Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells
José E. Capó‐AponteZheng WangM.A. AkinciJ. Mario WolosinKathryn S. PokornyPavel IserovishPeter S. Reinach
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Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium-chloride cotransporter (KCC) in mediating (1) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (2) increases in cell cycle progression; and (3) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G(0)/G(1)-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G(2)/M-phase and accelerated the transition from G(0)/G(1)-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (1) activation of the p44/42MAPK signaling pathway, (2) cell cycle progression, and (3) increases in cell proliferation.P27Kip1 is the one of the negative regulators of the cell cycle that plays an important role in regulating cell cycle and inhibiting cell proliferation by restraining cell in G1 phase. P27Kip1 downregulation maybe related to the occurrence of oral squamous cell carcinoma (OSCC). It was found that miR-155 significantly upregulated in OSCC tissue. Bioinformatics analysis revealed that miR-155 may bind with the 3'-UTR of p27Kip1. This study investigated the role of miR-155 in regulating p27Kip1 and affecting Tca8113 cell proliferation, cycle, and apoptosis.A total of 46 cases of OSCC patients received treatment in our hospital were enrolled to obtain tumor tissue. Another 25 normal oral mucosa samples were selected as control to detect the relationship between miR-155 and p27Kip1 expressions. Dual luciferase assay was adopted to confirm the targeted relationship between miR-155 and p27Kip1. Flow cytometry was applied to test cell apoptosis and cell cycle. CCK-8 assay was used to evaluate cell proliferation. Caspase-3 activity was detected by spectrophotometry.MiR-155 upregulated, while p27Kip1 declined in OSCC tissue compared with normal oral mucosa. Their expressions were related to TNM stage. MiR-155 targeted suppressed p27Kip1 expression. MiR-155 mimic and/or pEGFP-p27Kip1 transfection obviously declined p27Kip1 expression, blocked cell cycle in G1 phase, reduced cell proliferation, enhanced Caspase-3 activity, and increased cell apoptosis in Tca8113 cells.MiR-155 increased, while p27Kip1 reduced in OSCC tissue. Inhibition of miR-155 upregulated p27Kip1 expression, blocked cell cycle in G1 phase, weakened cell proliferation, and induced cell apoptosis.
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Objective To investigate the effect of miR-20b on cell proliferation and cell cycle in gastric cancer because of up-regulation of miR-20b in gastric cancer.Methods miR-20b mimics and its inhibitor were respectively transfected into MGC 803 gas-tric cancer cell and methyl thiazolyl tetrazolium （ MTT ） and fluorescence-activated cell sorting （ FACS ） were used to analyze cell growth and cell cycle.Western blot was used to explore the molecular basis of miR-20b.Results Compared with its control, cell growth was obvious elevated and the cell cycle transition was also increased from G 1 to S phase after miR-20b mimics transfection .After transfecting miR-20b inhibitor, cell growth was markedly decreased and cell cycle transition was also delayed from G 1 to S phase.Fur-thermore, miR-20b induced the expression of cyclin D1 （CCND1） and C-Myc, decreased the expressions of p21 and p15.Conclu-sions miR-20b was considered as a potential oncogene to modulate cell growth and cell cycle transition through regulating the expres -sion of cell cycle-related genes .
Key words:
微RNAs; MicroRNAs; Stomach neoplasms; Cell proliferation
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The effects of gamigeonsim-tang (GGT) on cellular proliferation and expression of cell cycle-related genes were investigated in human smooth muscle cell HISM. HISM cells were treated with an aqueous extract of GGT. Cellular proliferation was investigated by an immunocytometric analysis of PCNA expression and a flow cytometric analysis of the cell cycle progression. Reduced expression of PCNA and a significant accumulation of G1 phase cells were observed following treatment, indicating that GGT inhibits cellular proliferation of human smooth muscle cells. To explore whether GGT affects the transcription of cell cycle-regulating genes, we evaluated mRNA expression of p53, p21Waf1 PCNA, Cyclin D1, Cdc2, Histone H3, c-Myc, and c-Fos using a quantitative RT-PCR analysis. While increased expressions of two negative cell cycle regulators, p53 and p21Waf1 were found, reduced expressions of cell cycle stimulators, PCNA, c-Fos, and c-Myc, were identified following treatment. Taken together, our study demonstrates that GGT inhibits cellular proliferation of human smooth muscle cell through the up- and down-regulation of growth-inhibiting and growth-promoting genes, respectively.
Cyclin A
Cyclin B1
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Effects of all-trans retinoic acid on cell cycle and proliferation of MCF-7 and expression of restin
To study the role of all-trans retinoic acid(ATRA) in cell cycle, proliferation, differentiation and apoptosis of breast cancer cell line MCF-7 and the expression of restin gene. Methods: The cell cycle was observed by means of flow cytometry(FCM) , the cell proliferation was detected by MTT assay and the expression of restin mRNA was examined by RT-PCR. Results: Under the treatment by ATRA, the cell growth arrested at G1 phase and the proliferation was inhibited greatly. The level of restin mRNA was found to be up-regulated by ATRA in MCF-7 cell. Conclusion: G1 phase arrest and proliferation inhibition were involved in the process that ATRA induces MCF-7 cell differentiation and apoptosis, and restin maybe play an important role in this process, especially the cell apoptosis.
MCF-7
MTT assay
G1 phase
Tretinoin
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Abstract Zinc (Zn) plays a critical role in the growth of livestock, which depends on cell proliferation. In addition to modifying the growth associated with its effects on food intake, mitogenic hormones, signal transduction and gene transcription, Zn also regulates body weight gain through mediating cell proliferation. Zn deficiency in animals leads to growth inhibition, along with an arrest of cell cycle progression at G0/G1 and S phase due to depression in the expression of cyclin D/E and DNA synthesis. Therefore, in the present study, the interplay between Zn and cell proliferation and implications for the growth of livestock were reviewed, in which Zn regulates cell proliferation in several ways, especially cell cycle progression at the G0/G1 phase DNA synthesis and mitosis. During the cell cycle, the Zn transporters and major Zn binding proteins such as metallothioneins are altered with the requirements of cellular Zn level and nuclear translocation of Zn. In addition, calcium signaling, MAPK pathway and PI3K/Akt cascades are also involved in the process of Zn‐interfering cell proliferation. The evidence collected over the last decade highlights the necessity of Zn for normal cell proliferation, which suggests Zn supplementation should be considered for the growth and health of poultry.
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AIM: To investigate the effect of notch1 gene on the change of proliferation and cell cycle in human glioma U251 cell line.METHODS: The lentiviral vectors,which express notch1 shRNA or notch1 intracellular domain(NICD),were constructed and transfected into U251 cells,respectively.RT-PCR and Western blotting were applied to monitor the validity of down-regulation of notch1 expression and over-expression of NICD.MTT assay was performed to examine the cell proliferation.Flow cytometric analysis was used to detect the cell cycle.RESULTS: The lentiviral vectors,which expressed notch1 shRNA and NICD,were efficient in silencing notch1 expression and over-expression of NICD.Down-regulation of notch1 gene by RNAi inhibited the cell proliferation remarkably(P0.01),arrested cell cycle at G1 phase(P0.01) and decreased the cell number of S phase(P0.01).Over-expression of NICD enhanced the cell proliferation significantly(P0.01),promoted the cell cycle at G1 phase(P0.05) and increased the cell number of S phase(P0.01).CONCLUSION: notch1 gene,which leads to change the proliferation and cell cycle in human glioma U251 cell line,is likely to be potential molecular target for glioma in gene therapy.
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MicroRNAs (miRNAs/miRs) serve a key role in regulating the cell cycle and inducing tumorigenesis. Subgroup J of the avian leukosis virus (ALV-J) belongs to the family Retroviridae, subfamily Orthoretrovirinae and genus Alpharetrovirus that causes tumors in susceptible chickens. gga-miR-375 is downregulated and Yes-associated protein 1 (YAP1) is upregulated in ALV-J-induced tumors in the livers of chickens, and it has been further identified that YAP1 is the direct target gene of gga-miR-375. In the present study, it was found that ALV-J infection promoted the cell cycle and proliferation in DF-1 cells. As the cell cycle and cell proliferation are closely associated with tumorigenesis, further experiments were performed to determine whether gga-miR-375 and YAP1 were involved in these cellular processes. It was demonstrated that gga-miR-375 significantly inhibited the cell cycle by inhibiting G1 to S/G2 stage transition and decreasing cell proliferation, while YAP1 significantly promoted the cell cycle and proliferation. Furthermore, these cellular processes in DF-1 cells were affected by gga-miR-375 through the targeting of YAP1. Collectively, the present results suggested that gga-miR-375, downregulated by ALV-J infection, negatively regulated the cell cycle and proliferation via the targeting of YAP1.
YAP1
Hippo signaling pathway
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Retinoblastoma protein
G1 phase
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Objective To explore the mechanism of Qipiyin on treating psoriasis,and effects on cell cycle and proliferation.Methods The effect of Qipiyin and its components on HaCaT proliferation was measured through MTT method,and flow cytometry analysis was used to detect the content of DNA and cell cycle in various concentrations at different time points.Results Qipiyin could markedly inhibited the proliferation of HaCaT,and the effect was enhanced with concentration.The medicine could also disturbed the distribution of HaCaT cell cycle obviously,which showed that the cell percentage of HaCaT in G0/G1 phase increased and that in S phase decreased.Conclusion The mechanism of Qipiyin inhibiting the proliferation of keratinocyte may be related to the change of cell cycle.
HaCaT
MTT assay
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MicroRNA (miR)-106b-5p has been reported to act as both an oncogene and tumor suppressor in several tumors. The aim of the present study was to investigate the biological function of miR-106b-5p in osteosarcoma (OS). miR-106b-5p expression was observed to be significantly increased in OS tissues and cell lines. MTT assay and flow cytometry analysis determined that miR-106b-5p inhibitor transfection suppressed OS cell proliferation and induced cell cycle G0/G1 phase arrest. Furthermore, bioinformatics analysis and a luciferase reporter assay demonstrated that cyclin-dependent kinase inhibitor 1A (CDKN1A) was a potential target of miR-106b-5p. p21 protein expression was found to be significantly increased by miR-106b-5p downregulation in OS cells. Further analysis demonstrated that CDKN1A was downregulated in OS tissues and was negatively correlated with miR-106b-5p expression. Furthermore, upregulation of CDKN1A expression mimicked, whilst CDKN1A knockdown reversed the suppressive effects of miR-106b-5p inhibitor on OS cell proliferation and cell cycle progression. In summary, the present data suggested that miR-106b-5p promotes cell proliferation and cell cycle progression by directly targeting CDKN1A in OS.
MTT assay
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