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cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Zhang M, Xu JJ, Zhou RL, Zhang QY - PLoS ONE (2013)

Bottom Line: Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression.However, its transcriptional regulation mechanism is still unclear.The +10+292 promoter region was possessed the highest transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical laboratory, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, China.

ABSTRACT
Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

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shRNA targeting of CREB1 specifically inhibits its expression and leads to impairment of LAPTM4B gene expression.(A) MCF7 cell were transfected with control shRNA (Conrol-A) or CREB1-specific shRNA (RNAi). NC represented the MCF7 cell without any treatment. Whole cell lysates were subjected to western blot analysis for CREB1 protein. The intensity of gray value of western blot bands. The different superscript letters represent significantly different groups (p<0.05). (B) Analysis of LAPTM4B gene transcript by real-time PCR after 48 h of shRNA transfection. (*P<0.05, n = 3).
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pone-0057520-g005: shRNA targeting of CREB1 specifically inhibits its expression and leads to impairment of LAPTM4B gene expression.(A) MCF7 cell were transfected with control shRNA (Conrol-A) or CREB1-specific shRNA (RNAi). NC represented the MCF7 cell without any treatment. Whole cell lysates were subjected to western blot analysis for CREB1 protein. The intensity of gray value of western blot bands. The different superscript letters represent significantly different groups (p<0.05). (B) Analysis of LAPTM4B gene transcript by real-time PCR after 48 h of shRNA transfection. (*P<0.05, n = 3).

Mentions: Knockdown of CREB1 was performed by transient transfection of the shRNA targeting its mRNA. The CREB1 protein level in RNAi group decreased by approximately 50% compare with the control group: Control-A. However, the CREB1 protein level in Control-A group was higher than the NC group (Fig. 5A). Real-time PCR results showed that the level of LAPTM4B mRNA was significantly decreased in RNAi group compared with the group of Control-A (Fig. 5B). And the LAPTM4B mRNA in Control-A group was slightly increased compare with NC group.


cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Zhang M, Xu JJ, Zhou RL, Zhang QY - PLoS ONE (2013)

shRNA targeting of CREB1 specifically inhibits its expression and leads to impairment of LAPTM4B gene expression.(A) MCF7 cell were transfected with control shRNA (Conrol-A) or CREB1-specific shRNA (RNAi). NC represented the MCF7 cell without any treatment. Whole cell lysates were subjected to western blot analysis for CREB1 protein. The intensity of gray value of western blot bands. The different superscript letters represent significantly different groups (p<0.05). (B) Analysis of LAPTM4B gene transcript by real-time PCR after 48 h of shRNA transfection. (*P<0.05, n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585361&req=5

pone-0057520-g005: shRNA targeting of CREB1 specifically inhibits its expression and leads to impairment of LAPTM4B gene expression.(A) MCF7 cell were transfected with control shRNA (Conrol-A) or CREB1-specific shRNA (RNAi). NC represented the MCF7 cell without any treatment. Whole cell lysates were subjected to western blot analysis for CREB1 protein. The intensity of gray value of western blot bands. The different superscript letters represent significantly different groups (p<0.05). (B) Analysis of LAPTM4B gene transcript by real-time PCR after 48 h of shRNA transfection. (*P<0.05, n = 3).
Mentions: Knockdown of CREB1 was performed by transient transfection of the shRNA targeting its mRNA. The CREB1 protein level in RNAi group decreased by approximately 50% compare with the control group: Control-A. However, the CREB1 protein level in Control-A group was higher than the NC group (Fig. 5A). Real-time PCR results showed that the level of LAPTM4B mRNA was significantly decreased in RNAi group compared with the group of Control-A (Fig. 5B). And the LAPTM4B mRNA in Control-A group was slightly increased compare with NC group.

Bottom Line: Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression.However, its transcriptional regulation mechanism is still unclear.The +10+292 promoter region was possessed the highest transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical laboratory, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, China.

ABSTRACT
Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

Show MeSH
Related in: MedlinePlus