Puerarin inhibits hepatocellular carcinoma invasion and metastasis through miR-21-mediated PTEN/AKT signaling to suppress the epithelial-mesenchymal transition

Brazilian Journal of Medical and Biological Research (2020) 53(4): e8882, http://dx.doi.org/10.1590/1414-431X20198882 ISSN 1414-431X Research Article 1/12 Puerarin inhibits hepatocellular carcinoma invasion and metastasis through miR-21-mediated PTEN/AKT signaling to suppress the epithelial-mesenchymal transition Yuan Zhou0 0 -0 0 -0 0 -0 01*, Ruifeng Xue0 0 -0 0 -0 0 -0 02*, Jinglin Wang0 0 -0 0 -0 0 -0 10 , and Haozhen Ren0 0 -0 0 -0 0 -0 10 1 Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, China 2 Department of Hepatobiliary Surgery, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, Jiangsu Province, China Abstract Hepatocellular carcinoma (HCC) is one of the most common primary malignant tumors of the liver worldwide. Liver resection and transplantation are currently the only effective treatments; however, recurrence and metastasis rates are still high. Previous studies have shown that the epithelial-mesenchymal transition (EMT) is a key step in HCC invasion and metastasis. Inhibition of EMT has become a new therapeutic strategy for tumors. Recently, puerarin, a well-characterized component of traditional Chinese medicine, has been isolated from Pueraria radix and exerts positive effects on many diseases, particularly cancers. In this study, CCK-8, EdU immunofluorescence, colony formation, wound healing, and migration assays were used to detect the effects of puerarin on HCC cells. We further analyzed the relationship between puerarin and miR-21/PTEN/EMT markers in HCC cell lines. Our results showed that HCC cell proliferation, migration, invasion, tumor formation, and metastasis were reduced by puerarin in vitro and in vivo. Additionally, puerarin inhibited the EMT process of HCC by affecting the expression of Slug and Snail. Moreover, oncogenic miR-21 was inhibited by puerarin, coupled with an increase in the tumor suppressor gene PTEN. Increasing miR-21 expression or decreasing PTEN expression reversed the inhibition effects of puerarin in HCC. These data confirmed that puerarin affects HCC through the miR-21/PTEN/EMT regulatory axis. Overall, puerarin may represent a chemopreventive and/or chemotherapeutic agent for HCC treatment. Key words: Hepatocellular carcinoma; Metastasis; Epithelial-mesenchymal transition; PTEN; miR-21 Introduction Across the globe, hepatocellular carcinoma (HCC) is still a major health problem, as it is the fifth most common tumor in men and the seventh in women (1). Even with the development of early diagnosis and treatments, the survival rate of HCC patients has not been notably improved in recent decades (2). Unrestricted cell proliferation and distal organ metastasis are the major obstacles to clinical cancer treatment (3,4). Therefore, new treatment strategies for HCC are urgently needed. Recently, increasing research on traditional Chinese medicine has shown good therapeutic effects against HCC (5). Puerarin is a type of flavonoid extracted from the dried roots of Pueraria lobata. Puerarin may be an effective Correspondence: Haozhen Ren: <> *These authors contributed equally to this study. Received July 4, 2019 | Accepted October 10, 2019 Braz J Med Biol Res | doi: 10.1590/1414-431X20198882 preventive compound against various nervous system diseases by regulating apoptosis and the inflammatory response (6). Puerarin inhibits the formation and activation of Nlrp3 inflammasome, which is induced by high glucose (7). In addition, puerarin exerts antioxidative effects and scavenges reactive oxygen species in cells. Previous experiments have shown that puerarin can also treat a variety of liver diseases, such as liver fibrosis and nonalcoholic fatty liver disease (8). The molecular mechanisms of the anti-tumor effect of puerarin have been determined in different types of cancers, such as colon cancer, breast cancer, and hepatocellular carcinoma (9,10). The results showed that high concentrations of puerarin can inhibit the growth of breast cancer, and miR-21 in suppressing hepatocellular carcinoma invasion induce apoptosis of HT-29 colon cancer cells. Although the anti-tumor effect of puerarin in HCC has been verified (11), the molecular mechanism of puerarin in HCC is still unclear (12). Epithelial-mesenchymal transition (EMT) is an essential step during the progression of metastasis in the vast majority of cancers. In the EMT process, epithelial cells acquire a mesenchymal phenotype (13). During tumor invasion and metastasis, the abnormal activation and inactivation of numerous signaling pathways are observed and are responsible for regulating the EMT process (14). Previous studies have suggested that the activation of the AKT/GSK3b signaling pathway promotes metastasis by regulating the EMT of cancer cells (15). Moreover, activated AKT can promote an increased expression of Snail through phosphorylating GSK-3b. As a transcriptional downregulator of E-cadherin, Snail further alleviates the EMT process in HCC (16). In addition to Snail, Slug is another significant regulatory protein of EMT. Zeb1 is a master regulator of EMT; its deregulation has been observed in multiple cancers, and it can be induced by Slug (17). Slug is also controlled by AKT/GSK-3b (16). Furthermore, PTEN (phosphate and tensin homolog), deleted on chromosome ten and a well-defined cancer suppressor, is controlled by miR-21 and sequentially inactivates AKT (18). miR-21 negatively correlates with prognosis in patients with HCC (19). A previous study showed that puerarin can block paraquat-induced oxidative stress by inhibiting miR-21 expression, leading to the attenuation of paraquat-induced lung fibrosis (20). Based on previous research, we hypothesized that puerarin may interfere with HCC progression by inhibiting miR-21. In this study, we aimed to investigate the anti-tumor function of puerarin in HCC treatment and reveal its specific mechanism. Material and Methods Animals Male BALB/c nu/nu mice (4–5 weeks old) were purchased from Shanghai Institute of Material Medicine, Chinese Academy of Science and housed under specific pathogen-free conditions. All animals received humane care according to the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86–23, revised 1985). All animal procedures were approved by the Animal Research Ethics Committee of Nanjing Medical University. Animal studies For examining the roles of puerarin in HCC treatment, 1
107 Bel-7402 cells or Huh7 cells in 0.2 mL of sterile PBS were injected subcutaneously into the right side of each male nude mouse. After 45 days, the mice were Braz J Med Biol Res | doi: 10.1590/1414-431X20198882 2/12 sacrificed and the tumors were excised and weighed. The tumor nodules formed on the liver surfaces were counted. HCC cell lines All cell lines (Bel-7402 (...truncated)