Huo Y et al. (2022), F-circEA1 regulates cell proliferation and apop...

ECB-ART-48919

Hum Cell 2022 Jan 01;351:260-270. doi: 10.1007/s13577-021-00628-7.

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F-circEA1 regulates cell proliferation and apoptosis through ALK downstream signaling pathway in non-small cell lung cancer.

Huo Y , Lv T , Ye M , Zhu S , Liu J , Liu H , Song Y .

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Studies have confirmed that circular RNA (circRNA) has a stable closed structure, which plays an important role in the progression of tumors. Cancers with positive fusion genes can produce associated fusion circRNA (F-cirRNA). However, there are no reports concerning a role for F-circRNA of the echinoderm microtubule associated-protein like 4-anaplastic lymphoma kinase variant 1 (EML4-ALK1) in non-small cell lung cancer (NSCLC). Our study confirmed the existence of fusion circEA1 (F-circEA1) in NCI-H3122 cells (carrying the EML4-ALK1 gene), F-circEA1 was expressed both in the cytoplasm and nucleus as determined by fluorescence in situ hybridization (FISH) and Sanger sequencing. CCK8 and transwell assays showed that F-circEA1 was beneficial to cell proliferation, metastasis, and invasion. Overexpression of F-circEA1 can also promote cell proliferation, migration and invasion in A549 and SPCA1 cells (non-small cell lung cancer cell line not carrying the EML4-ALK1 gene). Interference with F-circEA1, induced cell cycle arrest and promoted apoptosis as determined by flow cytometry, and increased drug sensitivity to crizotinib in H3122 cells. F-circEA1 directly affected the expression of parental gene EML4-ALK1. Further research found that F-circEA1 can affect the downstream signaling pathway of ALK. In vivo, the growth rate of xenogeneic tumors was reduced and the protein expression level of EML4-ALK1 was significantly decreased in transplanted tumors measured by immunohistochemistry (IHC) after interference with F-circEA1. In conclusion, F-circEA1 can be considered as a proto-oncogene that regulates cell proliferation and apoptosis by affecting the expression of the parental gene EML4-ALK1 and its ALK downstream signaling pathway in non-small cell lung cancer.

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	Fig. 1. Identification of F-circEA1. a EML4-ALK1 and GAPDH were amplified from the cDNA of H3122, A549 and HBE cells, the RNAs of the cells were treated with and without RNase R, respectively, EML4-ALK1 was only expressed in H3122. b Sanger sequencing revealed the fusion site of EML4-ALK1. c F-circEA1 and GAPDH were detected and amplified from the PCR products of the RNase R-treated, without the RNase R-treated, and the genomic DNA, respectively. F-circEA1 was resistant to RNase R treatment. d Sanger sequencing showed the junction site of F-cricEA1. e FISH and f RNA nuclear and cytoplasmic isolation assay showed that the subcellular distribution of F-circEA1 mainly existed in the cytoplasm. (n = 3). (Scale bar = 20 μm)
	Fig. 2. F-circEA1 promotes the proliferation, migration and invasion of H3122 cells. a FISH showed F-circEA1 overexpression in H3122. (Scale bar = 20 μm). b Agarose gel electrophoresis revealed F-circEA1 overexpression in H3122. c Sanger sequencing showed the junction site of overexpressed F-cricEA1. d CCK8 assays tested the proliferation of H3122 cells after inhibiting or e overexpressing of F-circEA1. (n = 3). f Images and quantification of the migration and invasion with F-circEA1 interference or g overexpression in H3122 cells. (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 3. F-circEA1 promotes the proliferation, migration and invasion of A549 and SPCA1 cells. a The proliferation of A549 and b SPCA1 cells with F-circEA1 overexpression. (n = 3). c Images and quantification of migration and invasion with F-circEA1 overexpression in A549 and d SPCA1 cells. (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 4. Interference with F-circEA1 induces cell cycle arrest, promotes apoptosis and improves the sensitivity to crizotinib in H3122 cells. a Different stages of the cell cycle and b the apoptosis rate was quantified in H3122 cells after interference with F-circEA1. (n = 3). c The effect of overexpression of or interference with F-circEA1 on apoptosis-related proteins. (n = 3). d Cell viability was assessed with different concentration of crizotinib after cells were subjected to F-circEA1 interference. (n = 5). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 5. F-circEA1 promotes the expression of EML4-ALK1 in H3122 cells. a The mRNA expression of EML4-ALK1 was measured after F-circEA1 interference or b overexpression. (n = 3). c The protein levels of EML4-ALK1 were detected after F-circEA1 interference or d overexpression. (n = 3). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 6. F-circEA1 activates EML4-ALK1 associated downstream signaling pathways in H3122 cells. a The mRNA expression of the signaling factors were determined after transfection with F-circEA1 interference or b overexpression plasmid. (n = 3). c The protein expression of the signaling factors were detected after F-circEA1 interference or d overexpression. (n = 3). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 7. Interference with F-circEA1 inhibited subcutaneous xenograft growth and the protein level of EML4-ALK1 in the tumors. a No obvious differences in body weights of the nude mice were seen after interference with F-circEA1 (n = 5). b Tumor volumes and c weights were reduced after interference with F-circEA1 (n = 5). d EML4-ALK1 protein expressed in the cytoplasm and cell membrane, and EML4-ALK1 protein level decreased in the shF-circEA1 group (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)

References [+] :

Ambrogio, p130Cas mediates the transforming properties of the anaplastic lymphoma kinase. 2005, Pubmed

	Fig. 1. Identification of F-circEA1. a EML4-ALK1 and GAPDH were amplified from the cDNA of H3122, A549 and HBE cells, the RNAs of the cells were treated with and without RNase R, respectively, EML4-ALK1 was only expressed in H3122. b Sanger sequencing revealed the fusion site of EML4-ALK1. c F-circEA1 and GAPDH were detected and amplified from the PCR products of the RNase R-treated, without the RNase R-treated, and the genomic DNA, respectively. F-circEA1 was resistant to RNase R treatment. d Sanger sequencing showed the junction site of F-cricEA1. e FISH and f RNA nuclear and cytoplasmic isolation assay showed that the subcellular distribution of F-circEA1 mainly existed in the cytoplasm. (n = 3). (Scale bar = 20 μm)
	Fig. 2. F-circEA1 promotes the proliferation, migration and invasion of H3122 cells. a FISH showed F-circEA1 overexpression in H3122. (Scale bar = 20 μm). b Agarose gel electrophoresis revealed F-circEA1 overexpression in H3122. c Sanger sequencing showed the junction site of overexpressed F-cricEA1. d CCK8 assays tested the proliferation of H3122 cells after inhibiting or e overexpressing of F-circEA1. (n = 3). f Images and quantification of the migration and invasion with F-circEA1 interference or g overexpression in H3122 cells. (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 3. F-circEA1 promotes the proliferation, migration and invasion of A549 and SPCA1 cells. a The proliferation of A549 and b SPCA1 cells with F-circEA1 overexpression. (n = 3). c Images and quantification of migration and invasion with F-circEA1 overexpression in A549 and d SPCA1 cells. (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 4. Interference with F-circEA1 induces cell cycle arrest, promotes apoptosis and improves the sensitivity to crizotinib in H3122 cells. a Different stages of the cell cycle and b the apoptosis rate was quantified in H3122 cells after interference with F-circEA1. (n = 3). c The effect of overexpression of or interference with F-circEA1 on apoptosis-related proteins. (n = 3). d Cell viability was assessed with different concentration of crizotinib after cells were subjected to F-circEA1 interference. (n = 5). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 5. F-circEA1 promotes the expression of EML4-ALK1 in H3122 cells. a The mRNA expression of EML4-ALK1 was measured after F-circEA1 interference or b overexpression. (n = 3). c The protein levels of EML4-ALK1 were detected after F-circEA1 interference or d overexpression. (n = 3). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 6. F-circEA1 activates EML4-ALK1 associated downstream signaling pathways in H3122 cells. a The mRNA expression of the signaling factors were determined after transfection with F-circEA1 interference or b overexpression plasmid. (n = 3). c The protein expression of the signaling factors were detected after F-circEA1 interference or d overexpression. (n = 3). (P < 0.05, P < 0.01, **P < 0.005)
	Fig. 7. Interference with F-circEA1 inhibited subcutaneous xenograft growth and the protein level of EML4-ALK1 in the tumors. a No obvious differences in body weights of the nude mice were seen after interference with F-circEA1 (n = 5). b Tumor volumes and c weights were reduced after interference with F-circEA1 (n = 5). d EML4-ALK1 protein expressed in the cytoplasm and cell membrane, and EML4-ALK1 protein level decreased in the shF-circEA1 group (n = 5). (Scale bar = 20 μm). (P < 0.05, P < 0.01, **P < 0.005)