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Original Article Microrna-96-5P Induces the Epithelial

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Original Article Microrna-96-5P Induces the Epithelial Int J Clin Exp Pathol 2019;12(5):1897-1908 www.ijcep.com /ISSN:1936-2625/IJCEP0092831 Original Article microRNA-96-5p induces the epithelial-mesenchymal transition to promote the metastasis of hepatocellular carcinoma by post-transcriptionally downregulating Talin 1 Hongying Tang, Yi Liu, Wei Cheng, Zili He, Ning Zhou Laboratory of Hepatobiliary Molecular Oncology, Department of Hepatopancreatobiliary Surgery, Hunan Provincial People’s Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, P. R. China Received February 20, 2019; Accepted March 27, 2019; Epub May 1, 2019; Published May 15, 2019 Abstract: Numerous microRNAs (miRNAs) have been shown to play an important regulatory role in the progression of hepatocellular carcinoma (HCC). miR-96-5p, a cancer-related microRNA, was previously reported to inhibit cell apoptosis in HCC, but the function and underlying mechanism of miR-96-5p’s involvement in HCC metastasis and progression still remain unknown. In this study, we showed that a significant up-regulation of miR-96-5p in HCC tissues and cell lines, and its increased expression, are associated with microvascular invasion and with the TNM stages of HCC patients. Gain-of-function assays revealed that miR-96-5p induced the epithelial-mesenchymal tran- sition (EMT) to promote the migration and invasion of HCC in vitro. The expression of TLN1 (Talin 1) is significantly decreased in HCC tissues and is inversely correlated to miR-96-5p levels. Notably, through a luciferase reporter assay and a Western blot analysis, TLN1 was confirmed to be a direct target gene of miR-96-5p. Furthermore, results of cell functional assays revealed that the over-expression of TLN1 partially reverses the promotive effects of miR-96-5p overexpression on the migration, invasion, and EMT of HCC. Overall, data from the present study demonstrate that miR-96-5p induces EMT to promote the migration and invasion of HCC by post-transcriptionally downregulating TLN1, indicating that the miR-96-5p/TLN1 axis might provide a potential therapeutic target for the treatment of HCC. Keywords: HCC, miR-96-5p, EMT, TLN1, treatment Introduction lying molecular mechanisms of HCC metastasis and to develop novel strategies for the early Hepatocellular carcinoma (HCC) is among the diagnosis and treatment of HCC. most fatal malignancies and is the third leading cause of cancer-related death throughout the microRNAs (miRNAs) are a class of evolution- world [1]. The carcinogenesis and development arily conserved small noncoding RNAs com- of HCC are the result of complex and multi-step posed of 15~22 nucleotides that usually bind processes associated with epigenetic and ge- with the 3’-untranslated regions (3’-UTRs) of netic changes [2]. There are no obvious symp- target genes to suppress their expression [5]. toms during the early stages of HCC, and HCC As post-transcriptional regulators of gene ex- patients are commonly diagnosed at the ad- pression, miRNAs have broad effects on innu- vanced or unresectable stages. Although re- merous biological processes, including differ- markable improvements have been made in entiation, metastasis, apoptosis, and the cell surgical techniques and perioperative manage- cycle [6]. Recently, the aberrant expression of ment, the prognosis of HCC patients after hep- miRNAs has been shown to play an important atectomy is still unsatisfactory because of the role in the pathogenesis and tumorigenicity of high rates of recurrence and metastasis [3, 4]. various human malignancies [7-9]. For exam- Therefore, it is essential to elucidate the under- ple, the up-regulation of miR-1271 promotes miR-96-5p induces EMT to promote migration and invasion in HCC non-small-cell lung cancer cell proliferation and China. These cell lines were maintained in invasion by targeting HOXA5 (homeobox A5) Dulbecco’s Modified Eagle’s Medium (DMEM, [10]. miR-152 inhibits tumor cell growth by Gibco, CA, USA) supplemented with 10% fetal directly targeting RTKN (Rhotekin) in HCC [11]. bovine serum (FBS, Gibco, CA, USA), 100 μg/ml The overexpression of miR-613 inhibits the pro- streptomycin (Sigma, USA), and 100 U/ml peni- liferation and invasion of renal cell carcinoma cillin (Sigma, USA) at 37°C in a humidified cells by targeting FZD7 (Frizzled class receptor chamber containing 5% CO2. 7) [12]. miR-101-3p upregulation suppresses cell invasion and proliferation and enhances Cell transfection chemotherapeutic sensitivity in salivary gland adenoid cystic carcinoma by directly targeting The miR-96-5p mimic (5’-UUUGGCACUAGCAC- Pim-1 (Pim-1 proto-oncogene, serine/threonine AUUUUUGCU-3’) and the corresponding mimic kinase) [13]. negative control (mimic NC, 5’-CAAUAACCAU- GACUUGACCU-3’), as well as the TLN1 express- miR-96-5p is one member of the miR-183-96- ing vector and the corresponding negative con- 182 cluster, and it is located on human chro- trol vector (NC vector) were designed and syn- mosome 7q32.2. Accumulating evidence has thesized by RiboBio Co., Ltd (Guangzhou, Chi- suggested that miR-96-5p is upregulated and na). Huh7 and HCCLM3 cells were transfected plays a key role in different types of cancer, with 50 nmol/L miR-96-5p mimic and mimic NC including breast [14], colorectal [15], and ovar- and/or 1 μg TLN1 expressing vector and NC ian cancer [16]. Additionally, a previous study vector by utilizing the Lipofectamine 2000 tr- has demonstrated that miR-96-5p inhibits cell ansfection reagent (Invitrogen, Carlsbad, CA, apoptosis by targeting the caspase-9 gene in USA) according to the manufacturer’s proto- HCC [17]. However, an understanding of the cols. At 48 h post-transfection, the transfection exact role and mechanism of miR-96-5p on efficiency was evaluated in each experiment by HCC progression and metastasis is still need- qRT-PCR or Western blot analysis. ed. Recently, Fang et al. demonstrated that TLN1 (Talin 1) correlates with the malignancy Patients and tissue samples potential of the HCC MHCC-97 L cell [18]. Fur- This study was approved by the Ethics Board of ther, emerging studies have suggested that Hunan Provincial People’s Hospital (Changsha, TLN1 is significantly downregulated in HCC tis- China), and it complied with the Declaration of sues compared with adjacent non-tumor tis- Helsinki. All patients or the legal guardians of sues, and low TLN1 expression is associated the patients agreed to participate in this study. with HCC progression and a poor prognosis Informed consent was obtained from each par- [19]. TLN1 knockdown induces the epithelial- ticipant. HCC tissue specimens were obtained mesenchymal transition and promotes migra- from 51 patients who underwent a surgical tion and invasion in SK-Hep-1 cells and HepG2 resection of their HCC lesions at the Departme- cells [19]. Interestingly, a bioinformatics analy- nt of Hepatopancreatobiliary Surgery between sis predicts that miR-96-5p is able to bind TLN1 January 2007 and December 2010. None of mRNA 3’-UTR. Thus, we suspected miR-96-5p the patients had received prior radiotherapy or might have an important role in HCC progres- chemotherapy. Fresh HCC tissues were imme- sion by inhibiting TLN1 expression. In this study, diately snap-frozen in liquid nitrogen and stored we explored the possibility that miR-96-5p at -80°C until use. The pathological diagnosis might be involved in HCC cell progression and was based on the World Health Organization metastasis, and we partially elucidated the criteria, and the tumor differentiation grades underlying molecular mechanism. were based on the classification proposed by Materials and methods Edmondson and Steiner [20]. Tumor stage was determined in conformity with the TNM classifi- Cell lines and cell culture cation system proposed by the 2010 Interna- tional Union Against Cancer [21]. After their sur- The human normal liver cell line (L02) and geries, the patients were monitored until De- human liver cancer cell lines (Hep3B, Huh7, and cember 31, 2016. Overall survival (OS) was HCCLM3) were obtained from the Cell Bank of defined as the time between surgery and death the Chinese Academy of Sciences, Shanghai, or between surgery and the last observation 1898 Int J Clin Exp Pathol 2019;12(5):1897-1908 miR-96-5p induces EMT to promote migration and invasion in HCC time. The OS data were censored at the last using a Quick Change mutagenesis kit (Takara, follow-up for the surviving patients. Japan), and was also cloned into a pmirGLO vector to produce mutant (MUT)-TLN1 reporter Reverse transcription-quantitative polymerase plasmid. For the luciferase reporter assay, co- chain reaction (qRT-PCR) transfection with WT or MUT-TLN1 reporter plasmid and miR-96-5p mimic and mimic NC TRIzol solution (Invitrogen, Carlsbad, CA, USA) were introduced into Huh7 and HCCLM3 cells was used to extract total RNA from cell lines by using the Lipofectamine 2000 transfection and frozen tumor specimens according to the reagent. Luciferase activities were measured at manufacturer’s protocols. Synthesis of comple- 48 h post-transfection by a Dual-Luciferase mentary DNA (cDNA) was performed by a Taq- Assay kit (Promega, Madison, WI, USA), accord- Man™ MicroRNA Reverse Transcription Kit (Ap- ing to the manufacturer’s protocols. plied Biosystems, Foster City, CA, USA) or Pri- meScript Reverse Transcriptase reagent Kit Cell migration assay with gDNA Eraser Kit (Takara, Osaka, Japan). U6 small nuclear 1 (U6) or glyceraldehyde 3- A wound-healing assay was performed to evalu- phosphate dehydrogenase (GAPDH) was used ate cell migration. Briefly, Huh7 and HCCLM3 as an internal control. qPCR was performed cells (3×105 per/well) transfected with miR-96- using a 7900 Real-Time PCR System (Applied 5p mimic and mimic NC and/or TLN1 express- Biosystems, Foster City, CA, USA) with Hieff™ ing vector and NC vector were seeded into six- ® qPCR SYBR Green Master Mix (Yisheng Bio- well culture plates and cultured with DMEM Technology Co., Ltd; Shanghai, China), in accor- medium and FBS to form a tight monolayer.
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