LncRNA RSU1P2-microRNA let-7a-Testis-Expressed Protein 10 axis modulates tumorigenesis and cancer stem cell-like properties in liver cancer

ABSTRACT LncRNAs exert important functions in the modulation of tumorigenesis and cancer stem cell-like properties in liver cancer. However, the role of LncRNA Ras suppressor protein 1 pseudogene 2 (RSU1P2) in modulating tumorigenesis and cancer stem cell-like properties in liver cancer is still not known. In this study, the expression of LncRNA RSU1P2 was significantly elevated in liver cancer tissues and cells. Besides, knockdown of RSU1P2 repressed cell viability, invasion, epithelial-mesenchymal transition (EMT) of liver cancer cells and the expressions of cancer stem cell-related genes, whereas facilitated the apoptosis of liver cancer cells. In addition, LncRNA RSU1P2 can interact with microRNA let-7a (let-7a), and repress let-7a expression. Testis-Expressed Protein 10 (Tex10) was identified to be a target of let-7a, and let-7a repressed Tex10 expression. Finally, RSU1P2 knockdown suppressed tumor volume, tumor weight, and EMT in a xenograft model. Therefore, LncRNA RSU1P2 promotes tumorigenesis and cancer stem cell-like properties in liver cancer through let-7a/Tex10 pathway.


Introduction
Liver cancer is the fourth most common cancer deaths in digestive system in the United States [1]. It has been estimated that new cases will reach to 42,810 and the deaths will reach to 30,160 in the United States according to the 2020 Cancer Statistics [2]. In China, liver cancer is the second cause of cancer death in male and the third in female, and liver cancer is mainly caused by nonalcoholic steatohepatitis, hepatitis C virus and hepatitis B virus [3]. The progression of liver cancer is complex which is related with liver cancer cells proliferation, migration and invasion and cancer stem cell-like properties [4,5]. According to previous reports, cancer stem cells are in charge of liver cancer growth and metastasis, as well as related with the recurrence of liver cancer [6,7]. Although many high or low expressed genes involved in tumorigenesis and cancer stem celllike properties of liver cancer were identified, the underlying molecular mechanisms for liver cancer are not fully understood [5,8]. Therefore, underlying mechanisms that regulate liver cancer tumorigenesis and cancer stem cell-like properties should be identified to develop efficient strategies for the treatment of liver cancer.
Many studies have found that a variety of long non-coding RNAs (LncRNAs), such as LncRNA plasmacytoma variant translocation 1 (PVT1), LncRNA pituitary tumor-transforming 3, pseudogene (PTTG3P) and LncRNA n335586, can promote the proliferation, migration, invasion and cancer stem cell-like properties in liver cancer [9][10][11]. LncRNA Ras suppressor protein 1 pseudogene 2 (RSU1P2) is a newly discovered LncRNA that acts as a cancer-promoting gene in cervical cancer, and LncRNA RSU1P2 can act as a competitive endogenous RNA (ceRNA) against let-7a to promote its downstream molecules insulin-like growth factor 1 receptor (IGF1R), N-myc and EphA4 expressions, thereby promoting tumorigenesis and epithelial-mesenchymal transition (EMT) in cervical cancer [12]. However, the effect of LncRNA RSU1P2 in liver cancer progression is still unrevealed.
Let-7a is a disease-associated microRNA (miRNA) that can modulate biological functions involving the proliferation, migration, and invasion, EMT and cancer stem cell-like properties in cancers, including lung cancer, nasopharyngeal carcinoma, and breast cancer [13][14][15]. According to previous reports, let-7a is considered as being reduced in the progression of cancers, and let-7a can modulate the oncogenes expressions and facilitate colon cancer and pancreatic cancer tumorigenesis [16,17]. In addition, researchers have shown that let-7a can suppress the sphere formation efficiency of liver cancer cells, EMT, renewal of cancer stem cells and Wnt-β-catenin pathway in liver cancer [18]. EMT can contribute to the invasion and metastasis of liver cancer [19,20]. Therefore, let-7a might exert a vital function in modulating tumorigenesis and cancer stem celllike properties in liver cancer.
Testis-Expressed Protein 10 (Tex10) was firstly reported to regulate ribosome biogenesis in eukaryotes [21]. Later, Tex10 has been shown to link arginine methylation to desumoylation and modulate transcriptional activity [22]. In addition, Tex10, as an important pluripotency factor, exerts vital function in self-renewal of embryonic stem cells, pluripotency maintenance, embryo development, and reprogramming [23,24]. Importantly, Tex10 has been found to be elevated in liver cancer cells and liver cancer stem cells, as well as Tex10 overexpression facilitates cancer stem cell properties by activating STAT3 pathway [25].
This study assumes that LncRNA RSU1P2 is elevated in liver cancer tissues and cells, and acts as a cancer-promoting gene in liver cancer. We aim to investigate the promoting role of LncRNA RSU1P2 in liver cancer cells proliferation, invasion, EMT, and cancer stem cell-like properties, and its underlying mechanism, which might provide potential targets for liver cancer treatment.

Sample selection
Liver cancer tissues and tumor adjacent tissues (35 pairs) were obtained from liver cancer patients (25 male and 10 female patients; aged from 45 to 69 years old) when they underwent hepatectomy at The First Affiliated Hospital of Shantou University Medical College from January 2019 to October 2019, which were confirmed by two histopathologists. Informed consent was obtained from all patients. All liver cancer patients did not receive pre-operative treatments, such as immunotherapy, targeted therapy, and chemotherapy [26]. All liver cancer tissues and tumor adjacent tissues were stored at −80°C. This experiment was approved by the Ethics Committee of The First Affiliated Hospital of Shantou University Medical College.

Colony formation assay
Since liver cancer cells can proliferate in suspension, the malignant proliferation ability can be reflected by the ability of colony formation in plates [30]. After transfection for 48 h, HCCLM3 and HepG2 cells were counted and cells (2 × 10 3 / well) were put into each well of 6-well plates with DMEM (Gibco) for colony formation assay for 2 weeks. The colonies of HCCLM3 and HepG2 cells (approximately 50 or more cells) were fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) for half an hour. Finally, the colonies of HCCLM3 and HepG2 cells were stained with crystal violet (0.5%; Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) for half an hour at 25°C. The colonies of HCCLM3 and HepG2 cells were observed with a light microscope (Olympus, Tokyo, Japan).

Transwell cell invasion assay
For the observation of cell invasion ability, 0.2 ml HCCLM3 and HepG2 cells (1 × 10 5 cells) were added in the upper chambers of 8 μM pore-sized Transwell (Corning, NY, USA) which were precoated with 1:8 matrigel. HCCLM3 and HepG2 cells were incubated in Transwell for 24 h. Then, HCCLM3 and HepG2 cells remained in the upper chambers were removed. Cells invaded to lower chambers were immobilized with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and then stained with crystal violet (0.3%; Beyotime Biotechnology, Nantong, China). Invasive cells were counted by a light microscope (Olympus, Tokyo, Japan) [32].

Xenograft model for assess the effect of RSU1P2 on tumor growth and EMT
Male BALB/c nude mice (4-5 weeks, weighing 18-20 g) were kept in a 12 h light/12 h dark condition with free access to water and food. Nude mice were randomly divided into sh-NC and sh-RSU1P2 groups, with five mice in each group. To establish xenograft tumor model, HCCLM3 cells were transfected with sh-NC and sh-RSU1P2 (GenePharma, Shanghai, China). Briefly, HCCLM3 cells (1 × 10 5 ) were put to each well of 24-well plates and cultured for 24 h. sh-NC or sh-RSU1P2 (1 μg) was added into cells and incubated for 72 h. Then, HCCLM3 cells (1 × 10 6 cells, 200 μL) were injected into the right flank of nude mice subcutaneously [35]. Tumor volume was recorded every six days and calculated by the formula: volume = (length × width 2 )/2. Mice were euthanized by an overdose of 100 mg/kg sodium pentobarbital through intravenous injection. Tumor tissues were collected from sacrificed mice at day 30. This animal experiment was approved by the Animal Care and Use Committee of The First Affiliated Hospital of Shantou University Medical College (SYXK(Yue)2019-0079).

Immunohistochemical (IHC) staining
Tumor tissue sections were treated with 1% H 2 O 2 , blocked with 1% FBS (Gibco, CA, USA), and immunostained with anti-E-cadherin (1:1000; Cell Signaling Technology, MA, USA) or anti-Vimentin (1:500; Sigma-Aldrich, MO, USA) for 12 h at 4°C. After that, tumor tissue sections were incubated with a secondary antibody (1:500; Cell Signaling Technology) for 2 h at 25°C. All sections were developed by 3,3'-diaminobenzidine (DAB) kit (Thermo Scientific, CA, USA) [36]. SPSS 18.0 (Chicago, USA) was performed for data analysis, and data were presented as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used to analyze differences among multiple groups. Student's t-test was used to analyze differences between two groups. Significance levels were set at p value less than 0.05 [37].

Results
We aim to investigate the promoting role of LncRNA RSU1P2 in liver cancer and the underlying mechanism of LncRNA RSU1P2 in liver cancer cells proliferation, invasion, EMT and cancer stem cell-like properties in vitro and tumorigenesis in vivo. We proved that let-7a expression was regulated by LncRNA RSU1P2, and let-7a could target to Tex10. We verified that LncRNA RSU1P2/let-7a/Tex10 pathway promoted liver cancer cell proliferation, invasion, EMT and the expression of cancer stem cell-related genes in vitro and confirmed our hypothesis in vivo.

Discussion
Cancer-related LncRNAs are usually aberrantly expressed in cancer tissues and cancer cells, which play critical roles in regulating tumorigenesis and cancer stem cell-like properties [38,39]. Various LncRNAs are aberrantly expressed in liver cancer tissues and cells and act as oncogenes or tumor suppressors, thereby affecting multiple biological processes [40,41]. Although some LncRNAs are well identified in liver cancer, it is valuable to clarify potential meaningful LncRNAs in modulating the tumorigenesis and cancer stem cell-like properties in liver cancer [42,43]. In this study, LncRNA RSU1P2 was raised in cancer tissues from liver cancer patients and liver cancer cell lines. RSU1P2 knockdown remarkably decreased RSU1P2 expression in HepG2 and HCCLM3 cells. Thus, we first revealed that LncRNA RSU1P2 was elevated in liver cancer, which suggested that the up-regulation of LncRNA RSU1P2 might exert important function in the progression of liver cancer.
Previous researches have shown that abnormally expressed LncRNAs are vital regulators in controlling the proliferation, invasion, EMT, and cancer stem cell-like properties in liver cancer [44][45][46][47]. For example, LncRNA CRNDE was increased in liver cancer tissues and liver cancer cells, and knockdown of CRNDE restrained growth, metastasis of liver cancer tumors, suppressed liver cancer cell EMT and Wnt/βcatenin pathway [48]. LncRNA DANCR was raised in liver cancer tissues and liver cancer cells, and DANCR overexpression facilitated the proliferation and metastasis of liver cancer cells [49]. LncRNA AGAP2-AS1 was increased in liver cancer tissues and liver cancer cells, and AGAP2-AS1 overexpression facilitated cell proliferation, invasion, and migration, EMT of liver cancer [50]. In this study, RSU1P2 knockdown remarkably reduced the cell viability, suppressed the colony formation of liver cancer cells, decreased invasive cells number, inhibited expressions of cancer stem cell-related genes and Vimentin protein expression, increased the apoptosis of liver cancer cells and up-regulated E-cadherin protein expression in vitro. These findings indicated that LncRNA RSU1P2 functioned as a cancer-promoting gene in liver cancer. Until now, no other studies have verified the role of LncRNA RSU1P2 in controlling tumorigenesis and cancer stem cell-like properties in liver cancer, which will provide theoretical basis for liver cancer research.
Mounting evidences have provided evidences that LncRNAs play their roles in liver cancer via acting as a ceRNA to sponge miRNAs [51][52][53][54]. In this study, RSU1P2 expression was elevated whereas let-7a was remarkably restrained in liver cancer tissues and cells, and let-7a level was negatively related with RSU1P2 expression in liver cancer tissues. Moreover, it was identified that let-7a was a target of RSU1P2 in HCCLM3 and HepG2 cells. Since let-7a suppress the proliferation of liver cancer cells, EMT, renewal of cancer stem cells and Wnt-β-catenin pathway in liver cancer [18]. We therefore considered that LncRNA RSU1P2 acted as an oncogene in liver cancer by negatively regulating let-7a.
Online bioinformatics prediction tools are meaningful in helping researchers clarify the underlying molecular mechanism within miRNAs and their downstream targets [55]. Previous reports have shown that let-7a has binding sites with its downstream molecules and can target to 3' UTRs of these molecules, such as BCL2L1, STAT3 and RTKN [56][57][58]. According to the prediction of StarBase (http:// starbase.sysu.edu.cn/), there were binding sites between let-7a and 3' UTRs of Tex10 mRNA. So, we assumed that Tex10 might be a target of let-7a. In this study, inverse correlation between let-7a and Tex10 level was identified in liver cancer tissues. In addition, let-7a overexpression prominently down-regulated Tex10 expression in HCCLM3 and HepG2 cells. Luciferase reporter assay identified that let-7a targeted 3' UTR of Tex10 mRNA. Therefore, we first confirmed that let-7a acted as the upstream molecule to regulate Tex10 by binding to 3' UTRs of Tex10, and the methods used in this study were consistent with previous reports [59,60]. Moreover, there was a positive correlation between RSU1P2 and Tex10 in liver cancer tissues, and RSU1P2 positively regulated Tex10 mRNA and protein expressions in HCCLM3 and HepG2 cells. Therefore, we assumed that LncRNA RSU1P2 acted as an oncogene in liver cancer by promoting Tex10 via sponging let-7a.
According to the findings of previous report, highly expressed Tex10 in liver cancer cells and liver cancer stem cells can promote cancer stem cell properties [25]. Although the ability of Tex10 to promote self-renewal of stem cells have been confirmed, Tex10 is still a pluripotency factor that lacks concern and in-depth understanding [23,24]. Thus, the underlying mechanism of Tex10 in liver cancer is needed to be clarified. In this study, Tex10 restoration abolished the inhibition effect of RSU1P2+ let-7a on cell viability, proliferation, invasion, EMT of liver cancer cells, and the expressions of cancer stem cell-related genes. Therefore, our findings suggested that Tex10 was involved in LncRNA RSU1P2/let-7a mediated promotion of the tumorigenesis and cancer stem cell-like properties in liver cancer. Thus, we have revealed the role of LncRNA RSU1P2/let-7a/Tex10 pathway in controlling tumorigenesis and cancer stem cell-like properties in liver cancer, which may supply potential therapeutic targets for liver cancer treatment.
Wnt/β-catenin pathway, an important driver of tissue stem cells in mammals, can promote cancer progression and metastasis through controlling the growth and biology of stem cells [61]. In liver cancer treatment, targeting Wnt/β-catenin pathway components can achieve anticancer effect by reducing hepatocarcinogenesis and the maintenance of liver cancer stem cells [62]. LncRNAs/miRNAs are important modulators in regulating cancer stemness and cancer cell proliferation by enhancing Wnt/β-catenin pathway [63,64]. Xiang et al have shown that highly expressed Tex10 facilitates EMT and stemness of ESCC cells through activating Wnt/β-catenin pathway [65], indicating Tex10 might exert its promotion function in cancers through Wnt/β-catenin pathway. In this study, RSU1P2 facilitated Wnt1 and β-catenin expressions, RSU1P2+ let-7a down-regulated Wnt1 and βcatenin expressions, and RSU1P2+ let-7a+Tex10 abolished the inhibition effect on Wnt1 and βcatenin protein expressions, indicating RSU1P2/ let-7a/Tex10 regulated Wnt/β-catenin pathway in liver cancer.

Conclusion
LncRNA RSU1P2 functions as a cancer-promoting gene in liver cancer and promotes tumorigenesis in vivo. Besides, LncRNA RSU1P2 facilitates liver cancer cells proliferation, invasion, EMT, and cancer stem cell-like properties through let-7a/Tex10 pathway in vitro.

Disclosure statement
No potential conflict of interest was reported by the author(s).