• BMB Reports
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• 제47권5호
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• pp.262-267
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• 2014

Bcl-2 interacting cell death suppressor (Bis) has been shown to have anti-apoptotic and anti-stress functions. Recently, increased Bis expression was reported to correlate with glioma aggressiveness. Here, we investigated the effect of Bis knockdown on the acquisition of the invasive phenotype of A172 glioma cells, induced by 12-O-Tetradecanoylphorbol-3-acetate (TPA), using a Transwell assay. Bis knockdown resulted in a significant decrease in the migration and invasion of A172 cells. Furthermore, Bis knockdown notably decreased TPA-induced matrix metalloproteinase-9 (MMP-9) activity and mRNA expression, as measured by zymography and quantitative real time PCR, respectively. A luciferase reporter assay indicated that Bis suppression significantly down-regulated NF-${\kappa}B$-driven transcription. Finally, we demonstrated that the rapid phosphorylation and subsequent degradation of $I{\kappa}B-{\alpha}$ induced by TPA was remarkably delayed by Bis knockdown. These results suggest that Bis regulates the invasive ability of glioma cells elicited by TPA, by modulating NF-${\kappa}B$ activation, and subsequent induction of MMP-9 mRNA.

• Molecules and Cells
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• 제37권12호
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• pp.857-864
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• 2014

Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the antiapoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADPribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• Molecules and Cells
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• 제42권3호
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• pp.218-227
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• 2019

This study was designed to determine the effects of the long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) on vascular smooth muscle cell (VSMC) apoptosis and extracellular matrix (ECM) disruption in a murine abdominal aortic aneurysm (AAA) model. After injection of PVT1-silencing lentiviruses, AAA was induced in Apolipoprotein E-deficient ($ApoE^{-/-}$) male mice by angiotensin II (Ang II) infusion for four weeks. After Ang II infusion, mouse serum levels of pro-inflammatory cytokines were analysed, and aortic tissues were isolated for histological, RNA, and protein analysis. Our results also showed that PVT1 expression was significantly upregulated in abdominal aortic tissues from AAA patients compared with that in controls. Additionally, Ang II treatment significantly increased PVT1 expression, both in cultured mouse VSMCs and in AAA murine abdominal aortic tissues. Of note, the effects of Ang II in facilitating cell apoptosis, increasing matrix metalloproteinase (MMP)-2 and MMP-9, reducing tissue inhibitor of MMP (TIMP)-1, and promoting switching from the contractile to synthetic phenotype in cultured VSMCs were enhanced by overexpression of PVT1 but attenuated by knockdown of PVT1. Furthermore, knockdown of PVT1 reversed Ang II-induced AAA-associated alterations in mice, as evidenced by attenuation of aortic diameter dilation, marked adventitial thickening, loss of elastin in the aorta, enhanced aortic cell apoptosis, elevated MMP-2 and MMP-9, reduced TIMP-1, and increased pro-inflammatory cytokines. In conclusion, our findings demonstrate that knockdown of lncRNA PVT1 suppresses VSMC apoptosis, ECM disruption, and serum pro-inflammatory cytokines in a murine Ang II-induced AAA model.

• Molecules and Cells
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• 제44권1호
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• pp.13-25
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• 2021

Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. We report here that ana3m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. We propose that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation.

• The Korean Journal of Physiology and Pharmacology
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• 제27권2호
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• pp.157-165
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• 2023

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and current therapeutic strategies are limited in their effectiveness. The expressions of Rab5 and the M2 tumor-associated macrophage marker CD163 in tissues were detected by Western blot. The migration and invasion of cells were determined using a Transwell assay. The expressions of the exosome markers were evaluated by Western blot. The polarization of human macrophages (THP-1) was determined by incubation of THP-1 cells with conditioned medium or exosomes collected from MDA-MB-231 cells with indicated transfections or by a coculture system of THP-1 and MDA-MB-231 cells. The M1 and M2 macrophage markers were evaluated by qRT-PCR. The expression of Rab5 in TNBC was significantly higher than that in normal breast tissue. Rab5 expressions in triple-negative and luminal A breast cancer were higher than those in other molecular subtypes. Higher CD163 expression was observed in triple-negative breast cancer and in triple-negative and luminal B subtypes. Rab5 knockdown suppressed but Rab5 overexpression promoted the migration and invasion capacity of MDA-MB-231 cells. The levels of CD63 and CD9 in the medium of Rab5 knockdown cells were lower than those in control cells, whereas higher levels of CD63 and CD9 were observed in Rab5 overexpression cells. Rab5 knockdown decreased the excretion but did not alter the diameter of the exosomes. Knockdown of Rab5 facilitated the anti-tumor polarization of macrophages, which was partially reversed by Rab5 overexpression. Therefore, Rab5 is expected to be a potential therapeutic target for triple-negative breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제13권2호
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• pp.633-636
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• 2012

Purpose: PRIL (proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family and modulates death ligand-induced apoptosis. Here, we investigated the effect of PRIL on cellular characteristics relating to tumor progression in human gastric cancer. Method: Recombinant lentivirus containing PRIL siRNA was constructed and then infected MGC803 and SGC7901 gastric cancer cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colony formation and cell cycle analysis were used to study the effect of PRIL knockdown on gastric cancer cell proliferation. Results: PRIL expression in lentivirus infected cells was significantly reduced as evidenced by quantitative real-time PCR. Cell viability and colony formation of MGC803 and SGC7901 cells were significantly hampered in PRIL knock-down cells. Moreover, the cell cycle was arrested at G2/M phase, elucidating the mechanism underlying the inhibitory effect of siRNA on cell proliferation. Conclusions: Our study indicated that PRIL functions in promoting cell growth, and lentivirus-mediated PRIL gene knockdown might be a promising strategy in the treatment of gastric cancer.

• The Korean Journal of Physiology and Pharmacology
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• 제13권6호
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• pp.483-489
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• 2009

Despite the potential importance of the human regulator of calcineurin 1 (RCAN-1) gene in the modulation of cell survival under stress, little is known about its role in death-inducing signal pathways. In this study, we addressed the effects of RCAN1.4 knockdown on cellular susceptibility to apoptosis and the activation of death pathway proteins. Transfection of siRNAs against RCAN1.4 resulted in enhanced Fas- and etoposide-induced apoptosis, which was associated with increased expression and translocation of Bax to mitochondria. Our results suggest that enhanced expression and activation of p53 was responsible for the upregulation of Bax and the increased sensitivity to apoptosis, which could be reversed by p53 knockdown. To explain the observed upregulation of p53, we propose a downregulation of the ubiquitin ligase HDM2, probably translationally. These findings show the importance of appropriate RCAN1.4 expression in the modulation of cell survival and reveal a link between RCAN1.4 and p53.

• BMB Reports
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• 제48권8호
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• pp.467-472
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• 2015

Heat shock increases skin temperature during sun exposure and some evidence indicates that it may be involved in skin aging. The antioxidant response mediated by the transcription factor NF-E2-related factor 2 (Nrf2) is a critically important cellular defense mechanism that serves to limit skin aging. We investigated the effects of heat shock on collagenase expression when the antioxidant defense system was downregulated by knockdown of Nrf2. GSH and collagenases were analyzed, and the expression of inducible Nrf2, HO-1, and NQO1 was measured. HS68 cells were transfected with small interfering RNA against Nrf2. Heat shock induced the downregulation of Nrf2 in both the cytosol and nucleus and reduced the expression of HO-1, GSH, and NQO1. In addition, heat-exposed Nrf2-knockdown cells showed significantly increased levels of collagenase protein and decreased levels of procollagen. Our data suggest that Nrf2 plays an important role in protection against heat shock-induced collagen breakdown in skin. [BMB Reports 2015; 48(8): 467-472]

• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.5975-5979
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• 2012

Background: To investigate the influence of telomerase activity, apoptosis, radiosensitivity of cervical cancer after siRNA-mediated knockdown of telomerase RNA and evaluate in vivo growth with gene interference. Methods: We studied siRNA-targeting-telomerase RNA transfection into the Hela cell line. Expression of hTERC mRNA was detected by RT-PCR and telomerase activity was measured by the TRAP assay. Growth inhibition was determined by MTT assay and radiosensitivity of the cervical cancer cells was examined by colony formation assay. In addtion, effects of hTERC inhibition in vivo were studied by injection of siRNA-transfected Hela cells into nude mice. Results: The hTERC siRNA effectively downregulated the expression of hTERC mRNA and also reduced the telomerase activity to 30% of the untreated control vlaue. The viability of hTERC siRNA transfected Hela cells was reduced by 44.7% after transfection. After radiation treatment, the radiosensitivity of Hela cells with hTERC knockdown was increased. In vivo, the tumors developing from the hTERC siRNA-transfected cells were of reduced size, indicating that the hTERT siRNA also depressed the tumorigenic potential of the Hela cells. Conclusions: Our results supported the concept of siRNA-mediated inhibition of telomerase mRNA which could inhibit the expression of hTERC and telomerase activity. Furthermore, radiosensitivity was upregulated after knockdown the hTERC in vivo and in vitro.