Radiation Enhances the Epithelial– Mesenchymal Transition of A549 Cells via miR3591-5p/USP33/PPM1A

Abstract

Background/Aims: Radiotherapy plays a critical role in lung cancer treatment. Radiation can activate transforming growth factor-β (TGF-β) signaling and induce the epithelial-mesenchymal transition (EMT), which may lead to distant metastases. MicroRNAs (miRNAs) have been suggested to affect radiotherapy in lung cancer. Methods: miRNA Next-Generation Sequencing was performed to investigate the effects of irradiation on the miRNA profile of lung cancer A549 cells. The functions of identified miRNA on the radiation induced EMT and TGF-β activation in A549 cells were then explored. Protein expression was evaluated by western blotting. Immunofluorescence staining was performed to detect the localization of Snail. Luciferase Assay was used to determine the target gene regulated by the identified miRNA. Results: Radiation time-dependently induced EMT in A549 lung cancer cells as indicated by the changes of morphology, the expression of EMT marker proteins (E-cadherin, α-SMA and Vimentin) and the nuclear localization of Snail. Moreover, miR-3591-5p was identified as the most significant increased miRNA in response to radiation, and further experiments indicated that miR-3591-5p was required for radiation induced EMT and TGF-β/ Smad2/3 activation. Ubiquitin Specific Peptidase 33 (USP33) was a downstream target of miR-3591-5p as predicted by TargetScan and validated by 3’ untranslated regions (UTRs) Luciferase Assay. USP33 could deubiquitinate PPM1A (protein phosphatase, Mg2+/Mn2 + dependent 1A), a phosphatase for Smad2/3. Ectopic expression of USP33 or PPM1A partially abolished the effects of miR-3591-5p on EMT and TGF-β/ Smad2/3 activation. Conclusion: The present study revealed the critical role of miR-3591-5p/USP33/PPM1A in radiation-induced EMT via TGF-β signaling and may suggest novel radiation sensitise strategies for lung cancer.

HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells

“…Significantly higher expression in stage III colon cancers from patients with recurrence and associated with poorer DFS 229 .miR-34b-5p−2.20Downregulated in breast tumors 166,230 .Tumor suppressormiR-34c-5p−2.36Downregulated in breast tumors 231 Tumor suppressormiR-3591-5p−2.88No reports for BCa. Expression increased after radiation of A549 NSCLC cells and Ubiquitin Specific Peptidase 33 (USP33) was a downstream target of miR-3591-5p 232 .miR-3663-5p−2.96No reports for BCa. Expression increased in human nonalcoholic fatty liver disease (NAFLD) 233 .miR-3912-3p−0.72No reports for breast or other cancers.miR-424-5p−1.18Increased in serum of BCa patients with resistance to dovitinib 234 .…”

Epithelial to Mesenchymal Transition: A Mechanism that Fuels Cancer Radio/Chemoresistance

“…These data, produced by Ghosh et al, clearly indicate that the expression profile of microRNAs is a significant indicator of a therapy-resistant gene expression pattern [100]. This list can be extended with miR-3591-5p, which is the most significantly increased miRNA in response to radiation [102].…”

“…Several pieces of evidence revealed that, if EMT is experimentally induced in cultured epithelial carcinoma cells, they will display cisplatin-resistance and radio-resistance [25]. Moreover, if, in a pure epithelial cancer cell culture, radio-resistance is induced [101,102], the resulting gene expression profile will resemble an EMT phenotype. A novel hypothesis suggests that EMT cells release membranous vesicles with a diameter of 40 to 100 nm, called exosomes, which are seen as functional mediators of a tumor-stroma interaction and of EMT.…”

“…This EMT trans-differentiation pathway is achieved through STAT3 and Slug [104]. A sequential set of chemotherapy treatments or a series of irradiation treatments of pure epithelial cancer cell cultures induce resistant cells that also share the EMT phenotype [101,102,104,107]. These cells were not contacted by any external humoral factors or co-cultured with mesenchymal cells before.…”

miR‐522 stimulates TGF‐β/Smad signaling pathway and promotes osteosarcoma tumorigenesis by targeting PPM1A

“…Increasing studies are pointing toward the involvement of PPM1A in the regulation of cell proliferation, migration, invasion, and differentiation . It is evident from several investigations that PPM1A emerges as a cancer suppressor in human malignancy . For instance, PPM1A is a RelA phosphatase with tumor suppressor‐like activity .…”