Yun, Hye Hyeon
(Department of Biochemistry, College of Medicine, The Catholic University of Korea)
,
Baek, Ji-Ye
(Department of Biochemistry, College of Medicine, The Catholic University of Korea)
,
Seo, Gwanwoo
(The Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea)
,
Kim, Yong Sam
(Genome Editing Research Center, KRIBB)
,
Ko, Jeong-Heon
(Genome Editing Research Center, KRIBB)
,
Lee, Jeong-Hwa
(Department of Biochemistry, College of Medicine, The Catholic University of Korea)
The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these prot...
The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.
The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.
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제안 방법
We next measured the promoter activity of BIS and HSP70 using a luciferase reporter assay in BIS WT and BIS-KO-A549 cells. The aim of the reporter assay was to ensure that the promoter activity mediated through HSF1-HSE interaction was not influenced by BIS depletion, and to further determine if BIS specifically regulated its own promoter without affecting other HSF1-target gene via an HSF1-independent mechanism. Fig.
Then, quantitative real-time PCR (qRT-PCR) was performed using SYBR premix Ex Taq (Takara Biotechnology, Shiga, Japan) with specific primerson CFX96 Connect Real-Time PCR Detection System (Biorad, Hércules, CA, USA).
Therefore, the present study is designed to investigate the consequence of BIS depletion on the HSF1 activity as a transcriptional factor using BIS depletion strategy. Our results showed that activation of HSF1 by several stresses was not affected by BIS knockout or knockdown as determined by the endogenous HSP70 and HSP27 mRNAs as well as HSP70 promoter activity.
Our findings were inconsistent with a previous report showing that BIS knockdown using adenovirus was accompanied by decreases in HSP70 and HSP27 expression as determined by western blots of T98G glioblastoma cells [27]. Thus, to determine if the lack of a functional association of BIS with HSF1 activity was the specific phenotypes of A549 cells, we suppressed HSF1 or BIS expression in U87 glioblastoma cells and examined the HSP70 and HSP27 mRNA levels upon heat shock stress. As shown inFig.
To construct the open reading frame of WT BIS gene and the corresponding region of BIS gene with 14 bp deletion, PCR was performed with the cDNA from WT A549 and BIS-KO A549 cells as a template, respectively. After verification of the correct sequences, the PCR product was then digested and cloned into XhoI and EcoRI sites of pEGFP-C1 (Promega).
To exclude the possibility that our results were due to the cellular features acquired during the selection process of BIS-KO A549 cells, which were not relevant with BIS depletion, we transiently suppressed BIS expression in WT A549 cells using siRNA and examined the induction of HSP70 and HSP27 mRNA. Fig.
데이터처리
Statistical significance between two groups was analyzed by Student’s t test.
이론/모형
Wild type (WT) A549 and BIS-KO A549 cells were transfected with a BIS or HSP70 promoter for 24 h and then exposed to heat shock. The activity reporter was measured using a Dual-Luciferase reporter assay system (Promega). After normalization with renilla activity, the promoter activities of each constructs are presented as fold change of luciferase activities relative to that from pGL3 basic plasmid, which was taken as 1.
The relative values for BIS, HSP70, HSP27or HSF1 mRNA were calculated after normalizing the Ct value to β-actin levels from the same sample using the ddCt method.
성능/효과
Therefore, the present study is designed to investigate the consequence of BIS depletion on the HSF1 activity as a transcriptional factor using BIS depletion strategy. Our results showed that activation of HSF1 by several stresses was not affected by BIS knockout or knockdown as determined by the endogenous HSP70 and HSP27 mRNAs as well as HSP70 promoter activity. However, the expression of the BIS gene that also contains HSEs in its promoter region was reduced specifically in the BIS knockout (KO) cells.
45-fold compared to WT A549 cells. These results indicated that BIS depletion inhibited the constitutive as well as inducible expression of the BIS mRNA without alteration of HSP70 and HSP27 mRNA levels.
후속연구
Therefore, it is also possible that BIS might participate in an alternative transcriptional program that leads to long-term cell survival, which is supported by the constitutive high expression of HSF1 and BIS in various type of human cancer [3,4,44,45]. However, to investigate the mechanistic network between BIS andHSF1 in the pro-growth program in cancer cells, further studies should be performed under more detailed and diverse experimental conditions.
Our findings show that neither promoter activity nor mRNA stability of BIS was affected by BIS depletion. Therefore, the nuclear or cellular events linking these two processes, including splicing process, should be further studied to investigate the molecular mechanism by which BIS controls the autoregulatory loop. Targeting the auto-regulatory pathway is an efficient strategy to suppress BIS expression, which is aberrantly expressed in most cancers.
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