Thymosin Beta-4 Knockdown in IEC-6 Normal Intestinal Epithelial Cells Induces DNA Re-replication Via Downregulating Emi1

Abstract

Thymosin β4 (Tβ4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tβ4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3β (GSK-3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4 .

Recombinant adeno-associated virus carrying thymosin β4suppresses experimental colitis in mice

“…Tβ 4 can promote the regeneration of the cornea, skin, heart, and intestine epithelial cells[1,46,47]; nevertheless, PCNA immunostaining in the current study revealed that Tβ 4 attenuated the accelerated proliferation of colonic mucosal cells following DSS or TNBS treatment. One possible explanation may be that the attenuation of mucosal damage by Tβ 4 reduces the compensatory regeneration secondary to tissue damage, which surpasses the proliferation-promoting effect mediated by Tβ 4 .…”

Thymosin β4 induces proliferation, invasion, and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

“…Consistently, recombinant human Tβ4 has been shown to promote lymphocyte proliferation (Li et al 2007), and its treatment also enhances endothelial cell proliferation (Xu et al 2013). Reversely, shRNA-mediated knockdown of Tβ4 significantly suppresses growth of the small intestine in rats, colorectal cancer stem cells, and bladder cancer cells (Chao et al 2014;Wang et al 2012;Ricci-Vitiani et al 2010). Thus, Tβ4 might play diverse roles under different conditions and its significance in cell proliferation could vary according to the origin of cells.…”

Thymosin β4 dynamics during chicken enteroid development

“…The peaks corresponding sum of MRM transitions of SDKPDMAEIEK (acetylate N-terminus), NPLPSK, ETIEQEK of Tβ4 and the corresponding enteroid peptides and the enterocytes was reported by Nemolato et al using immunohistochemical localization that showed its heterogeneous expression associated with different phases of development [31]. An experimental knock down of Tβ4 gene was reported to increase DNA re-replication in intestinal epithelial cells [32]. However, a well-studied function of Tβ4 is that, it regulates both polymerization and depolymerization of cytoskeletal actin and sequesters the monomeric G-actin, preventing its assembly into filamentous F-actin [33].…”