1.Maternal heterozygous mutation in CHEK1 leads to mitotic arrest in human zygotes.
Beili CHEN ; Jianying GUO ; Ting WANG ; Qianhui LEE ; Jia MING ; Fangfang DING ; Haitao LI ; Zhiguo ZHANG ; Lin LI ; Yunxia CAO ; Jie NA
Protein & Cell 2022;13(2):148-154
2.Palbociclib induces cell cycle arrest and senescence of human renal tubular epithelial cells
Liuwei HUANG ; Yanting SHEN ; Chongbin LIU ; Caizhen LI ; Jun WANG
Journal of Southern Medical University 2020;40(12):1784-1792
OBJECTIVE:
To investigate the effect of palbociclib on cell cycle progression and proliferation of human renal tubular epithelial cells.
METHODS:
Human renal tubular epithelial cell line HK-2 was treated with 1, 5, 10, and 20 μmol/L of palbociclib, and the changes in cell proliferation and viability were examined by cell counting and CCK8 assay. EDU staining was used to assess the proliferation of HK-2 cells following palbiciclib treatment at different concentrations for 5 days. The effect of palbociclib on cell cycle distribution of HK-2 cells was evaluated using flow cytometry. SA-β-Gal staining and C12FDG senescence staining were used to detect senescence phenotypes of HK-2 cells after palbociclib treatment at different concentrations for 5 days. The relative mRNA expression levels of P16, P21, and P53 and the genes associated with senescence-related secretion phenotypes were detected by RT-PCR, and the protein expressions of P16, P21 and P53 were detected by Western blotting.
RESULTS:
Palbociclib inhibited HK-2 cell proliferation and induced cell cycle arrest in G1 phase. Compared with the control cells, HK-2 cells treated with high-dose (10 μmol/L) palbociclib exhibited significantly suppressed cell proliferation activity, and the inhibitory effect was the most obvious on day 5 (
CONCLUSIONS
Palbociclib induces HK-2 cell senescence by causing cell growth arrest and delaying cell cycle progression.
Cell Cycle
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Cell Cycle Checkpoints
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Cellular Senescence
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Epithelial Cells
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Humans
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Piperazines/pharmacology*
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Pyridines/pharmacology*
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Tumor Suppressor Protein p53/genetics*
3.Effect of miR-101 on biological characteristics of colorectal cancer cell line SW620.
Yan LIU ; Yan-Xia LU ; Min ZHOU ; Lin ZHENG ; Xue-Nong LI
Journal of Southern Medical University 2016;36(7):990-996
OBJECTIVETo investigate the effect of miR-101 on biological behaviors of colorectal cancer cell line SW620.
METHODSCCK-8 method, colony formation assay, cell cycle analysis and apoptosis analysis were applied to assess the effects of miR-101 on cell proliferation, invasion and apoptosis of SW620 cells.
RESULTSOver-expression of miR-101 in SW620 cells significantly suppressed the cell proliferation and attenuated the colony-forming ability of the cells. Flow cytometry showed that over-expression of miR-101 in SW620 cells caused obvious cell cycle arrest in G2/M and 1/ phases, and significantly increased the cell apoptosis rate.
CONCLUSIONOver-expression of miR-101 can inhibit the proliferation, cause cell cycle arrest and promote apoptosis of colorectal cancer SW620 cells.
Apoptosis ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness
4.Overexpression of connexin 40 (Cx40) inhibits the proliferation of H9c2 cardiomyocytes in rats by cell cycle arrest.
Yuanyuan REN ; Jie YANG ; Minxin WEI ; Chao SU
Chinese Journal of Cellular and Molecular Immunology 2023;39(8):714-720
Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.
Rats
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Humans
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Animals
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Cyclin D1/genetics*
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Transfection
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Myocytes, Cardiac
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HEK293 Cells
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Cell Cycle Checkpoints/genetics*
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Cell Proliferation/genetics*
;
Lentivirus/genetics*
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Genetic Vectors/genetics*
;
Gap Junction alpha-5 Protein
5.Progress in the Study of Spindle Assembly Checkpoint in Lung Cancer.
Xinchen QIN ; Yao ZHANG ; Haijie YU ; Lijuan MA
Chinese Journal of Lung Cancer 2023;26(4):310-318
Spindle assembly checkpoint (SAC) is a protective mechanism for cells to undergo accurate mitosis. SAC prevented chromosome segregation when kinetochores were not, or incorrectly attached to microtubules in the anaphase of mitosis, thus avoiding aneuploid chromosomes in daughter cells. Aneuploidy and altered expression of SAC component proteins are common in different cancers, including lung cancer. Therefore, SAC is a potential new target for lung cancer therapy. Five small molecule inhibitors of monopolar spindle 1 (MPS1), an upstream component protein of SAC, have entered clinical trials. This article introduces the biological functions of SAC, summarizes the abnormal expression of SAC component proteins in various cancers and the research progress of MPS1 inhibitors, and expects to provide a reference for the future development of lung cancer therapeutic strategies targeting SAC components.
.
Humans
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Cell Cycle Proteins/metabolism*
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Spindle Apparatus/metabolism*
;
Protein Serine-Threonine Kinases/metabolism*
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M Phase Cell Cycle Checkpoints/genetics*
;
Lung Neoplasms/metabolism*
6.miR-18a enhances the radiosensitivity of nasopharyngeal carcinoma cells through inducing autophagy.
Li Hong CHANG ; Zhou Zhou YAO ; Hong Wei BAO ; Yue LI ; Xiao Hong CHEN ; Xiao Ping LAI ; Zi Zhen HUANG ; Ge Hua ZHANG
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2021;56(7):736-745
Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.
Apoptosis
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Autophagy
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Cell Line, Tumor
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Cell Proliferation
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G2 Phase Cell Cycle Checkpoints
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Humans
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MicroRNAs/genetics*
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Nasopharyngeal Carcinoma/genetics*
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Nasopharyngeal Neoplasms/genetics*
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Radiation Tolerance
7.Expression of peroxisome proliferators-activated receptor in glioma and its effect on the growth of human glioma cells.
Yan SHI ; Wenkang LUAN ; Tao TAO ; Jiajia WANG ; Jin QIAN ; Qingsheng DONG ; Ning LIU ; Yongping YOU
Chinese Journal of Medical Genetics 2014;31(3):317-321
OBJECTIVETo study the expression of peroxisome proliferators-activated receptor (PPAR) in human glioma tissue and its influence on tumor growth.
METHODSExpression of PPAR mRNA in glioma tissue was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequently, MTT (3-(4, 5)-dimethylthiahiazo(-z-y1)-3, 5-di-phenytetrazoliumromide) assay, flow cytometry, reactive oxygen species assay kit and Western blotting were used to assay U87 cells with agonist activity of PPAR.
RESULTSThe data demonstrated that the expression of PPAR in glioma was low and negatively correlated with its pathological grade. Activation of PPAR suppresses tumor cell proliferation, delays the cell cycle at G1 phrase, and induces apoptosis and accumulation of reactive oxygen species (ROS) in U87 cells.
CONCLUSIONThe expression of PPAR mRNA in human glioma was low. PPAR protein plays a critical role in the progression of glioma via the PPAR signal pathway.
Apoptosis ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression ; Glioma ; genetics ; metabolism ; physiopathology ; Humans ; PPAR alpha ; genetics ; metabolism ; Signal Transduction
8.Spindle assembly checkpoint complex-related genes TTK and MAD2L1 are over-expressed in lung adenocarcinoma: a big data and bioinformatics analysis.
Zhu LIU ; Zeqin GUO ; Lili LONG ; Yanpei ZHANG ; Yuwen LU ; Dehua WU ; Zhongyi DONG
Journal of Southern Medical University 2020;40(10):1422-1431
OBJECTIVE:
To screen the key genes related to the prognosis of lung adenocarcinoma through big data analysis and explore their clinical value and potential mechanism.
METHODS:
We analyzed GSE18842, GSE27262, and GSE33532 gene expression profile data obtained from the Gene Expression Omnibus (GEO). Bioinformatics methods were used to screen the differentially expressed genes in lung adenocarcinoma tissues and KEGG and GO enrichment analysis was performed, followed by PPI interaction network analysis, module analysis, differential expression analysis, and prognosis analysis. The expressions of MAD2L1 and TTK by immunohistochemistry were verified in 35 non-small cell lung cancer specimens and paired adjacent tissues.
RESULTS:
We identified a total of 256 genes that showed significant differential expressions in lung adenocarcinoma, including 66 up-regulated and 190 down-regulated genes. Thirty-two up-regulated core genes were screened by functional analysis, and among them 29 were shown to significantly correlate with a poor prognosis of patients with lung adenocarcinoma. All the 29 genes were highly expressed in lung adenocarcinoma tissues compared with normal lung tissues and were mainly enriched in cell cycle pathways. Seven of these key genes were closely related to the spindle assembly checkpoint (SAC) complex and responsible for regulating cell behavior in G2/M phase. We selected SAC-related proteins TTK and MAD2L1 to test their expressions in clinical tumor samples, and detected their overexpression in lung adenocarcinoma tissues as compared with the adjacent tissues.
CONCLUSIONS
Seven SAC complex-related genes, including TTK and MAD2L1, are overexpressed in lung adenocarcinoma tissues with close correlation with the prognosis of the patients.
Adenocarcinoma of Lung/genetics*
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Big Data
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Cell Cycle Proteins/genetics*
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Computational Biology
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Gene Expression Profiling
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Gene Expression Regulation, Neoplastic
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Humans
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Lung Neoplasms/genetics*
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M Phase Cell Cycle Checkpoints
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Mad2 Proteins/genetics*
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Protein-Serine-Threonine Kinases/genetics*
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Protein-Tyrosine Kinases/genetics*
9.Essential functions of iron-requiring proteins in DNA replication, repair and cell cycle control.
Protein & Cell 2014;5(10):750-760
Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these iron-requiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.
Animals
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Cell Cycle Checkpoints
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DNA
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metabolism
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DNA Repair
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DNA Replication
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Hemeproteins
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genetics
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metabolism
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Iron
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chemistry
;
metabolism
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Iron-Sulfur Proteins
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genetics
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metabolism
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Ribonucleotide Reductases
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genetics
;
metabolism
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Yeasts
;
metabolism
10.Glucose-6 phosphatase catalytic subunit inhibits the proliferation of liver cancer cells by inducing cell cycle arrest.
Xue LIN ; Xuan Ming PAN ; Zi Ke PENG ; Kai WANG ; Ni TANG
Chinese Journal of Hepatology 2022;30(2):213-219
Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.
Catalytic Domain
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Cell Cycle Checkpoints
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Cell Line, Tumor
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Cell Proliferation
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Gene Expression Regulation, Neoplastic
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Glucose-6-Phosphatase/metabolism*
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Humans
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Liver Neoplasms/genetics*