Endoglycosidase-Mediated Incorporation of ¹⁸O into Glycans for Relative Glycan Quantitation

Stable isotopic labeling coupled with mass spectrometry analysis is a promising method of detecting quantitative variations in glycans, which may result in aberrant glycosylation in many disorders and diseases. Although various isotopic labeling methods have been used for relative glycan quantitation, enzymatic 18O labeling, which offers advantages for glycomics similar to those by protease-catalyzed 18O labeling for proteomics, has not been developed yet. In this study, endoglycosidase incorporated 18O into the N-glycan reducing end in 18O-water as N-glycans were released from glycoproteins, rendering glycan reducing-end 18O labeling (GREOL) a potential strategy for relative glycan quantitation. This proposed method provided good linearity with high reproducibility within 2 orders of magnitude in dynamic range. The ability of GREOL to quantitatively discriminate between isomeric hybrid N-glycans and complex N-glycans in glycoproteins was validated due to the distinct substrate specificities of endoglycosidases. GREOL was also used to analyze changes in human serum N-glycans associated with hepatocellular carcinoma

DataSheet4_Maackiain Modulates miR-374a/GADD45A Axis to Inhibit Triple-Negative Breast Cancer Initiation and Progression.ZIP

Breast cancer ranks as the leading cause of death in lethal malignancies among women worldwide, with a sharp increase of incidence since 2008. Triple negative breast cancer (TNBC) gives rise to the largest proportion in breast cancer-related deaths because of its aggressive growth and rapid metastasis. Hence, searching for promising targets and innovative approaches is indispensable for the TNBC treatment. Maackiain (MA), a natural compound with multiple biological activities, could be isolated from different Chinese herbs, such as Spatholobus suberectus and Sophora flavescens. It was the first time to report the anti-cancer effect of MA in TNBC. MA could suppress TNBC cell proliferation, foci formation, migration, and invasion. MA also exerted a significant inhibitory effect on tumor growth of TNBC. Furthermore, MA could induce apoptosis with an increase of GADD45α and a decrease of miR-374a. In contrast, overexpressing miR-374a would result in at least partly affecting the proapoptotic effect of MA and suppressing GADD45α stimulated by MA. These results reveal the anti-TNBC effect of MA in vitro and in vivo, providing evidence for its potential as a drug candidate utilized in TNBC therapy.</p

DataSheet2_Maackiain Modulates miR-374a/GADD45A Axis to Inhibit Triple-Negative Breast Cancer Initiation and Progression.ZIP

Breast cancer ranks as the leading cause of death in lethal malignancies among women worldwide, with a sharp increase of incidence since 2008. Triple negative breast cancer (TNBC) gives rise to the largest proportion in breast cancer-related deaths because of its aggressive growth and rapid metastasis. Hence, searching for promising targets and innovative approaches is indispensable for the TNBC treatment. Maackiain (MA), a natural compound with multiple biological activities, could be isolated from different Chinese herbs, such as Spatholobus suberectus and Sophora flavescens. It was the first time to report the anti-cancer effect of MA in TNBC. MA could suppress TNBC cell proliferation, foci formation, migration, and invasion. MA also exerted a significant inhibitory effect on tumor growth of TNBC. Furthermore, MA could induce apoptosis with an increase of GADD45α and a decrease of miR-374a. In contrast, overexpressing miR-374a would result in at least partly affecting the proapoptotic effect of MA and suppressing GADD45α stimulated by MA. These results reveal the anti-TNBC effect of MA in vitro and in vivo, providing evidence for its potential as a drug candidate utilized in TNBC therapy.</p

DataSheet8_Maackiain Modulates miR-374a/GADD45A Axis to Inhibit Triple-Negative Breast Cancer Initiation and Progression.ZIP

Breast cancer ranks as the leading cause of death in lethal malignancies among women worldwide, with a sharp increase of incidence since 2008. Triple negative breast cancer (TNBC) gives rise to the largest proportion in breast cancer-related deaths because of its aggressive growth and rapid metastasis. Hence, searching for promising targets and innovative approaches is indispensable for the TNBC treatment. Maackiain (MA), a natural compound with multiple biological activities, could be isolated from different Chinese herbs, such as Spatholobus suberectus and Sophora flavescens. It was the first time to report the anti-cancer effect of MA in TNBC. MA could suppress TNBC cell proliferation, foci formation, migration, and invasion. MA also exerted a significant inhibitory effect on tumor growth of TNBC. Furthermore, MA could induce apoptosis with an increase of GADD45α and a decrease of miR-374a. In contrast, overexpressing miR-374a would result in at least partly affecting the proapoptotic effect of MA and suppressing GADD45α stimulated by MA. These results reveal the anti-TNBC effect of MA in vitro and in vivo, providing evidence for its potential as a drug candidate utilized in TNBC therapy.</p

DataSheet5_Maackiain Modulates miR-374a/GADD45A Axis to Inhibit Triple-Negative Breast Cancer Initiation and Progression.ZIP

Breast cancer ranks as the leading cause of death in lethal malignancies among women worldwide, with a sharp increase of incidence since 2008. Triple negative breast cancer (TNBC) gives rise to the largest proportion in breast cancer-related deaths because of its aggressive growth and rapid metastasis. Hence, searching for promising targets and innovative approaches is indispensable for the TNBC treatment. Maackiain (MA), a natural compound with multiple biological activities, could be isolated from different Chinese herbs, such as Spatholobus suberectus and Sophora flavescens. It was the first time to report the anti-cancer effect of MA in TNBC. MA could suppress TNBC cell proliferation, foci formation, migration, and invasion. MA also exerted a significant inhibitory effect on tumor growth of TNBC. Furthermore, MA could induce apoptosis with an increase of GADD45α and a decrease of miR-374a. In contrast, overexpressing miR-374a would result in at least partly affecting the proapoptotic effect of MA and suppressing GADD45α stimulated by MA. These results reveal the anti-TNBC effect of MA in vitro and in vivo, providing evidence for its potential as a drug candidate utilized in TNBC therapy.</p

Characteristics of the included studies.

<p>Characteristics of the included studies.</p

LNA-anti-miR-182 and -381 induces <i>LRRC4</i> up-regulation and <i>BRD7</i> down-regulation.

<p>(A) qRT-PCR showing down-regulation of miR-182 and miR-381 in U251 cells after LNA-anti-miRs transfection. * <i>p</i><0.05. (B) LNA-mediated miR-182 and -381 silencing restored endogenous levels of <i>LRRC4</i> protein and decreased BRD7 expression. U251 cells were transfected with either LNA-scrambled, LNA-anti-miR-182 or -381 for 48 h. <i>LRRC4</i> and <i>BRD7</i> expression was assessed by Western blot. GAPDH was used as a loading control. (C) qRT-PCR confirmed re-expression of <i>LRRC4</i> and decreased <i>BRD7</i> expression after LNA-anti-miR-182 and -381 transfection. (D) Ectopic <i>LRRC4</i> expression decreased endogenous levels of <i>BRD7</i> protein in U251 cells. <i>LRRC4</i> and <i>BRD7</i> expression were assessed by Western blot (left) and gray image scanning (right). * <i>p</i><0.05 compared with mock (control). (E) 5-Aza-dC restored endogenous levels of <i>LRRC4</i> protein and decreased that of <i>BRD7</i> expression in U251, SF126, and SF767 cells. <i>LRRC4</i> and <i>BRD7</i> expressions were assessed by Western blot (left) and gray image scanning (right). * <i>p</i><0.05 compared with LNA-scrambled control.</p

Disturbing miR-182 and -381 Inhibits BRD7 Transcription and Glioma Growth by Directly Targeting <i>LRRC4</i>

<div><p>Inactivated <i>LRRC4</i> has been clinically detected in gliomas, and promoter hypermethylation has been implicated as the mechanism of inactivation in some of those tumors. Our previous researches indicated that <i>LRRC4</i> is a target gene of miR-381, the interaction of miR-381 and <i>LRRC4</i> is involved in glioma growth. In this study, we demonstrate that <i>LRRC4</i> is a target gene of the other microRNA, miR-182. We found that the high expression of miR-182 and miR-381 in gliomas are involved in pathological malignant progression. The silencing of miR-182 and miR-381 inhibited the proliferation <i>in vitro</i> and growth of glioma cell with <i>in vivo</i> magnetic resonance imaging by intracranial transplanted tumor model in rats. We also demonstrated that BRD7, a transcriptional cofactor for p53, is highly expressed and negatively correlated with <i>LRRC4</i> expression in gliomas. Disturbing miR-182 and miR-381 affected transcriptional regulation of the <i>BRD7</i> gene. This finding was verified by ectopic overexpression of <i>LRRC4</i> or restoration of endogenous <i>LRRC4</i> expression by treatment with the DNA demethylating agent 5-Aza-dC. Taken together, miR-182 and miR-381 may be a useful therapeutic target for treatment of glioma.</p></div

BikDDA eliminated TNBC cells more powerfully than BikDD.

<p>A. S124A and previous mutant sites T33D and S35D didn’t locate in the pro-apoptotic region of Bik. B. MDA-MB-231 cells were transfected for 28h with the same amount of pUK21, pUK21-BikDD or pUK21-BikDDA. Apoptotic cells were monitored by Annexin V/PI staining and flow-cytometry analysis. The right-lower or right upper quadrant of each plot showed early apoptotic or late apoptotic cells. C. Histogram directly showed the enhanced apoptosis-inducing activity of BikDDA. D. The cytotoxicity of BikDD and BikDDA were analyzed by Cell counting kit and MTT assay (setting at 100% in vector group). E. Western blotting was performed to confirm the prolonged half-life of BikDDA in 231 cells. F. MEK1/2 inhibitor UO126 and selective ERK1/2 inhibitor FR180204 increased the stability of BikDD in 231 cells.</p

<i>LRRC4</i> is a target gene of miR-182.

<p>(A) Schema of the interaction sites between miR-182 and the 3′-UTRs of <i>LRRC4</i> (B) (B) Luciferase assay of U251 glioma cells co-transfected with pMIR-REPORT–WT/mutant 3′-UTR <i>LRRC4</i> and miR-182 or scrambled control as indicated. * <i>p</i><0.05. (C) Western blot showing the protein expression of <i>LRRC4</i> after miR-182 was transfected into U251/L cells for 48 h. miR-182 mimics inhibited the protein expression of <i>LRRC4</i>. GAPDH was used as a loading control. (D) qRT-PCR showing the mRNA level of <i>LRRC4</i> after miR-182 mimic was transfected into U251/L cells for 48 h. miR-182 mimic down-regulated the mRNA level of <i>LRRC4</i>. * <i>p</i><0.05.</p