MED12 exerts an emerging role in actin-mediated cytokinesis via LIMK2/cofilin pathway in NSCLC

Abstract

Background Mediator complex subunit 12 (MED12) is an essential hub for transcriptional regulation, in which mutations and overexpression were reported to be associated with several kinds of malignancies. Nevertheless, the role of MED12 in non-small cell lung cancer (NSCLC) remains to be elucidated. Methods MED12 mutation was detected by Next-generation sequencing. The expression of MED12 in 179 human NSCLC tissue samples and 73 corresponding adjacent normal lung tissue samples was measured by immunohistochemistry (IHC). CRISPR-Cas9 was used to knock out MED12 in PC9 and SPC-A1 cells. MED12 rescued stable cell lines were generated by lentivirus infection. We traced cell division process by live cell imaging. The molecular mechanism of aborted cytokinesis resulted by MED12 knockout was investigated by RNA-seq. Effects of MED12 deletion on the proliferation of NSCLC cells were determined by MTT assay and Colony-formation assay in vitro and xenograft tumor model in nude mouse. Cell senescence was measured by SA-β-gal staining. Results In our study, no MED12 exon mutation was detected in NSCLC samples, whereas we found that MED12 was overexpressed in human NSCLC tissues, which positively correlated with the tumor volume and adversely affected patient survival. Furthermore, knockout MED12 in NSCLC cell lines resulted in cytokinesis failure, displayed a multinuclear phenotype, and disposed to senescence, and become non-viable. Lack of MED12 decreased the proliferative potential of NSCLC cells and limited the tumor growth in vivo. Mechanism investigations revealed that MED12 knockout activated LIMK2, caused aberrant actin cytoskeleton remodeling, and disrupted the abscission of intercellular bridge, which led to the cytokinesis failure. Reconstitution of exogenous MED12 restored actin dynamics, normal cytokinesis and cell proliferation capacity in MED12 knockout cells. Conclusions These results revealed a novel role of MED12 as an important regulator for maintaining accurate cytokinesis and survival in NSCLC cells, which may offer a therapeutic strategy to control tumor growth for NSCLC patients especially those highly expressed MED12. Electronic supplementary material The online version of this article (10.1186/s12943-019-1020-4) contains supplementary material, which is available to authorized users.

Cofilin: A Promising Protein Implicated in Cancer Metastasis and Apoptosis

“…Phosphorylation of cofilin regulated by LIMK can abrogate actin depolymerization activities and enhances stabilization of actin filament, inhibits the lamellipodium formation and tumor cell mobilization (Wioland et al, 2017 ). The overexpression of LIMK1 phosphorylated cofilin and supressed the cancer metastasis by suppressing of lamellipodium formation, while mutated LIMK1 increases the motility of tumor cells (Meyer et al, 2005 ; Li Z. et al, 2014 ), similar results were observed in studies of LIMK2 (Collazo et al, 2014 ; Xu et al, 2019 ). Nonetheless, conflicting results have been observed regarding the role of LIMK.…”

GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma

“…NK-92 cells were grown in 75% alpha-MEM supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 2 mM l-glutamine, 10 mL/L penicillin-streptomycin, 10 ng/mL recombinant human IL-2 (rIL-2), and 5 mg/mL plasmocin to prevent mycoplasma contamination. All cells were maintained at 37 °C in a humidified 5% CO2 incubator [ 25 , 26 ].…”

SARS-CoV-2 membrane protein causes the mitochondrial apoptosis and pulmonary edema via targeting BOK

“…Knockout status of expanded single-cell clones was validated by gene sequences and WB analysis of immunoblotting with BAK antibody (1:1000, #ab32371, Abcam, Cambridge, England), BAX antibody (1:1000, #2772 s, CST, USA) or BOK antibody (1:1000, #ab233072, Abcam, Cambridge, England). The rescue expression of BOK followed Xu et al [ 22 ]. The sgRNA sequences for CRISPR/Cas 9 knockout were listed in Supplementary Table 1 .…”