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ERManI is a target of miR-125b and promotes transformation phenotypes in hepatocellular carcinoma (HCC).

Pan S, Cheng X, Chen H, Castro PD, Ittmann MM, Hutson AW, Zapata SK, Sifers RN - PLoS ONE (2013)

Bottom Line: By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway.These phenotypical changes occurred in the absence of alterations in global glycoprotein secretion or ER-stress status.Together, these results revealed a novel post-transcriptional regulatory mechanism for ERManI and implied that this molecule contributes to the regulation of carcinogenesis in HCC independent of its function in glycoprotein quality control.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
The MAN1B1 gene product, designated ER alpha-1, 2-mannosidase (ERManI), is an enzyme localized in the Golgi complex of mammalian cells. By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway. In the present study, we identified that a conserved motif within the 3'UTR of ERManI is a target of miR-125b, a microRNA frequently down-regulated in numerous types of cancers, including hepatocellular carcinoma (HCC). As predicted, the expression of ERManI is significantly elevated in HCC, as measured by immunohistochemistry in a liver spectrum tissue microarray. Additional analyses using several hepatoma cell lines demonstrated that the elevated ERManI inversely correlates with a diminished intracellular concentration of miR-125b. Moreover, functional studies indicated that RNAi-mediated knock-down of endogenous ERManI was sufficient to inhibit proliferation, migration, and invasion of hepatoma cells. These phenotypical changes occurred in the absence of alterations in global glycoprotein secretion or ER-stress status. Together, these results revealed a novel post-transcriptional regulatory mechanism for ERManI and implied that this molecule contributes to the regulation of carcinogenesis in HCC independent of its function in glycoprotein quality control.

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ERManI knockdown does not induce ER-stress.A. Autoradiography of total glycoproteins isolated from extracts and culture media of PLC/PRF5 cells after transfection with control siRNA or two separate ERManI siRNAs (#1 and #2). B. Western blotting of indicated the proteins in extracts of PLC/PRF5 cells following transfection with either control siRNA or ERManI-specific siRNA #1. C. PLC/PRF5 cells were transfected with either control siRNA (Ctrl) or two separate ERManI-specific siRNAs (#1 and #2). 48hr post-transfection, the cells were subjected to metabolic labeling with 35S-methionine and chased at 3hr and 5hr. The image shows the autoradiography of A1AT isolated from cell extracts (IC) or culture media (EC) of the PLC/PRF5 cells at indicated time points.
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pone-0072829-g004: ERManI knockdown does not induce ER-stress.A. Autoradiography of total glycoproteins isolated from extracts and culture media of PLC/PRF5 cells after transfection with control siRNA or two separate ERManI siRNAs (#1 and #2). B. Western blotting of indicated the proteins in extracts of PLC/PRF5 cells following transfection with either control siRNA or ERManI-specific siRNA #1. C. PLC/PRF5 cells were transfected with either control siRNA (Ctrl) or two separate ERManI-specific siRNAs (#1 and #2). 48hr post-transfection, the cells were subjected to metabolic labeling with 35S-methionine and chased at 3hr and 5hr. The image shows the autoradiography of A1AT isolated from cell extracts (IC) or culture media (EC) of the PLC/PRF5 cells at indicated time points.

Mentions: We have recently reported that ERManI functions as a “gate keeper” in the Golgi complex to facilitate the retention and recycling of misfolded glycoproteins escaped from the ER [31]. Lowering the endogenous level of ERManI allows misfolded glycoproteins to escape ERAD and be secreted [31]. It is possible that such effects will influence the overall intracellular level of glycoproteins, which subsequently changes the ER-stress status and indirectly affects cell proliferation and migration. As the first step to test this possibility, we used PLC/PRF5 cells to determine whether knockdown of endogenous ERManI affects overall glycoprotein secretion. For this, total glycoproteins were isolated from the 35S-labelled cells and media after transfection with a control siRNA or siRNAs targeting ERManI. The isolated proteins were analyzed using autoradiography. Unexpectedly, no significant changes in the overall amount of intracellular and extracellular glycoproteins were observed in the presence or absence of ERManI knockdown (Figure 4A), implying that the function of ERManI is selective, and does not affect the secretion of the majority of endogenous glycoproteins. Consistent with this notion, no significant differences in the level of major ER-stress markers, such as BiP, XBP-1, phosph-eIF2α, etc (Figure 4B) were detected, indicating that the overall ER-stress level remained the same upon ERManI knockdown. All together, these results support the notion that the function of ERManI in regulating cell transformation is independent of its function in regulating glycoprotein quality control.


ERManI is a target of miR-125b and promotes transformation phenotypes in hepatocellular carcinoma (HCC).

Pan S, Cheng X, Chen H, Castro PD, Ittmann MM, Hutson AW, Zapata SK, Sifers RN - PLoS ONE (2013)

ERManI knockdown does not induce ER-stress.A. Autoradiography of total glycoproteins isolated from extracts and culture media of PLC/PRF5 cells after transfection with control siRNA or two separate ERManI siRNAs (#1 and #2). B. Western blotting of indicated the proteins in extracts of PLC/PRF5 cells following transfection with either control siRNA or ERManI-specific siRNA #1. C. PLC/PRF5 cells were transfected with either control siRNA (Ctrl) or two separate ERManI-specific siRNAs (#1 and #2). 48hr post-transfection, the cells were subjected to metabolic labeling with 35S-methionine and chased at 3hr and 5hr. The image shows the autoradiography of A1AT isolated from cell extracts (IC) or culture media (EC) of the PLC/PRF5 cells at indicated time points.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733964&req=5

pone-0072829-g004: ERManI knockdown does not induce ER-stress.A. Autoradiography of total glycoproteins isolated from extracts and culture media of PLC/PRF5 cells after transfection with control siRNA or two separate ERManI siRNAs (#1 and #2). B. Western blotting of indicated the proteins in extracts of PLC/PRF5 cells following transfection with either control siRNA or ERManI-specific siRNA #1. C. PLC/PRF5 cells were transfected with either control siRNA (Ctrl) or two separate ERManI-specific siRNAs (#1 and #2). 48hr post-transfection, the cells were subjected to metabolic labeling with 35S-methionine and chased at 3hr and 5hr. The image shows the autoradiography of A1AT isolated from cell extracts (IC) or culture media (EC) of the PLC/PRF5 cells at indicated time points.
Mentions: We have recently reported that ERManI functions as a “gate keeper” in the Golgi complex to facilitate the retention and recycling of misfolded glycoproteins escaped from the ER [31]. Lowering the endogenous level of ERManI allows misfolded glycoproteins to escape ERAD and be secreted [31]. It is possible that such effects will influence the overall intracellular level of glycoproteins, which subsequently changes the ER-stress status and indirectly affects cell proliferation and migration. As the first step to test this possibility, we used PLC/PRF5 cells to determine whether knockdown of endogenous ERManI affects overall glycoprotein secretion. For this, total glycoproteins were isolated from the 35S-labelled cells and media after transfection with a control siRNA or siRNAs targeting ERManI. The isolated proteins were analyzed using autoradiography. Unexpectedly, no significant changes in the overall amount of intracellular and extracellular glycoproteins were observed in the presence or absence of ERManI knockdown (Figure 4A), implying that the function of ERManI is selective, and does not affect the secretion of the majority of endogenous glycoproteins. Consistent with this notion, no significant differences in the level of major ER-stress markers, such as BiP, XBP-1, phosph-eIF2α, etc (Figure 4B) were detected, indicating that the overall ER-stress level remained the same upon ERManI knockdown. All together, these results support the notion that the function of ERManI in regulating cell transformation is independent of its function in regulating glycoprotein quality control.

Bottom Line: By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway.These phenotypical changes occurred in the absence of alterations in global glycoprotein secretion or ER-stress status.Together, these results revealed a novel post-transcriptional regulatory mechanism for ERManI and implied that this molecule contributes to the regulation of carcinogenesis in HCC independent of its function in glycoprotein quality control.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
The MAN1B1 gene product, designated ER alpha-1, 2-mannosidase (ERManI), is an enzyme localized in the Golgi complex of mammalian cells. By functioning as a "gate keeper" to prevent the inappropriate secretion of misfolded glycoproteins, it plays a critical role in maintaining protein homeostasis in the mammalian secretory pathway. In the present study, we identified that a conserved motif within the 3'UTR of ERManI is a target of miR-125b, a microRNA frequently down-regulated in numerous types of cancers, including hepatocellular carcinoma (HCC). As predicted, the expression of ERManI is significantly elevated in HCC, as measured by immunohistochemistry in a liver spectrum tissue microarray. Additional analyses using several hepatoma cell lines demonstrated that the elevated ERManI inversely correlates with a diminished intracellular concentration of miR-125b. Moreover, functional studies indicated that RNAi-mediated knock-down of endogenous ERManI was sufficient to inhibit proliferation, migration, and invasion of hepatoma cells. These phenotypical changes occurred in the absence of alterations in global glycoprotein secretion or ER-stress status. Together, these results revealed a novel post-transcriptional regulatory mechanism for ERManI and implied that this molecule contributes to the regulation of carcinogenesis in HCC independent of its function in glycoprotein quality control.

Show MeSH
Related in: MedlinePlus