Astragaloside IV suppresses development of hepatocellular carcinoma by regulating miR-150-5p/β-catenin axis

Highlights

• Astragaloside IV contributed to the enhancement of miR-150−5p level in HCC cells.

• AS-IV modulated miR-150−5p or β-catenin levels to facilitate HCC cells apoptosis in HCC.

• MiR-150−5p directly targeted and negatively regulated β-catenin in HCC.

• MiR-150−5p regulated cell apoptosis and tumor growth through β-catenin in HCC.

Abstract

Hepatocellular carcinoma (HCC), a common malignant tumor, has been regarded as a leading cause of cancer-related deaths globally. Astragaloside IV (AS-IV) was reported to participate in the regulation of multiple tumors. However, the role of AS-IV in HCC was still unclear in HCC. Bioinformatics analysis and function or mechanism experiments including RT-qPCR, MTT assay, flow cytometry, Western blot, luciferase reporter assay and xenografts assays were applied to investigate the function of AS-IV, miR-150−5p and CTNNB1. We discovered that AS-IV treatment was supposed to significantly increase miR-150−5p level. In addition, AS-IV accelerated cell apoptosis by inducing miR-150−5p in vitro and in vivo. Furthermore, AS-IV increased cell apoptosis rate through reducing β-catenin level in vitro and in vivo. In detail, AS-IV triggered a decline of Bax and a rise of Bcl-2 in HCC cells and xenograft tissues. In mechanism, we validated the combination between miR-150−5p and CTNNB1. Moreover, miR-150−5p could negatively regulate CTNNB1 level by binding to its3’UTR. Finally, rescue assay demonstrated that CTNNB1 overexpression partially rescued the inhibitive effect on tumor growth and promotive influence on cell apoptosis caused by miR-150−5p amplification. The up-regulation of miR-150−5p induced by AS-IV suppressed the progression of HCC by repressing β-catenin, providing a new molecular target for the utilization of AS-IV In the treatment of HCC.

Introduction

Liver cancer has been regarded as one of the most common malignancies globally (Gravitz, 2014). Data from 2018 Global Cancer Statistics showed that nearly 841,000 people were diagnosed with liver cancer, and 782,000 liver cancer mortalities were appeared all over the world. Particularly, patients in China occupied approximately a half of all the newly diagnosed cases (Bray et al., 2018). Accounting for more than 80 % of all liver cancer, Hepatocellular carcinoma (HCC) is extremely difficult to be cured. Known factors like chronic hepatitis b, hepatitis c virus infection, aflatoxin exposure, smoking, obesity and diabetes were closely related to the initiation and development of HCC (Goossens and Hoshida, 2015; Kew, 2013; Lee and Ahn, 2016; Liu et al., 2018; Petrick et al., 2016), but the pathology of HCC remained fully unknown.

A traditional Chinese herb, Astragaloside IV (AS-IV) is a category of natural triterpene glycoside, which could be extracted from astragalus membranaceus Bge (AM) (Ren et al., 2013). Functionally, AS-IV was reported to regulate the initiation or progression of diseases (Cao et al., 2019; Li et al., 2017a). Additionally, AS-IV displayed a variety of pharmacological effects including improving cardiac cell energy metabolism, preventing cell proliferation, and accelerating cell apoptosis (Li et al., 2017b). As examples, AS-IV inhibited cell proliferation of vulvar squamous cell carcinoma cells by targeting TGF-β/Smad signaling pathway (Zhao et al., 2019). AS-IV/lncRNA-TUG1/TRAF5 regulatory network participated in the modulation of podocyte apoptosis in diabetic nephropathy rats (Lei et al., 2018). Moreover, AS-IV was reported to regulate the level of miRNAs in several diseases. For instances, AS-IV inhibits EMT process and promotes chemotherapeutic sensitivity of colorectal cancer cells via miR-134/CREB1 signaling (Ye et al., 2017). AS-IV restrains hypoxia-induced injury of cardiomyocytes through reducing miR-23a and miR-92a levels (Gong et al., 2018). Hence, we anticipated that AS-IV also affects miRNAs level in HCC. In the current study, we discovered that different concentration of AS-IV significantly changed miR-150−5p levels. Therefore, we aimed to explore the relation of AS-IV and miR-150−5p in HCC.

MicroRNAs (miRNAs), a category of non-coding RNAs (ncRNAs), is unable to encode protein with 20–24 nucleotides in length and frequently participates in the regulation of tumors (Rupaimoole and Slack, 2017; Vishnoi and Rani, 2017). For example, miR-299−5p targeted CDK family to facilitate cell proliferation and progression in osteosarcoma (Zhang et al., 2018). MiR-195−5p/NOTCH2 affected M2-like TAM polarization by inducing EMT process and modulating IL-4 secretion to in colorectal cancer (Lin et al., 2019). Most recently, miR-150−5p has been identified as a tumor suppressor in non-small cell lung cancer, gastric cancer and glioma (Quan et al., 2019; Sakr et al., 2016; Zeng et al., 2019). On the top pf that, miR-150−5p was reported to inhibit tumorigenesis and metastasis of HCC by targeting c-Myb (Lan et al., 2019). Nevertheless, the relation between AS-IV and miR-150−5p remained unknown in HCC.

Mechanistically, the mature miRNA is capable of binding with the 3’ untranslated region (3’UTR) of the target mRNA(s) to silence or degrade target mRNA (s) (Seo et al., 2019). With the assistance of biological analysis, β-catenin (or CTNNB1), a famous regulator for tumor progression, was confirmed to serves as a target gene of miR-150−5p, which has been widely reported to activate Wnt pathway in diverse cancers (Guan et al., 2018). In detail, aberrant accumulation of β-catenin in the nucleus was capable to up-regulate several proto-oncogenes such as c-Myc and Cyclin D1, which was deemed as the basis for carcinogenesis or progression (Hong et al., 2015). A multitude of studies claimed β-catenin could play a role in HCC cells proliferation and apoptosis (Cao et al., 2015; Puliga et al., 2017). Besides, β-catenin was reported to be targeted by several miRNAs including miR-197 and miR-320a in HCC (Hu et al., 2018; Lu et al., 2017). Nevertheless, the relation between miR-150−5p and β-catenin was unclear in HCC.

In present study, we planned to investigate the specific function and regulatory mechanism of AS-IV in HCC. We found that AS-IV suppressed tumor progression by up-regulating miR-150−5p to reduce β-catenin in HCC, implying that AS-IV may provide a novel insight for HCC treatment.