α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy
Authors: Cheng-Yu Gu
Yuan-Fei Peng
Jia Fan
Jian Zhou
Bo Hui
Ying-Hong Shi
Hua Yang
Wei-Ren Liu
Zhen-Bin Ding
Shuang-Jian Qiu
Journal: PloS one (2013)
Discipline: Multidisciplinary
Keywords: RNA interference; hepatocellular carcinoma; HCC-specific; miRNA; shRNA
Affiliations: Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, PR China.

Abstract:  Background: RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment. Methods: Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo. Results: The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo. Conclusions: An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC. 

Retracted: No

PubPeer comments:
 Figure 2C. * Red boxes: The left and right GAPDH panels look unexpectedly similar. * Blue boxes: Two lanes in the left Beclin 1 panel appear to look very similar. Sharp background vertical transitions are visible between lanes. ![file] , 
 Dear Dr. Elisabeth M Bik, I am Yuanfei Peng, the first author of this paper. I am very sorry I did not give a prompt response because I am involved in the battle of the novel coronavirus disease (COVID-19) in China. Thank you very much for pointing out the issues of Figure 2C in our published paper. These comments are very important and helpful to us. Issue 1: u201cRed boxes: The left and right GAPDH panels look unexpectedly similar.u201d Thank you very much for pointing out this mistake. The right GAPDH panel is indeed a duplicate of the left GAPDH panel. It was a mistake in preparation of figure, which is inadvertent. We are so sorry for the misplacement of the images and deeply regret any inconvenience this mistake has caused for readers. Please accept our sincere apology for this mistake. Attached please find the corrected Figure 2 (File-1) in which the correct corresponding GAPDH image of the right panel has been incorporated into Figure 2C. We will contact the editor of the journal and correct the figure as soon as possible. Issue 2: u201cBlue boxes: Two lanes in the left Beclin 1 panel appear to look very similar. Sharp background vertical transitions are visible between lanes. Thank you for pointing out this issue. We also peruse this figure again and found the lane 2 and lanes 6 are puzzlingly similar. We had not paid attention to this during the figure preparation. We have rechecked the original picture. Attached please find the raw figure of the Beclin1 blot (File-2) (the original file is TIF and bigger than 1MB, we upload the file with full size to cloud disk: https: pan.baidu.com s 1tH86KGZNho-VId62JAkZMw, access code: 56yz). The lanes were truly very similar in shape but they still demonstrated some difference in my opinion. When we change the display to see the details (enlarge to 1200x and darken the blot), we can see the height of left part of the blots and the background shadows (up and down) are different (attached please find the screenshot of the picture, File-3). The vertical transitions are not visible between lanes, when viewed in inverse to clearly illustrate it (File-4A). The u201cvertical transitionu201d like changes may be caused by the background disturbances and compression during figure preparation. A bubble water stain results in a right vertical transition like changes, while the specks of background may cause the other vertical transition like changes (File-4A). An inappropriate compression may also contribute to the vertical transition like changes. The blot is too long for figure illustration (File-4B), so we perform a horizontal compression to make it shorter to put all the blots in one figure (File-4B). It appears that the compression makes vertical transition like change (File-4C). We are sorry for the inappropriate compression manipulation and the image quality here. The quality of the picture is not high. It is a little dirty. Whatever, they looked very similar and caused confusion without the aid of the raw picture. The aim of this experiment is to determine the optimal MOI (multiplicity of infection) value of the vector. The experiment had ever been repeated and the results as depicted in current figure are reproducible. For your reference, we attach another image of repeated experiment. Pilot study was performed immediately after the vector (lentivirus) was harvested in the virology lab. Attached pleased find the original film (File-5, File-5 with marker) (the original file is TIF and bigger than 1MB, we upload the file with full size to cloud disk: https: pan.baidu.com s 1tH86KGZNho-VId62JAkZMw, access code: 56yz). If necessary, we would like to replace the current figures with the repeated one. Thank you for your kind comments. Thank you again for helping us to find and correct the mistakes. We appreciate your valuable comments and your efforts to ensure the accuracy of scientific records and the integrity of scientific research. We again apologize for the issues and we will pay more attention to the accuracy in the future. We will send an erratum to the Journal to revise our mistake as soon as possible. Please donu2019t hesitate to let us know if you have any questions. Best regards, Yuanfei Peng ![file] ![file] ![file] ![file] ![file] ![file] ,