High-metastatic cancer cells derived exosomal miR92a-3p promotes epithelial-mesenchymal transition and metastasis of low-metastatic cancer cells by regulating PTEN/Akt pathway in hepatocellular carcinoma
Authors: Jian Wu
Yunhao Chen
Rongliang Tong
Xiaode Feng
Qiyang Cheng
Jingbang Wu
Xianlei Cai
Junru Chen
Hua Liu
Wenxuan Wu
Yuejie Lu
Beng Yang
Zhen Lv
Hanxi Yu
Kejiong Liang
Jiating Hu
Changbiao Li
Shusen Zheng
Journal: Oncogene (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Exosomes; Hepatocellular carcinoma; Metastasis; miR-92a-3p; Epithelial-mesenchymal transition
Affiliations: Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China., Department of gynecology and obstetrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China., Department of orthopedics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China., Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China. .

Abstract:  Exosomes play an important role in intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). However, cellular communication between heterogeneous HCC cells with different metastatic potentials and the resultant cancer progression are not fully understood in HCC. Here, HCC cells with high-metastatic capacity (97hm and Huhm) were constructed by continually exerting selective pressure on primary HCC cells (MHCC-97H and Huh7). Through performing exosomal miRNA sequencing in HCC cells with different metastatic potentials (MHCC-97H and 97hm), many significantly different miRNA candidates were found. Among these miRNAs, miR-92a-3p was the most abundant miRNA in the exosomes of highly metastatic HCC cells. Exosomal miR92a-3p was also found enriched in the plasma of HCC patient-derived xenograft mice (PDX) model with high-metastatic potential. Exosomal miR-92a-3p promotes epithelial-mesenchymal transition (EMT) in recipient cancer cells via targeting PTEN and regulating its downstream Akt/Snail signaling. Furthermore, through mRNA sequencing in HCC cells with different metastatic potentials and predicting potential transcription factors of miR92a-3p, upregulated transcript factors E2F1 and c-Myc were found in high-metastatic HCC cells promote the expression of cellular and exosomal miR-92a-3p in HCC by directly binding the promoter of its host gene, miR17HG. Clinical data showed that a high plasma exosomal miR92a-3p level was correlated with shortened overall survival and disease-free survival, indicating poor prognosis in HCC patients. In conclusion, hepatoma-derived exosomal miR92a-3p plays a critical role in the EMT progression and promoting metastasis by inhibiting PTEN and activating Akt/Snail signaling. Exosomal miR92a-3p is a potential predictive biomarker for HCC metastasis, and this may provoke the development of novel therapeutic and preventing strategies against metastasis of HCC. 

Retracted: No

PubPeer comments:
 Figure 5e: the Akt immunoblot has been reused and presented as something else (different cell type). Figure 6c: Same problem with the Gapdh immunoblot. In addition the p-Akt and Akt blots in Figure 6c do not origintae from the same gel run experiment due to the different band shape. ![file] , 
 Sorry, something was wrong with one of the illustrations. Here is the corrected one: ![file] , 
 Thank you for pointing these mistakes out. When we upload our result, we misplace the WB pictures accidentally and unconsciouslyuff08The bands of AKT in figure 5e AND GAPDH in Fig 6cuff09among so many immunoblot pictures. It is not a duplicated behavior but a mistake. The primary pictures of AKT immunoblot in figure 5e and GAPDH immunoblot in figure 6c were attached in the uploaded picture. Figure 5e: We perform this Akt immunoblot on 03 28 2019, and the Akt immunoblot band of 97h and Huh7 cells was pointed out by red frame and blue frame. Figure 6c: we perform this GAPDH immunoblot on 05 11 2020, and the GAPDH immunoblot band of Huh7 and 97h cells was pointed out by red frame and blue frame, besides, we perform this GAPDH immunoblot replication assay in the meanwhile. The expression level of total-Akt in figure 5e and GAPDH in figure 6c in each group(97h and Huh7) is similar, this episode wonu2019t affect the result and conclusion of our study. As for the different band shape of p-Akt and Akt in Figure 6c, we perform these two immunoblots in two separate gel rather than use striping buffer and incubate primary antibodies in the same membrane from one gel. This is why the band shape is different! We apologize for our mistakes and appreciate your comment.![file] , 
 Thank you for your reply. This is duplication of data, and might be a honest mistake. However, the data you provided do not prove anything. Could you please show picture of the blots including MW markers? Runnig samples you want to compare on different gels is NOT accepted. , 
 #4 Thank you for your response, and we take your comments very seriously. The akt immunoblot with MW weight maker is provided in the uploaded picture, we used Prestained Protein Ladder from ThermoFisher (cat no: 26616). And as for GAPDH immunoblot, we do not have that picture with MW marker. But in the previous western blot assay, in order to determine the certain location of GAPDH in our western blot system, we have taken the GAPDH immunoblot with MW weight marker. This can indicate the location of the GAPDH that we take in our assay. That GAPDH blot picture with MW weight marker is attached in the right side of the uploaded picture. As for running different gels, actually, we have ever tried to incubate one membrane with Akt and p-Akt primary antibodies, but the results are not good enough to be published. Firstly, we performed western blot and detected the expression of Akt protein in the membrane. After that, we used striping buffer to wash membrane and incubate it with p-Akt primary antibody. However, when we tried to detect the p-Akt protein in the membrane, we found the remaining signal of Akt protein was too strong, which greatly interrupted the detection of p-akt signal. Therefore, we choose to loaded same amount of protein samples in the same batch of precast gel, and performed western blot at the same time. All steps are the same. And due to the expression of Akt is similar, we just want to compare p-Akt expression in one gel. We think this method is reasonable and its result is reliable. Thank you. ![file] ,