Construction of the plasmid coding for the expression of the EGFP-M-IL-2(88Arg, 125Ala) fusion protein and the anti-tumor effects exerted by the fusion protein in HeLa-60 cells
Authors: Bin Wang
Zhiyong Yan
Guangcan Shao
Tongmei Wang
Dongmeng Qian
Ming Hu
Haitao Wang
Journal: Oncology Letters (2015)
Discipline: CLINICAL MEDICINE
Keywords: apoptosis; expression plasmid; fusion protein; human interleukin-2; melittin

Abstract:  Gene therapy is a promising therapeutic option for the treatment of various cancers, and tumor-targeted plasmids encoding toxic protein genes are potential tools for gene therapy. In the present study, a recombinant plasmid containing the genes for the toxic protein melittin and interleukin-2 (IL-2) was constructed. Melittin and IL-2 are known to play key roles in immunoregulation and cancer therapy, but they each possess defects that limit the clinical application of these proteins. The present study aimed to construct a novel recombinant expression plasmid, pLEGFP-C1-M-IL-2(88Arg, 125Ala), and to improve the biological activity of IL-2 and melittin. The M-IL-2(88Arg, 125Ala) gene was excised from the pPICZŒ±A/M-IL-2(88Arg, 125Ala) plasmid by polymerase chain reaction (PCR). The pLEGFP-C1 plasmid carrying the enhanced green fluorescent protein (EGFP) gene was used as a shuttle plasmid. Subsequent to digestion, the M-IL-2(88Arg, 125Ala) gene was subcloned into the pLEGFP-C1 vector to build the pLEGFP-C1-M-IL-2(88Arg, 125Ala) eukaryotic expression plasmid, which was identified by restriction enzyme digestion and gene sequencing. Confocal microscopy was used to determine the transfection efficiency subsequent to the plasmid being transfected into the cervical cancer HeLa cell line. The cells transfected with the pLEGFP-C1-M-IL-2(88Arg, 125Ala) plasmid demonstrated a decreased transfection efficiency compared with the cells transfected with the pLEGFP-C1 plasmid. The cellular expression of M-IL-2(88Arg, 125Ala) was detected by reverse transcription PCR and western blot analysis. Finally, cell counting kit-8 and apoptosis assays were performed to investigate the effects of the expression of the M-IL-2(88Arg, 125Ala) fusion protein on HeLa cells and to analyze the antitumor activity of the protein. In conclusion, a recombinant eukaryotic pLEGFP-C1-M-IL-2(88Arg, 125Ala) expression plasmid containing the M-IL-2(88Arg, 125Ala) fusion gene was constructed and the M-IL-2(88Arg, 125Ala) fusion protein was successfully expressed in HeLa cells. Furthermore, the M-IL-2(88Arg, 125Ala) fusion protein was able to inhibit HeLa cell proliferation and induce apoptosis in the tumor cells. These findings may offer an alternative method for anticancer therapy. The present study has provided a basis for future studies into the M-IL-2(88Arg, 125Ala) fusion gene. 

Retracted: No

PubPeer comments:
 This paper was found during a systematic search of papers in this journal with possible duplicated images. Fifteen papers in this journal (n=98 examined) were found to contain inappropriately duplicated images. These papers have all been reported to the journal publisher in 2017. The editors promised to  investigate promptly but no action has been taken as of today. Figure 4. Green boxes highlight an area of similarity between two different panels.  , 
 Corrigendum [10 March 2021] . ---------------------------------------- Following the publication of the above article, an interested reader drew to the authorsu2019 attention that the 24 h, A and the 48 h, C panels shown in Fig. 4 appeared to contain overlapping data. After having re-examined their original data, the authors have realized that this Figure was assembled incorrectly. The corrected version of Fig. 4 is presented opposite. Note that this inadvertent error did not have a major impact on the results or the conclusions reported in this paper. Four of the named authors (Guangcan Shao, Dongmeng Qian, Ming Hu and Tongmei Wang) agree with this corrigendum; the other three authors proved to be uncontactable. These four authors would like to thank the Editor of *Oncology Letters* for allowing them the opportunity to publish this corrigendum; furthermore, they apologize to the readership of the Journal for any inconvenience caused.  Figure 4. - The morphology of various HeLa cell treatment groups were observed at 24, 48 and 72 h subsequent to transfection. (A) Cells transfected with pLEGFP-C1-M-IL-2(88Arg, 125Ala). (B) pLEGFP-C1-transfected cells. (C) Lipofectamine 2000-transfected cells. (D) non-transfected cells. ,