Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma
Wing Chun TangChung ChuiSarwat FatimaStanton KokK.C. PakTian OuKin HuiMei Mei WongJohn WongSimon LawSai Wah TsaoAlfred K. LamPhilip BehGopesh SrivastavaA.S.C. ChanKwok W. HoJohnny Tang
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Abstract:
Esophageal squamous cell carcinoma (ESCC) shows high frequency and mortality in Asian regions, including China. Previous analysis of genomic DNA of ESCC using comparative genomic hybridization indicated that amplification of the chromosome 5p regions is a common event in ESCC cell lines and patient cases of Hong Kong Chinese origin, and the results suggested that the genes located in the chromosome 5p regions may play crucial roles in the molecular pathogenesis of ESCC. Our previous studies on ESCC confirmed the tumorigenic and overexpression properties of a novel gene JS-1 located in chromosome 5p15.2 upstream to delta-catenin. In the present study, another novel gene JK-1 which is located at 5p15.1 downstream to delta-catenin was characterized for its roles in the pathogenesis of ESCC. Thirteen ESCC cell lines and 30 surgical specimens of esophageal tumors were studied for the overexpression of JK-1 using multiplex RT-PCR analysis. The transforming capacity of overexpression of JK-1 was also investigated by transfecting NIH 3T3 and HEK 293 cells with the expression vector cloned with JK-1, followed by the soft agar and foci formation assays. JK-1 was overexpressed in 9/13 (69%) of the ESCC cell lines and 9/30 (30%) of the ESCC patient cases. Both NIH 3T3 and HEK 293 cells acquired the properties of anchorage-dependent and -independent growth when JK-1 was overexpressed. Most significantly, subcutaneous sarcomas were formed in all (3/3) the athymic nude mice after NIH 3T3 cells overexpressing JK-1 were injected subcutaneously. Our results thus indicated that JK-1 is commonly overexpressed in ESCC and has a prominent capacity to transform normal cells. Our overall results thus provide the first evidence that the overexpression of JK-1 and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC.Keywords:
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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.
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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.
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MTT assay
G1 phase
Tretinoin
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Radiotherapy is an important therapeutic strategy for the treatment of numerous types of malignant tumors, including glioma. However, radioresistance and anti‑apoptotic mechanisms decrease the efficacy of radiotherapy in many patients with glioma. BMI1 polycomb ring finger oncogene (Bmi‑1) is an oncogene associated with radioresistance in tumor cells. MicroRNA (miRNA)‑128a is a brain-specific miRNA, which suppresses Bmi‑1 expression. The present study investigated the effects of various radiation intensities on U‑87 MG glioma cells, as well as the role of reactive oxygen species (ROS), Bmi‑1, and miRNA‑128a in the cellular response to radiotherapy. The response of U‑87 MG cells following exposure to X‑ray radiation was assessed using a cell growth curve and inhibition ratio. Cell cycle distribution and the levels of intracellular ROS were evaluated by flow cytometry. The mRNA expression levels of Bmi‑1 and those of miRNA‑128a in U‑87 MG cells exposed to X‑ray radiation were evaluated by reverse transcription‑quantitative polymerase chain reaction. X‑ray radiation did not decrease the number of U‑87 MG cells; however, it did inhibit cellular growth in a dose‑dependent manner. Following exposure to X‑ray radiation for 24 h, cell cycle distribution was altered, with an increase in the number of cells in G0/G1 phase. The mRNA expression levels of Bmi‑1 were downregulated in the 1 and 2 Gy groups, and upregulated in the 6 and 8 Gy groups. The expression levels of miRNA‑128a were upregulated in the 1 and 2 Gy groups, and downregulated in the 8 Gy group. The levels of ROS were increased following exposure to ≥2 Gy, and treatment with N-acetyl cysteine was able to induce radioresistance. These results suggested that U‑87 MG cells exhibited radioresistance. High doses of X‑ray radiation increased the expression levels of Bmi‑1, which may be associated with the evasion of cellular senescence. miRNA‑128a and its downstream target gene Bmi‑1 may have an important role in the radioresistance of U‑87 MG glioma cells. In addition, ROS may be involved in the mechanisms underlying the inhibitory effects of X‑ray radiation in U‑87 MG cells, and the downregulation of ROS may induce radioresistance.
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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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B7 homolog 6 (B7‑H6) was recently discovered to act as a co‑stimulatory molecule. In particular, the expression of B7‑H6 has been found to play an important biological role in several types of tumors. The aim of the present study was to determine the role of B7‑H6 in cervical cancer. Immunohistochemistry was used to analyze the expression levels of B7‑H6 in cervical precancerous and cancerous tissues. Furthermore, the expression of B7‑H6 was knocked down in HeLa cells using short hairpin RNA and the effects of B7‑H6 on HeLa cell proliferation, migration and invasion were determined using Cell Counting Kit‑8, colony formation, wound healing and Transwell invasion assays, respectively. In addition, flow cytometry was used to analyze the levels of cell apoptosis and the cell cycle distribution. The results of the immunohistochemical staining revealed that the expression levels of B7‑H6 were upregulated in cervical lesions. Furthermore, the expression levels of B7‑H6 were positively associated with the clinical stage of the cervical lesions. B7‑H6 knockdown suppressed the invasive, migratory and proliferative abilities of HeLa cells, and promoted G1 cell cycle arrest and apoptosis. In conclusion, the findings of the present study suggested that B7‑H6 may serve as a novel oncogene and may hold promise as a potential therapeutic target for cervical cancer.
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It has been demonstrated that the KCa3.1, the intermediate‐conductance Ca 2+ ‐activated K + channel KCNN4, increases cell proliferation. It still remains to be determined how this occurs. Recently, we reported that wild‐type KCa3.1, a non‐conducting KCa3.1 or non‐trafficking KCa3.1 channel increased cell proliferation when transiently expressed in HEK cells (Millership et al., Am J. Physiol Cell Physiol 300:C792‐C802, 2011). In the present study, we have used chimeric channels of KCa3.1 (IK) and KCa2.2 (SK; has no affect on cell proliferation) to uncover the potential molecular portion of KCa3.1 that alters cell proliferation. Transient transfection of HEK cells with IK increased cell number after 3 days to 131±7% (n = 7) compared to mock‐transfected cells (n = 7) while SK had no effect on cell number (n = 4). HEK cells transfected with a chimeric channel of SK‐200IK (SK+S5‐pore‐ S6‐C‐terminus of IK) significantly increased cell number to 119±3% (n = 5) when compared with mock‐transfected cells, however, SK‐287IK (SK+C‐terminus of IK) did not increase cell count (n = 3). We are currently exploring other chimeric and mutated channels to further define the portion of KCa3.1 responsible for altered cell proliferation. At present, our data suggest that the S5‐pore‐S6 portion of KCa3.1 is crucial for modulation of cell proliferation. This work was funded by Department of Physiology at UoO (KLH) and NIH (DCD).
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Emerging evidence suggests the critical function of microRNAs in regulating the growth of cancer cells. In the present study, it was demonstrated that miR‑221‑3p was overexpressed in non‑small cell lung cancer (NSCLC) tissues and cell lines compared with that noted in the normal controls. Downregulation of miR‑221‑3p suppressed the proliferation, colony formation and invasion of NSCLC cells. To further understand the molecular mechanisms underlying the potential oncogenic function of miR‑221‑3p in NSCLC, the downstream targets of miR‑221‑3p were predicted using bioinformatic databases. The prediction suggested the cell cycle regulator p27 as one of the targets of miR‑221‑3p. Molecular experiments showed that miR‑221‑3p was able to bind with the 3'‑untranslated region (UTR) of p27 and decreased the expression of p27 in NSCLC cells. Consistent with the suppressive role of p27 in controlling cell cycle progression, overexpression of miR‑221‑3p decreased the expression of p27 and promoted cell cycle progression from G1 to S phase. Collectively, our findings identified miR‑221‑3p as a novel regulator of NSCLC cell growth via modulating the expression of p27.
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