BMAL1 Suppresses Proliferation, Migration, and Invasion of U87MG Cells by Downregulating Cyclin B1, Phospho-AKT, and Metalloproteinase-9.
BMAL1 通过下调 Cyclin B1 、 Phospho-AKT 和 Metalloproteinase-9 抑制 U87MG 细胞的增殖、迁移和侵袭。
- 作者列表："Gwon DH","Lee WY","Shin N","Kim SI","Jeong K","Lee WH","Kim DW","Hong J","Lee SY
:Several studies have shown that brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), an important molecule for maintaining circadian rhythms, inhibits the growth and metastasis of tumor cells in several types of cancer, including lung, colon, and breast cancer. However, its role in glioblastoma has not yet been established. Here, we addressed the function of BMAL1 in U87MG glioblastoma cells with two approaches-loss and gain of function. In the loss of function experiments, cell proliferation in U87MG cells transfected with small interfering RNA (siRNA) targeting BMAL1 was increased by approximately 24% (small interfering (si)-NC 0.91 ± 0.00 vs. si-BMAL1 1.129 ± 0.08) via upregulation of cyclin B1. In addition, cell migration and invasion of BMAL1 siRNA-treated glioblastoma cells were elevated by approximately 20% (si-NC 51.00 ± 1.53 vs. si-BMAL161.33 ± 0.88) and 209% (si-NC 21.28 ± 1.37 vs. si-BMAL1 44.47 ± 3.48), respectively, through the accumulation of phosphorylated-AKT (p-AKT) and matrix metalloproteinase (MMP)-9. Gain of function experiments revealed that adenovirus-mediated ectopic expression of BMAL1 in U87MG cells resulted in a 19% (Adenovirus (Ad)-vector 0.94± 0.03 vs. Ad-BMAL1 0.76 ± 0.03) decrease in cell proliferation compared with the control via downregulation of cyclin B1 and increased early and late apoptosis due to changes in the levels of BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cleaved caspase-3. Likewise, cell migration and invasion were attenuated by approximately 24% (Ad-vector 55.00 ± 0.00 vs. Ad-BMAL1 41.83 ± 2.90) and 49% (Ad-vector 70.01 ± 1.24 vs. Ad-BMAL1 35.55 ± 1.78), respectively, in BMAL1-overexpressing U87MG cells following downregulation of p-AKT and MMP-9. Taken together, our results suggest that BMAL1 acts as an anti-cancer gene by altering the proliferation, migration, and invasion of glioblastoma cells. Therefore, the BMAL1 gene could be a potential therapeutic target in the treatment of glioblastoma.
: 几项研究表明，脑和肌肉芳香烃受体核转运体样 1 (BMAL1)，维持昼夜节律的重要分子,在几种类型的癌症中抑制肿瘤细胞的生长和转移，包括肺癌、结肠癌和乳腺癌。然而，其在胶质母细胞瘤中的作用尚未确立。在此，我们用两种方法 -- 功能丧失和获得 -- 探讨了 BMAL1 在 U87MG 胶质母细胞瘤细胞中的功能。在功能丧失实验中，转染靶向 BMAL1 的小干扰 RNA (siRNA) 的 U87MG 细胞的细胞增殖增加了约 24% (小干扰 (si)-NC 0.91 ± 0.00 vs.通过上调细胞周期蛋白 b1 si-BMAL1 1.129 ± 0.08)。此外，BMAL1 siRNA 处理的胶质母细胞瘤细胞的细胞迁移和侵袭能力提高了约 20% (si-NC 51.00 ± 1.53 vs. si-BMAL161.33 ± 0.88) 和 209% (si-NC 21.28 ± 1.37 vs. si-BMAL1 44.47 ± 3.48)，分别通过磷酸化 AKT (p-AKT) 和基质金属蛋白酶 (MMP)-9 的积累。功能获得实验表明，腺病毒介导的 BMAL1 在 U87MG 细胞中的异位表达产生了 19% (腺病毒 (Ad) 载体 0.94 ± 0.03 vs。ad-BMAL1 (0.76 ± 0.03) 细胞增殖与对照相比，通过下调细胞周期蛋白 B1，增加早期和晚期细胞凋亡，由于 BCL2-associated X 蛋白水平的变化(BAX) 、 b细胞淋巴瘤 2 (BCL-2) 和 cleaved caspase-3。同样，细胞迁移和侵袭减弱约 24% (Ad-vector 55.00 ± 0.00 vs. Ad-BMAL1 41.83 ± 2.90) 和 49% (Ad-vector 70.01 ± 1.24 vs. Ad-BMAL1 35.55 ± 1.78)，分别在 BMAL1-overexpressing U87MG 细胞中下调 p-AKT 和 MMP-9。总之，我们的结果表明，BMAL1 通过改变胶质母细胞瘤细胞的增殖、迁移和侵袭而作为抗癌基因发挥作用。因此，BMAL1 基因有可能成为治疗胶质母细胞瘤的潜在靶点。
METHODS::Glioma growth can cause pervasive changes in the functional connectivity (FC) of brain networks, which has been associated with re-organization of brain functions and development of functional deficits in patients. Mechanisms underlying functional re-organization in brain networks are not understood and efforts to utilize functional imaging for surgical planning, or as a biomarker of functional outcomes are confounded by the heterogeneity in available human data. Here we apply multiple imaging modalities in a well-controlled murine model of glioma with extensive validation using human data to explore mechanisms of FC disruption due to glioma growth. We find gliomas cause both local and distal changes in FC. FC changes in networks proximal to the tumor occur secondary to hemodynamic alterations but surprisingly, remote FC changes are independent of hemodynamic mechanisms. Our data strongly implicate hemodynamic alterations as the main driver of local changes in measurements of FC in patients with glioma.
METHODS::Mutations in LZTR1, already known to be causal in familial schwannomatosis type 2, have been recently involved in a small proportion of patients with autosomal dominant and autosomal recessive Noonan syndrome. LZTR1 is also a driver gene in non syndromal glioblastoma. We report a 26-year-old patient with typical Noonan syndrome, and the dominantly transmitted c.850C > T (p.(Arg284Cys)) variant in LZTR1. An oligoastrocytoma was diagnosed in the patient at the age of 22 years; recurrence of the tumor occurred at age 26, as a ganglioblastoma. The patient had been transiently treated with growth hormone between ages 15 and 17. Considering the implication of LZTR1 in sporadic tumors of the nervous system, we hypothesize that gliomas are a possible complication of LZTR1-related Noonan syndrome. This report also supports a possible link between occurrence of a cerebral tumor in Noonan syndrome and a previous treatment with growth hormone.
METHODS:BACKGROUND:Susceptibility weighted imaging (SWI) provides vascular information and plays an important role in improving the diagnostic accuracy of preoperative glioma grading. Intratumoral susceptibility signal intensities (ITSS) obtained from SWI has been used in glioma grading. However, the current method for estimation of ITSS is semiquantitative, manual count-dependent, and includes hemorrhage as well as vasculature. PURPOSE:To develop a quantitative approach that calculates the vasculature volume within tumors by filtering out the hemorrhage from ITSS using R2 * values and connected component analysis-based segmentation algorithm; to evaluate the accuracy of the proposed ITSS vasculature volume (IVV) for differentiating various grades of glioma; and compare it with reported semiquantitative ITSS approach. STUDY TYPE:Retrospective. SUBJECTS:Histopathologically confirmed 41 grade IV, 19 grade III, and 15 grade II glioma patients.Field Strength/Sequence: SWI (four echoes: 5.6, 11.8, 18, 24.2 msec) along with conventional MRI sequences (T2 -weighted, T1 -weighted, 3D-fluid-attenuated inversion recovery [FLAIR], and diffusion-weighted imaging [DWI]) at 3.0T. ASSESSMENT:R2 * relaxation maps were calculated from multiecho SWI. The R2 * cutoff value for hemorrhage ITSS was determined. A segmentation algorithm was designed, based on this R2 * hemorrhage combined with connected component shape analysis, to quantify the IVV from all slices containing tumor by filtering out hemorrhages. Semiquantitative ITSS scoring as well as total ITSS volume (TIV) including hemorrhages were also calculated. STATISTICAL TESTS:One-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc tests were performed to see the difference among the three grades of the tumor (II, III, and IV) in terms of semiquantitative ITSS scoring, TIV, and IVV. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the three methods individually in discriminating between grades of glioma. RESULTS:One-way ANOVA showed that only the proposed IVV significantly differentiated different grades of gliomas having visible ITSS. ROC analysis showed that IVV provided the highest AUC for the discrimination of grade II vs. III (0.93), grade III vs. IV (0.98), and grade II vs. IV glioma (0.94). IVV also provided the highest sensitivity and specificity for differentiating grade II vs. III (87.44, 98.41), grade III vs. IV (97.15, 94.12), and grade II vs. IV (98.72, 92.31). DATA CONCLUSION:The proposed quantitative method segregates hemorrhage from tumor vasculature. It scores above the existing semiquantitative method in terms of ITSS estimation and grading accuracy. LEVEL OF EVIDENCE:4 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:225-233.