 method showed that the apoptosis rate and cell inhibition rate among the four groups were significantly different( F=2 749.420, 171.508, both P<0.05). The apoptosis rate of SKP1 interference group was lower than that of MPP+ group ((8.22±0.25)%, (15.30±0.21)%, P<0.05), while the cell inhibition rate of SKP1 interference group was lower than that of MPP+ group((26.31±3.73)%, (55.05±3.84)%, P<0.05). The apoptosis rate of SKP1 interference+ MG132 group ((9.49±0.07)%) was higher than that of SKP1 interference group, and the cell inhibition rate ((36.06±2.85)%) was higher than that of SKP1 interference group (both P<0.05). The results of immunoprecipitation method showed that P27 decreased after SKP1 immunoprecipitation. Conclusion:After SKP1 gene was interfered, the autophagy function of PD cells decreased, which may be related to parkin promoting α-syn ubiquitination, activating UBE1/ Parkin-mediated UPS pathway to degrade α-syn, and mediating P27 to inhibit apoptosis.

Inhibitory Effect and Mechanism of Jingulian Capsule on Human Breast Cancer MDA-MB-231 Cells / 中国实验方剂学杂志

Objective:To observe effect of Jingulian capsule on the proliferation of human breast cancer MDA-MB-231 cells and investigate its action mechanism against triple negative breast cancer （TNBC）. Method:The ingredients of Jingulian capsule were identified by ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）. The inhibitory effect of Jingulian capsule at different doses （0.125，0.25，0.5，1，and 2 g·L^-1） against the proliferation of MDA-MB-231 cells were detected by methyl thiazolyl tetrazolium （MTT） assay. After treatment for 24 h， the morphological changes in nuclear apoptosis of MDA-MB-231 cells were detected by Hoechst 33258 staining. The effect of different concentrations of Jingulian capsule on the apoptosis and cycle of MDA-MB-231 cells after different treatment time were determined by flow cytometry. The protein expression levels of Poly-ADP-ribose polymeras （PARP）， proto-oncogene c-Myc， cyclin B₁， and phosphorylated extracellular signal-regulated kinase （p-ERK） in each group were assayed by Western blot. Result:A total of 113 compounds in Jingulian capsule were identified by UPLC-MS/MS. As revealed by MTT assay，compared with blank group，Jingulian capsule （0.125，0.25，0.5，1，2 g·L^-1） significantly inhibited viability of MDA-MB-231 cells （<italic>P</italic><0.01）， with the half maximal inhibitory concentration （ IC₅₀） of（0.13±0.02）g·L^-1. According to flow cytometry，compared with the blank group，Jingulian capsule at 1 g·L^-1 significantly induced the apoptosis of MDA-MB-231 cells （<italic>P</italic><0.05）and Jingulian capsule at 0.5， 1 g·L^-1 obviously increased the number of MDA-MB-231 cells in S phase （<italic>P</italic><0.05，<italic>P</italic><0.01）. The results of Western blotting demonstrated that the protein expression levels of PARP，c-Myc，and cyclin B₁ in 0.5， 1 g·L^-1Jingulian capsule groups were remarkably down-regulated as compared with those in the blank group（<italic>P</italic><0.01）， and the protein expression level of p-ERK in 1 g·L^-1Jingulian capsule group was also down-regulated （<italic>P</italic><0.01）. Conclusion:Jingulian capsule is able to inhibit the proliferation of MDA-MB-231 cells，induce S phase cell cycle arrest， and promote their apoptosis， which may be related to the inactivation of the MAPK signaling pathway.

Silencing gtse1 gene expression induces apoptosis of hepatocarcinoma cells through p53 pathway / 肿瘤

Objective: To validate the high-expression of G2 and S phase-expressed protein 1 (GTSE1) gene expression in primary liver cancer tissues and cell lines, and further to study the effect of GTSE1 gene silencing on the apoptosis of liver cancer cells. Methods: The expression matrix data of 4 individual clinical studies on primary liver cancer from Gene Expression Omnibus (GEO) database were analyzed using R software, and the differentially expressed genes between liver cancer and adjacent tissues were screened. The high expression of GTSE 1, one of the up-regulated genes, was validated in 3 cases of liver cancer tissues and 3 stains of liver cancer cell lines by Western blotting. Hep-G2 and Bel-7402 cells were transfected with the specific siRNA targeting GTSE 1 gene, then the effects of GSTE1 gene expression down-regulation on the proliferation, apoptosis and 5-fluorouracil (5-FU) sensitivity of liver cancer cells were measured by CCK-8 and FCM, respectively. Furthermore, the content and sub-cellular localization of p53 as well as the expression alteration of Bcl-2 family members were detected by Western blotting. Results: 51 up-regulated and 146 down-regulated genes were found in all 4 studies. Among them, the up-regulation of GTSE1 gene expression was further verified in 3 clinical liver cancer tissue samples and 3 liver cancer cell lines Hep-G2, Bel-7402 and SMMC-7721 (all P < 0.001). The silencing efficiency of GTSE1 gene in Hep-G2 and Bel-7402 cells transfected with the specific siRNA was more than 70%. After the down-regulation of GTSE1 expression, the proliferation of Hep-G2 and Bel-7402 cells was significantly inhibited in a time-dependent manner (all P < 0.05), the cell cycle was arrested in G1 phase, the apoptotic rate was significantly increased, and the cell sensitivity to 5-FU was significantly enhanced (all P < 0.01). In GTSE1 expression down-regulated Hep-G2 and Bel-7402 cells, the levels of total p53 and phosphorylated p53 were up-regulated (all P < 0.05), and the nuclear portion of p53 was increased (both P < 0.01); while the expression levels of Bcl-2 family pro-apoptotic members Bak and Bax were significantly raised, and the anti-apoptotic member Bcl-2 expression were reduced (all P < 0.05), respectively. Conclusion: The down-regulation of GTSE1 expression promotes the apoptosis of liver cancer cells by activating p53 pathway, and enhances the response to anti-tumor drugs.

Effects and mechanism of bakuchiol-induced S-phase cell cycle arrest in human breast cancer MCF-7 cells / 中草药

Objective: To investigate the cell growth inhibitory effect and molecular mechanism of bakuchiol against human breast cancer MCF-7 cells. Methods: The growth inhibitory effect of bakuchiol on MCF-7 cells was tested by MTT assay. Flow cytometry was used to investigate the distribution of cell cycle and ROS generation. Fluorescence microscope was used to observe the change of cell nucleus. Western blotting was used to detect the expression of the protein related to cell cycle and MAPK family. The ROS scavenger and inhibitors of MAPK family were introduced to investigate the effect on the growth inhibitory rate and the levels of cell cycle related protein by bakuchiol. Results: Bakuchiol inhibited the cell growth on the MCF-7 cells in dose- and time-dependent manner, which showed stronger effect than that of 5-fluorouracil. Furthermore, bakuchiol induced S-phase arrest in MCF-7 cells via ROS generation. The production of ROS up-regulated p-p53 and p21 expression, and then decreased CDK2 and CyclinA2. The changes of bakuchiol on these proteins could be reversed by the ROS scavenger Trion, indicating that ROS was associated with bakuchiol-induced S-phase arrest. In addition, pretreatment with p38MAPK inhibitor SB203580 decreased bakuchiol-caused ROS generation, suggesting that the production of ROS was dependent on p38MAPK pathway. Conclusion: The proliferation inhibitory effect of bakuchiol on MCF-7 cells is related with S-phase cell cycle arrest, and ROS plays a role in the bakuchiol-induced S-phase arrest.

Effect of sodium arsenite on cell cycle of SH-SY5Y cells and intervention of MPST over-expression / 中国药理学通报

Aim To investigate the effects of sodium arsenite (NaAsO2) on the cell cycle and growth, and the intervention of MPST over-expression in the neural cells. Methods NaAsO2 was used to treat SH-SY5Y neuroblastoma cells for 48 h from the blank control (BC), empty vector control ( transfected with empty vector, NC ) and over-expression group ( lentiviral transfection with MPST,OP). The methods of CCK-8, crystal violet staining,flow cytometry and Western blot were used to examine cell viability,adherent rate,cell cycle and protein expression of p53,CDC25A,CyclinA and CDK2. Results The cell viability and adherent rate significantly decreased after treatment with NaAsO2 for 48 h,which was reversed in OP group (P <0. 01). Meanwhile, NaAsO2 also significantly increased the proportion of S phase cells and p53 protein expression, and down-regulated the protein levels of CDC25A,Cyc-lin A and CDK2 in BC and NC groups ( P < 0. 01), whereas the above changes of protein levels were significantly antagonized in OP group compared with NC group (P < 0. 05, P < 0. 01). Conclusions NaAsO2 inhibits the cell growth by inducing S-phase arrest and over-expression of MPST could reverse the noxious effects caused by NaAsO2 in SH-SY5Y cells.

Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells / 中国天然药物

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.

MicroRNA-186 targets SKP2 to induce p27(Kip1)-mediated pituitary tumor cell cycle deregulation and modulate cell proliferation

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27(Kip1), a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27(Kip1) expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27(Kip1) and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27(Kip1) expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27(Kip1)-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27(Kip1) expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27(Kip1) expression. In contrast, miR-186 expression positively associated with p27(Kip1) expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27(Kip1)-mediated cell cycle alternation.

Experimental study on the pancreatic cancer G1/S phase arrest byβ2-adrenergic antagonist / 西安交通大学学报(医学版)

Objective To investigate the effects of β2-adrenergic antagonist ICI118 ,551 on pancreatic cancer cell G1/S phase arrest and its action mechanism .Methods The cell cycle indexes were determined by the flow cytometry assay ;the expressions of Cyclin D1 and Cyclin E were analyzed by Western blot ;the activation of NF-κB was measured by electrophoretic mobility shift assay ;the proliferation of PanCa cells was determined by BALB/c athymic nude mice subrenal capsular assay .Results β2-adrenergic antagonist ICI118 ,551 significantly induced G1/S phase arrest compared with β1-adrenergic antagonist metoprolol in MIA PaCa-2 and BxPC-3 cell lines .ICI118 ,551 inhibited the expressions of Cyclin D1 and Cyclin E and reduced the activation of NF-κB .The proliferation of PanCa cells was strongly suppressed in the renal capsule xenografts in mice after ICI 118 ,551 treatment .Conclusion The blockage ofβ2-adrenoceptor markedly induces PanCa cells to arrest at G1/S phase and inhibits the proliferation of pancreatic cancer cells .

Screening of S-phase kinase-assuciated protein 2 in the cervical carcinoma HeLa cell / 中国肿瘤生物治疗杂志

@#[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.

Activation of liver X receptors induced pancreatic β cell cycle arrest by up-regulating the expression of p27 protein / 临床检验杂志

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.

MicroRNA-340 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma / 第二军医大学学报

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.

The effect of Skp2 gene silencing on the proliferation and apoptosis of SPC-A-1 lung cancer cells / 重庆医学

Objective To investigate the effect of Skp2 gene silencing on the proliferation and apoptosis of SPC-A-1 lung cancer cells .Methods Specific small hairpin RNA (shRNA ) targeting Skp2 gene was introduced into lung cancer cells by Lipo-fectamine 2000 and the positive clones were screened by G418 .The Skp2 mRNA and protein expression level of lung cancer cells were detected by RT-PCR and Western blot .M TT method and flow cytometry (FCM ) analysis were used to observe the effect of RNAi on proliferation of lung cancer cells .Cell apoptosis was analyzed by FCM .Results Transfected with Skp2 shRNA expression vector significantly reduced the expression of Skp2 protein in lung cancer cells .Inhibition efficiency was respectively (75 .3 ± 5 .1) , (70 .4 ± 3 .2) .P27 protein expression was increased significantly in lung cancer cells .The growth of lung cancer cells transfected with Skp2 shRNA was blocked ,with Gl phase cells increased and S phase cells decreased .The apoptosis rate of cancer cells was higher in Skp2 shRNA groups than in control groups .Apoptosis rates were (17 .5 ± 2 .8)% ,(15 .6 ± 3 .1)% in Skp2 shRNA groups .Conclusion Specific inhibition of the expression of Skp2 can slow down the growth of lung cancer cells ,increase cell apop-tosis .

Expression of Skp2 and its Relationship with VEGF and MVD in Colorectal Carcinoma / 胃肠病学

Background:In recent years,the incidence and mortality of colorectal carcinoma have been increasing rapidly. Diagnosing and treating cancer at gene level have become a new effective approach. Aims:To investigate the expression of S-phase kinase-associated protein 2(Skp2)and its relationship with vascular endothelial growth factor(VEGF)and tumor microvessel density(MVD)in colorectal carcinoma. Methods:Thirty-five colorectal carcinoma patients from March 2014 to March 2015 at People’s Hospital of Hubei University of Medicine were enrolled. Fifteen para-cancerous tissue samples were served as controls. The expressions of Skp2,VEGF and MVD value were determined by immunohistochemical SP, and their relationships with clinicopathological characteristics were analyzed. Results:The expression of Skp2 in colorectal carcinoma was significantly higher than that in para-cancerous tissue( P = 0. 000),and was correlated with tumor differentiation and lymph node metastasis(P 0. 05). Expression of VEGF and MVD value were significantly higher than those in para-cancerous tissue(P = 0. 019,P = 0. 002),and were correlated with lymph node metastasis and TNM stage(P < 0. 05). Expression of Skp2 was positively correlated with the expression of VEGF and MVD value in colorectal carcinoma( r = 0. 569,P = 0. 000;r = 0. 481,P = 0. 017 ). Conclusions:The abnormal high expression of Skp2 is involved in the angiogenesis of colorectal carcinoma. Skp2,VEGF expressions and MVD value may be served as important markers for malignancy degree of colorectal carcinoma.

Prognostic role of s‑phase kinase‑associated protein 2 in breast cancer: A meta‑analysis.

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.

Analysis and clinical significance of DNA ploidy, S-phase fraction and proliferating index in colorectal carcinoma / 中国医师进修杂志

ObjectiveTo study the changes and clinical significance of DNA ploidy,S-phase fraction(SPF),proliferating index(PI) in colorectal carcinoma.MethodsIn 40 cases of colorectal carcinoma (colorectal carcinoma group ),40 cases corresponding cancer-adjacent tissue (cancer-adjacent tissue group ),12 cases of precancerous lesions (precancerous lesions group) and 10 cases of normal colorectal mucosa coli (normal colorectal mucosa coli group),DNA ploidy,SPF and PI were detected with flow cytometry and compared.ResultsDNA diploid was 7 cases,DNA heteroploid was 33 cases in colorectal carcinoma group,35,5 cases in cancer-adjacent tissue group,10,2 cases in precancerous lesions group,10,0 case innormal colorectal mucosa coli group,there were significant differences among them(P＜ 0.01 ).SPF and PI in colorectal carcinoma group (35.36 ± 7.45,42.92 ± 6.81 ) were significantly higher than those in cancer-adjacent tissue group (20.82 ±5.51,31.34 ±4.88),precancerous lesions group (21.13 ± 5.07,31.70 ±5.59) and normal colorectal mucosa coli group ( 19.93 ± 3.73,32.01 ± 4.99),there were significant differences among them(P＜ 0.01 ).DNA ploidy was significantly correlated with Dukes staging (P=0.027) and the differentiation of colorectal carcinoma (P =0.030).ConclusionsDNA ploidy,SPF,PI may get some molecular biologycharacteristic and proliferation activity of colorectal carcinoma at molecule level,which may be helpful for treatment and evaluation of prognosis of colorectal carcinoma.

Expression and functional role of S phase kinase associated protein 2 (Skp2) in development of glioma / 解放军医学杂志

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.

Expression and functional role of S phase kinase associated protein 2 (Skp2) in development of glioma / 解放军医学杂志

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.