miR-612 suppresses the stemness of liver cancer via Wnt/β-catenin signaling

doi:10.1016/j.bbrc.2014.03.135

Biochemical and Biophysical Research Communications

Volume 447, Issue 1, 25 April 2014, Pages 210-215

https://doi.org/10.1016/j.bbrc.2014.03.135 Get rights and content

Highlights

•
miR-612 suppresses tumorsphere and clone formation of HCC cells.
•
miR-612 reduces drug resistance of HCC cells.
•
miR-612 suppresses tumorigenesis of HCC in NOD/SCID mice.
•
miR-612 inhibits an invasive frontier of HCC xenografts.
•
miR-612 suppresses Wnt/β-catenin signaling.

Abstract

Previous research showed that microRNA-612 (miR-612) has inhibitory effects on cell proliferation, migration, invasion, and metastasis of hepatocellular carcinoma (HCC). AKT2 was confirmed to be a direct target of miR-612, through which the epithelial–mesenchymal transition (EMT) and metastasis of HCC were inhibited. Our present findings reveal that miR-612 is able to suppress the stemness of HCC by reducing the number and size of tumorspheres as well as clone formation in soft agar, and to relieve drug resistance to cisplatin and 5-fluorouracil. In addition, miR-612 hampered the capacity of tumorigenesis in NOD/SCID mice and redistributed the tumor invasive frontier of miR-612-modulating cells. Finally, our findings suggest that Wnt/β-catenin signaling is required in the regulation of EMT-associated stem cell-like traits by miR-612.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer death globally [1]. Despite the improved understanding of tumor biology, the treatment efficacy in liver cancers has not improved greatly over the past decade. Cancer stem cells (CSCs) are defined as cells that possess the capacity to self-renew and differentiate into the heterogeneous lineages of cancer cells in a tumor [2]. The first conclusive evidence of the existence of CSCs was published in 1997 by Bonnet and Dick, who studied acute myelogenous leukemia [3]. More recently, CSCs have been isolated from many solid tumors, including HCC [4]. The existence of CSCs offers a reasonable explanation as to why current treatments targeting rapidly dividing cells fail to cure cancer patients. In fact, some evidence has directly attributed the therapeutic resistance to putative CSCs of solid tumors.

MicroRNAs (miRNAs) are endogenous noncoding RNAs that are post-transcriptional regulators of gene expression [5]. Emerging evidence suggests that several miRNAs may distinctively regulate the key biological properties of CSCs, including self-renewal and tumorigenesis [6]. In our previous study, miR-612 exhibited a pleiotropic inhibitory role on the HCC invasive-metastatic cascade, especially by suppressing cell proliferation, the epithelial–mesenchymal transition (EMT), local invasion, and distant colonization [7]. All these biological properties of miR-612 suggested to us that this miRNA could be a regulator of HCC stemness through an EMT-associated mechanism. Therefore, in this study we attempted to identify the possible roles of miR-612 in HCC stemness.

Section snippets

Cell lines

HCCLM3 was established by our institute and kept in the cell bank of the Liver Cancer Institute, Zhongshan Hospital, Shanghai. HepG2 was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (CAS). HCCLM3 cells have a relatively high metastatic potential and express a low endogenous level of miR-612, whereas HepG2 cells have a lower metastatic potential and express a higher endogenous level of miR-612. Both cell lines were cultured under standard conditions.

Oligonucleotides and transfection

Oligonucleotides

miR-612 suppresses tumorsphere and clone formation of HCC cells

Sphere-forming capability is a major property of normal stem cells as well as putative CSCs, and nonadherent tumorsphere assays are widely used to evaluate the self-renewal ability of CSCs. Therefore, we first evaluated tumorsphere formation of HCCLM3 and HepG2 cells after a 48-h treatment with miR-612 mimic (miR-612-o), miR-612 inhibitor (miR-612-i), and mock oligonucleotides (mock), respectively. We found that the number of tumorsphere (with a diameter larger than 50 μm) in miR-612-o-treated

Discussion

MicroRNAs are good molecular biomarkers for cancer diagnosis, prognosis, and therapy, and function as regulators of many oncobiological processes, such as CSC division and differentiation, tumorigenesis, EMT, and tumor metastasis [13]. The stemness of cancer cells has been proven to be regulated by the miRNA pathway [14].

The suppressive effects of miR-612 on HCC proliferation, EMT, metastatic colonization, and growth suggested that miR-612 could be a key regulator of the stemness of HCC. To

Acknowledgments

The authors thank all members of the Animal Department of the Liver Cancer Institute and Shanghai Medical School of Fudan University for nude mouse and NOD/SCID mouse maintenance, as well as xenograft establishment. We thank Prof. Li-Jian Hui (Shanghai Institutes for Biological Sciences, CAS) for kindly providing TOPflash reporter and Renilla luciferase constructs.

References (18)

H.B. El-Serag et al.
Hepatocellular carcinoma: epidemiology and molecular carcinogenesis
Gastroenterology
(2007)
Z.F. Yang et al.
Significance of CD90+ cancer stem cells in human liver cancer
Cancer Cell
(2008)
F. Ceteci et al.
Disruption of tumor cell adhesion promotes angiogenic switch and progression to micrometastasis in RAF-driven murine lung cancer
Cancer Cell
(2007)
S. Ma et al.
Identification and characterization of tumorigenic liver cancer stem/progenitor cells
Gastroenterology
(2007)
T.K. Lee et al.
CD24(+) liver tumor-initiating cells drive self-renewal and tumor initiation through STAT3-mediated NANOG regulation
Cell Stem Cell
(2011)
P.C. Hermann et al.
Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer
Cell Stem Cell
(2007)
P.B. Gupta et al.
Cancer stem cells: mirage or reality?
Nat. Med.
(2009)
D. Bonnet et al.
Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell
Nat. Med.
(1997)
M. Lagos-Quintana et al.
Identification of novel genes coding for small expressed RNAs
Science
(2001)

There are more references available in the full text version of this article.

Cited by (66)

Cancer stem cells: The important role of CD markers, Signaling pathways, and MicroRNAs
2024, Pathology Research and Practice
For the first time, a subset of small cancer cells identified in acute myeloid leukemia has been termed Cancer Stem Cells (CSCs). These cells are notorious for their robust proliferation, self-renewal abilities, significant tumor-forming potential, spread, and resistance to treatments. CSCs are a global concern, as it found in numerous types of cancer, posing a real-world challenge today. Our review encompasses research on key CSC markers, signaling pathways, and MicroRNA in three types of cancer: breast, colon, and liver. These factors play a critical role in either promoting or inhibiting cancer cell growth. The reviewed studies have shown that as cells undergo malignant transformation, there can be an increase or decrease in the expression of different Cluster of Differentiation (CD) markers on their surface. Furthermore, alterations in essential signaling pathways, such as Wnt and Notch1, may impact CSC proliferation, survival, and movement, while also providing potential targets for cancer therapies. Additionally, some research has focused on MicroRNAs due to their dual role as potential therapeutic biomarkers and their ability to enhance CSCs' response to anti-cancer drugs. MicroRNAs also regulate a wide array of cellular processes, including the self-renewal and pluripotency of CSCs, and influence gene transcription. Thus, these studies indicate that MicroRNAs play a significant role in the malignancy of various tumors. Although the gathered information suggests that specific CSC markers, signaling pathways, and MicroRNAs are influential in determining the destiny of cancer cells and could be advantageous for therapeutic strategies, their precise roles and impacts remain incompletely defined, necessitating further investigation.
To be or not to be: The double-edged sword roles of liver progenitor cells
2023, Biochimica et Biophysica Acta - Reviews on Cancer
Given the liver's remarkable and unique regenerative capacity, researchers have long focused on liver progenitor cells (LPCs) and liver cancer stem cells (LCSCs). LPCs can differentiate into both hepatocytes and cholangiocytes. However, the mechanism underlying cell conversion and its distinct contribution to liver homeostasis and tumorigenesis remain unclear. In this review, we discuss the complicated conversions involving LPCs and LCSCs. As the critical intermediate state in malignant transformation, LPCs play double-edged sword roles. LPCs are not only involved in hepatic wound-healing responses by supplementing liver cells and bile duct cells in the damaged liver but may transform into LCSCs under dysregulation of key signaling pathways, resulting in refractory malignant liver tumors. Because LPC lineages are temporally and spatially dynamic, we discuss crucial LPC subgroups and summarize regulatory factors correlating with the trajectories of LPCs and LCSCs in the liver tumor microenvironment. This review elaborates on the double-edged sword roles of LPCs to help understand the liver's regenerative potential and tumor heterogeneity. Understanding the sources and transformations of LPCs is essential in determining how to exploit their regenerative capacity in the future.
Dysregulation of non-coding RNAs mediates cisplatin resistance in hepatocellular carcinoma and therapeutic strategies
2022, Pharmacological Research
Citation Excerpt :
The mRNA and protein levels of cyclin D1 were significantly downregulated after miR-612-o treatment. Hence, overexpression of miR-612 triggered the Wnt/β-catenin signaling pathway by targeting AKT2 to suppress EMT-related stem cell-like traits in NOD/SCID mice, ultimately alleviating CDDP resistance [38]. Besides above upregulated miRNAs, some downregulated miRNAs also contributed to the HCC drug resistance process through related signaling pathways.
Hepatocellular carcinoma (HCC) is the fourth major contributor to cancer-related deaths worldwide, and patients mostly have poor prognosis. Although several drugs have been approved for the treatment of HCC, cisplatin (CDDP) is still applied in treatment of HCC as a classical chemotherapeutic drug. Unfortunately, the emergence of CDDP resistance has caused HCC patients to exhibit poor drug response. How to mitigate or even reverse CDDP resistance is an urgent clinical issue to be solved. Because of critical roles in biological functional processes and disease developments, non-coding RNAs (ncRNAs) have been extensively studied in HCC in recent years. Importantly, ncRNAs have also been demonstrated to be involved in the development of HCC to CDDP resistance process. Therefore, this review highlighted the regulatory roles of ncRNAs in CDDP resistance of HCC, elucidated the multiple potential mechanisms by which HCC develops CDDP resistance, and attempted to propose multiple drug delivery systems to alleviate CDDP resistance. Recently, ncRNA-based therapy may be a feasible strategy to alleviate CDDP resistance in HCC. Meanwhile, nanoparticles can overcome the deficiencies in ncRNA-based therapy and make it possible to reverse tumor drug resistance. The combined use of these strategies provides clues for reversing CDDP resistance and overcoming the poor prognosis of HCC.
Microvesicles – promising tiny players’ of cancer stem cells targeted liver cancer treatments: The interesting interactions and therapeutic aspects
2021, Pharmacological Research
Liver cancer is one of the most malignant cancers worldwide with poor prognosis. Intracellular mediators like microvesicles (MVs) and cancer stem cells (CSCs) are considered as potential candidates in liver cancer progression. CSCs receive stimuli from the tumor microenvironment to initiate tumor formation in which it’s secreted MVs play a noteworthy role. The phenotypic conversion of tumor cells during epithelial-to-mesenchymal transition (EMT) is a key step in tumor invasion and metastasis which indicates that the diverse cell populations within the primary tumor are in a dynamic balance and can be regulated by cell to cell communication via secreted microvesicles. Thus, in this review, we aim to highlight the evidences that suggest CSCs are crucial for liver cancer development where the microvesicles plays an important part in the maintenance of its stemness properties. In addition, we summarize the existing evidences that support the concept of microvesicles, the tiny particles have a big role behind the rare immortal CSCs which controls the tumor initiation, propagation and metastasis in liver cancer. Identifying interactions between CSCs and microvesicles may offer new insights into precise anti-cancer therapies in the future.
3,4-seco-lupane triterpene derivatives with cytotoxic activities from the leaves of Eleutherococcus sessiliflorus
2021, Natural Product Research
A new 3,4-seco-lupane triterpene, named sessiligenin (1), along with four known 3,4-seco-lupane triterpene derivatives (chiisanogenin 2, chiisanoside 3, divaroside 4, and sessiliside-A1 5) were isolated from the ethanol extract of the leaves of Eleutherococcus sessiliflorus (Rupr. & Maxim.) S.Y. Hu by silica gel column chromatography, and their structures were determined by spectroscopic data. Furthermore, all these compounds were tested for their cytotoxicities against cancer cell lines HepG2, B16-F10, Lewis and YAC-1, as well as normal cell lines NCTC1469 and HL-7702, and significant cytotoxicities had been found for this new compound (sessiligenin 1) which exhibited much lower cytotoxicities against normal cell lines NCTC1469 and HL-7702. It was deduced that the reduce of glycosyl from the structures of these 3,4-seco-lupane triterpenoids enhanced the cytotoxicities. Furthermore, with the complete removal of glycosyl group and the 11-hydroxyl and 3-carboxyl formed by the opening of the lactone ring, the cytotoxicity increased significantly.
Signaling pathways and microRNAs, the orchestrators of NANOG activity during cancer induction
2020, Life Sciences
Cancer stem cells (CSCs) are a small part of cancer cells inside the tumor that have similar characteristics to normal stem cells. CSCs stimulate tumor initiation and progression in a variety of cancers. Several transcription factors such as NANOG, SOX2, and OCT4 maintain the characteristics of CSCs and their upregulation is seen in many malignancies resulting in increased metastasis, invasion, and recurrence. Among these factors, NANOG plays an important role in regulating the self-renewal and pluripotency of CSCs and the clinical significance of NANOG has been suggested as a marker of CSCs in many cancers. The up and down-regulation of NANOG is associated with several important signaling pathways, including JAK/STAT, Wnt/β-catenin, Notch, TGF-β, Hedgehog, and several microRNAs (miRNAs). In this review, we will investigate the function of NANOG in CSCs and the molecular mechanism of its regulation by signaling pathways and miRNAs. We will also investigate targeting NANOG with different techniques, which is a promising treatment strategy for cancer treatment.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

miR-612 suppresses the stemness of liver cancer via Wnt/β-catenin signaling

Highlights

Abstract

Introduction

Section snippets

Cell lines

Oligonucleotides and transfection

miR-612 suppresses tumorsphere and clone formation of HCC cells

Discussion

Acknowledgments

Gastroenterology

Cancer Cell

Cancer Cell

Gastroenterology

Cell Stem Cell

Cell Stem Cell

Cancer stem cells: mirage or reality?

Nat. Med.

Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell

Nat. Med.

Identification of novel genes coding for small expressed RNAs

Science