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Gastrointestinal stromal tumors (GISTs) are common neoplasms of the gastrointestinal tract that can be treated successfully using C-kit target therapy and surgery; however, imatinib chemoresistance is a major barrier to success in therapy. The present study aimed to discover alternative pathways in imatinib-resistant GISTs. Long noncoding RNAs (lncRNAs) are newly discovered regulators of chemoresistance. Previously, we showed that the lncRNA HOTAIR was upregulated in recurrent GISTs. In this study, we analyzed differentially expressed lncRNAs after imatinib treatment and found that HOTAIR displayed the largest increase. The distribution of HOTAIR in GISTs was shifted from nucleus to cytoplasm after imatinib treatments. The expression of HOTAIR was validated as related to drug sensitivity through Cell Counting Kit-8 assays. Moreover, HOTAIR was associated strongly with cell autophagy and regulated drug sensitivity via autophagy. Mechanistically, HOTAIR correlated negatively with miRNA-130a in GISTs. The downregulation of miRNA-130a reversed HOTAIR-small interfering RNA-induced suppression of autophagy and imatinib sensitivity. We identified autophagy-related protein 2 homolog B (ATG2B) as a downstream target of miR-130a and HOTAIR. ATG2B downregulation reversed the effect of pEX-3-HOTAIR/miR-130a inhibitor on imatinib sensitivity. Finally, HOTAIR was shown to influence the autophagy and imatinib sensitivity of GIST cells in mouse tumor models. Our results suggested that HOTAIR targets the ATG2B inhibitor miR-130a to upregulate the level of cell autophagy so that promotes the imatinib resistance in GISTs.

Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2-mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2′-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.

CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased ∼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes. Keywords: CRISPR/Cas9, gene therapy, epidermolytic palmoplantar keratoderma, Krt9 gene (mouse), indel

// Rong-Rong Liu 1, * , Ya-Su Lv 1, * , Yue-Xiao Tang 1 , Yan-Fang Wang 1 , Xiao-Ling Chen 2 , Xiao-Xiao Zheng 1 , Shang-Zhi Xie 1 , Ying Cai 1 , Jun Yu 3 , Xian-Ning Zhang 1 1 Department of Cell Biology and Medical Genetics, Research Center for Molecular Medicine, National Education Base for Basic Medical Sciences, Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, 310058, China 2 Department of Biological Chemistry, Zhejiang Chinese Medical University, Hangzhou, 310053, China 3 Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Key Laboratory of Multi-Organ Transplantation of Ministry of Public Health, Hangzhou, 310003, China * These authors contributed equally to this work Correspondence to: Xian-Ning Zhang, e-mail: zhangxianing@zju.edu.cn Jun Yu, e-mail: Dr.yujun@gmail.com Keywords: eukaryotic translation initiation factor 5A2, reactive oxygen species, epithelial-mesenchymal transition, migration, hepatocellular carcinoma Received: September 24, 2015 Accepted: February 28, 2016 Published: March 23, 2016 ABSTRACT Eukaryotic translation initiation factor 5A2 ( eIF5A2 ) has been identified as a critical gene in tumor metastasis. Research has suggested that reactive oxygen species (ROS) serve as signaling molecules in cancer cell proliferation and migration. However, the mechanisms linking eIF5A2 and ROS are not fully understood. Here, we investigated the effects of ROS on the eIF5A2-induced epithelial-mesenchymal transition (EMT) and migration in six hepatocellular carcinoma (HCC) cell lines. Western hybridization, siRNA transfection, transwell migration assays, wound-healing assays, and immunofluorescence analysis were used. The protein levels of eIF5A2 in tumor and adjacent tissue samples from 90 HCC patients with detailed clinical, pathological, and clinical follow-up data were evaluated. Overexpression of eIF5A2 was found in cancerous tissues compared with adjacent tissues. We found that eIF5A2 overexpression in HCC was associated with reduced overall survival. Knockdown of eIF5A2 and intracellular reduction of ROS significantly suppressed the invasion and metastasis of HCC cells. Interestingly, N1-guanyl-1, 7-diaminoheptane (GC7) suppressed the intracellular ROS levels. After blocking the EMT, administration of GC7 or N-acetyl-L-cysteine did not reduce cell migration further. Based on the experimental data, we concluded that inhibition of eIF5A2 alters progression of the EMT to decrease the invasion and metastasis of HCC cells via ROS-related pathways.