Pan et al. Cancer Cell Int (2021) 21:161 https://doi.org/10.1186/s12935-021-01797-z Cancer Cell International PRIMARY RESEARCH Open Access MicroRNA-1182 and let-7a exert synergistic inhibition on invasion, migration and autophagy of cholangiocarcinoma cells through down-regulation of NUAK1 Xin Pan*, Gang Wang and Baoming Wang Abstract Background: Cholangiocarcinoma (CCA) is the second most common primary liver malignancy worldwide. Several microRNAs (miRNAs) have been implicated as potential tumor suppressors in CCA. This study aims to explore the potential efects of miR-1182 and let-7a on CCA development. Methods: Bioinformatics analysis was conducted to screen diferentially expressed genes in CCA, Western blot analysis detected NUAK1 protein expression and RT-qPCR detected miR-1182, let-7a and NUAK1 expression in CCA tissues and cell lines. Dual luciferase reporter gene assay and RIP were applied to validate the relationship between miR-1182 and NUAK1 as well as between let-7a and NUAK1. Functional experiment was conducted to investigate the role of miR-1182, let-7a and NUAK1 in cell migration, proliferation and autophagy. Then, the CCA cells that received various treatments were implanted to mice to establish animal model, followed by tumor observation and HE staining to evaluate lung metastasis. Results: CCA tissues and cells were observed to have a high expression of NUAK1 and poor expression of miR-1182 and let-7a. NUAK1 was indicated as a target gene of miR-1182 and let-7a. Importantly, upregulation of either miR-1182 or let-7a induced autophagy, and inhibited cell progression and in vivo tumor growth and lung metastasis; moreover, combined treatment of miR-1182 and let-7a overexpression presented with enhanced inhibitory efect on NUAK1 expression and CCA progression, but such synergistic efect could be reversed by overexpression of NUAK1. Conclusion: Taken together, the fndings suggest the presence of a synergistic antitumor efect of miR-1182 and let- 7a on the development of CCA via the down-regulation of NUAK1, providing novel insight into the targeted therapy against CCA. Keywords: MicroRNA-1182, Let-7a, NUAK1, Cholangiocarcinoma, Invasion, Migration, Autophagy Background and reparative biliary epithelial cell proliferation [1, 2]. As the second-most common primary hepatobiliary Te high mortality of CCA is explained by the extreme malignancy, cholangiocarcinoma (CCA) generally difculty associated with the diagnosis of CCA at an occurs as a result of long-standing infammation, injury early stage, along with high frequency of recurrence and metastasis and lack of efective chemotherapeu- tical, surgical or other treatment options [3]. Te risk *Correspondence: [email protected] Interventional Department, The Fourth Afliated Hospital of China factors of CCA include cirrhosis and viral hepatitis B Medical University, No. 4, Chongshan East Road, Huanggu District, and C [4]. On the other hand, overexpression of infam- Shenyang 110032, Liaoning, People’s Republic of China matory genes, cytokines or STAT3, could result in the © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pan et al. Cancer Cell Int (2021) 21:161 Page 2 of 16 development of CCA, especially intrahepatic cholan- and autophagy with aims of discovering a promising giocarcinoma (ICC). Additionally, strong correlations therapeutic strategy for CCA. have been identifed between CCA and genetic muta- tions in patients [5]. Several genes have been consid- Materials and methods ered as potential therapeutic targets in CCA secondary Ethical statement to their independent prognostic values, such as VDR, Tis study was carried out in strict accordance with the CYPA, CD147 and HER4 [6, 7]. Nevertheless, the ther- recommendations of the Guide for the Care and Use of apeutic and diagnostic potential of these genes requires Laboratory Animals of the National Institutes of Health. further large-scale investigations. Te protocol was approved by the Institutional Animal As a member of the human adenosine monophosphate- Care and Use Committee of Te Fourth Afliated Hospi- activated protein kinases family, NUAK1 (also known tal of China Medical University. as ARK5, AMPK-related kinase 5) has been observed to have a high expression in multiple human malignancies, Bioinformatics analysis and participates in tumor invasion, migration, survival Diferentially expressed genes (DEGs) were screened and progression [8, 9]. For example, NUAK1 is highly using CAA-related gene expression profle downloaded expressed in non-small cell lung cancer (NSCLC) and from the Gene Expression Omnibus (GEO) database knockdown of this gene served as a block in lung metas- (https ://www.ncbi.nlm.nih.gov/geo/) and R language tasis and invasive ability in a xenograft mouse model [8]. "limma" package with threshold set as |logFC|> 2 and Moreover, NUAK1 is a valuable molecular biomarker p < 0.05. DEGs were presented in a heat map by using for gastric cancer progression, since its suppression can “pheatmap” package. Subsequently, the expression of prevent cell invasion, lymph node metastasis, pathologi- NUAK1 in the CCA samples collected in Te Cancer cal stage and histological diferentiation [10]. miRNAs Genome Atlas (TCGA) was analyzed based on the UAL- are endogenous noncoding RNAs that serve as modula- CAN database (http://ualca n.path.uab.edu/index .html). tors of gene expression and their dysregulation has been Next, CCA related genes were retrieved in the MalaC- linked with the development of infammatory-driven car- ards database (http://www.malac ards.org/), and their cinogenesis [11]. Numerous studies have emphasized the interaction with NUAK1 was further analyzed in the tumor suppressor or promoter role of miRNAs, which STRING database (https ://strin g-db.org/). Finally, the might function in the tumorigenic process of malignant RNA22 database (https ://cm.jefe rson.edu/rna22 /Preco tumors such as CCA [3, 12–14]. In pancreatic cancer, mpute d/) and miRGator database (http://mirga tor.kobic NUAK1 is specifcally up-regulated and the down-regu- .re.kr/) were performed to predict the miRNAs that reg- lation by miR-96 is capable of impeding the proliferation, ulated NUAK1. Te intersection of the frst 80 miRNAs migration and invasion of MIA PaCa-2 pancreatic can- from the result obtained from the above two databases cer cells [15]. In addition, NUAK1 level has been shown was analyzed using the Venn diagram website (http:// to be signifcantly increased in ICC tissues and cell lines bioin forma tics.psb.ugent .be/webto ols/Venn/). while miR-145 expression was signifcantly decreased; moreover, NUAK1 was also identifed as a direct target Study subjects of miR-145 regulation, aiding the prevention of ICC pro- A total of 56 patients confrmed as hilar CCA by surgery gression [16]. Autophagy may be related to the develop- and pathology from January 2018 to December 2018 in ment of CCA [17]. As autophagy is essential in numerous Te Fourth Afliated Hospital of China Medical Univer- physiologies, ranging from immune response to neuronal sity were enrolled as study subject, including 34 males health, its activation is key in the pathogenesis of CCA and 22 females, aged 35–78 years old. According to progression [18]. Pietri Puustinen et al. demonstrated tumor nodes metastasis (TNM) stage of Union for Inter- the presence of a close correlation between NUAK1 and national Cancer Control, there are 22 cases of stage I-II autophagy [19], whilst Chen et al. suggested that the inhi- and 34 cases of III-IV. According to the degree of difer- bition of NUAK1 suppressed autophagy as indicated by entiation, 20 cases had poor diferentiation, 19 cases had the decreased expression of MMP-2, matrix metallopro- moderate diferentiation, and 17 cases had high difer- teinase (MMP)-2 and MMP-9 [8]. However, the interac- entiation. Meanwhile, 32 cases of bile duct tissues were tion between NUAK1 and autophagy remains unclear. obtained from patients with hepatic trauma specimen Tese fndings led to the hypothesis that there existed a and served as control. promising correlation between NUAK1 and other miR- Human normal biliary epithelial cell line HIBEPIC NAs in CCA. Terefore, the present study focused on and CCA cell lines (CCC-5, HCC-9810, Huh28) from exploring the correlations between the expression of BeNa Culture Collection (Beijing, China) were detected miR-1182 and let-7a and CCA cell migration, invasion by mycoplasma and identifed by short tandem repeat Pan et al. Cancer Cell Int (2021) 21:161 Page 3 of 16 (STR) genotyping (Additional fle 1: Table S1). Cells were New York, California, USA). Afterwards, 4 μg of the tar- cultured in dulbecco’s modifed eagle medium (DMEM) get plasmid or 2 ug miRNA and 10 μL Lipofectamine medium (QBC939) or a Roswell Park Memorial Institute 2000 were diluted by 250 μL serum free Opti-MEM (RPMI) 1640 medium (HCCC-9810 and Huh28) contain- (Gibco), placed for 5 min and mixed, with the mixture ing 10% serum at 37 °C in a 5% CO 2 incubator and were added to the plate well.