Introduction
Within worldwide, HCC is the fifth most common malignant tumors and the third cause of cancer-related death currently. Despite advances in HCC treatment in recent years, the survival rate is still poor [
1]. The recurrence and metastasis of HCC in the liver is one of the obstacles affecting its therapeutic effect. Therefore, it is urgent to further explore the molecular pathological mechanism underlying cancer progression and metastasis, and it may provide a new therapeutic strategy for improving the prognosis of HCC patients.
MicroRNA (miRNA) is a small endogenous non-coding RNA molecule consisting of approximately 21–25 nucleotides. The miRNA is linked to the 3’-UTR of the target gene, resulting in decreased mRNA expression or transcriptional inhibition of protein [
2]. Currently, growing studies have shown that miRNA, as a proto-oncogene or tumor suppressor gene, mediates cell proliferation, apoptosis, invasion and metastasis through various signaling pathways, thus playing a crucial regulation role in tumor development and prognosis [
3]. As a member of the miRNA family, miR-6875 has been reported to be highly expressed in early breast cancer patients [
4]. Kijima [
5] demonstrated that poor tumor immunotherapy effect and prognosis occurred in metastatic colorectal cancer patients with high expression of miR-6875. These studies suggest that miR-6875 may play an important role in the development of tumors. Furthermore, our previous studies have shown that miR-6875-3p expression was significantly increased in HCC tissues compared with para-carcinoma tissues using microarray analysis (
P < 0.05, Additional file
1: Figure S1). However, its biological functions and correlation with HCC prognosis have not been reported yet and need to be further explored.
BTG2, also known as PC3/APRO1/TIS21, is a member of the BTG/TOB family. Located on chromosome 1q32.1, this gene encodes 158 amino acids [
6]. Studies have found that the expression of BTG2 is decreased in various tumor tissues such as prostate cancer, renal cell carcinoma and HCC [
7‐
9], and it has been confirmed to be associated with poor prognosis of breast cancer, bladder cancer and lung cancer [
10‐
12]. There is evidence that BTG2, as a tumor suppressor gene, plays an vital role in regulating the differentiation, proliferation, apoptosis and migration of tumor cells [
13]. Using miRNA target prediction algorithms, BTG2 was detected to be one of the potential target genes of miR-6875-3p.
The role of miR-6875-3p in the invasion and metastasis of HCC was analyzed in this experiment. The results indicated that miR-6875-3p expression was abnormally elevated in HCC tissues, and was positively correlated with low expression of BTG2, vascular invasion, poor differentiation, TNM staging and prognosis. In vitro and in vivo experiments showed that miR-6875-3p regulates EMT, enhances stem-like cell characteristics, and promotes the proliferation, invasion and metastasis of HCC cells. We also found that miR-6875-3p played it biological function by modulating the BTG2/FAK/Akt signaling pathway.
Material and methods
Cell cultures
The HCC cell lines (HL7702, Huh7, SNU-449, HepG2, HCCLM3, BEL-7404, MHCC97H) were bought from the American Type Culture Collection. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) containing 10% foetal bovine serum(Gibco, USA) in a 37 °C, 5% CO2 incubator.
Patient specimens
This study was approved by the Ethics Committees of the Second Affiliated Hospital of Jilin University. A total of 108 HCC samples were obtained from patients with HCC aged from 36 to 77 years, all of whom underwent radical surgical resection at the Second Affiliated Hospital of Jilin University between January 2010 and December 2012. None of these patients underwent chemotherapy or radiotherapy before surgery. Tumorous and adjacent non-cancerous tissues were frozen in liquid nitrogen after the surgical resection for further examination. Tumour stage was classified according to pTNM classification advocated by the International Union against Cancer and were followed up until June 2018. We obtained written informed consent from all of the patients in accordance with the Declaration of Helsinki.
In situ hybridization (ISH)
ISH analysis was performed as the method described before [
14]. Antisense oligonucleotide probes for miR-6875-3p (Exiqon Inc., Woburn, MA, USA) were used for ISH.
Immunohistochemistry
Immunohistochemical analysis was performed as previously described [
14]. Tumorous and adjacent non-cancerous tissue sections were hybridized with diluted primary antibody against BTG2 (1:200, Santa Cruz, USA) at 4 °C overnight. The positive cells were counted under a microscope (Olympus Vanox-T, Hamburg, Germany) and analyzed according to published protocols.
Western blot
Cells and tissues were washed once with ice-cold phosphate-buffered saline (PBS) containing 100 mM sodium orthovanadate and solubilized in lysis buffer. The supernatant was collected after centrifugation. Then the protein concentration was measured using a BCA Protein Quantitation Assay (Pierce, USA). A total of 20 μg of each protein sample was separated on a 10% gradient polyacrylamide gel and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in a primary antibody against BTG2, E-cadherin,α-catenin, N-cadherin, vimentin, β-actin, p-FAK(Tyr576, Tyr925), FAK, p-Akt, and Akt (Cell Signaling, USA), overnight at 4 °C. After incubation in a secondary antibody for 2 h, the targeted proteins in the membrane were detected using an enhanced chemiluminescence system. The intensities of the bands were qualified by Image J (National Institutes of Health, Bethesda, MD, USA).
Quantitative real-time RT-PCR
Total RNA from the cells or tissues was isolated with TRIzol Reagent (Invitrogen) and reverse transcribed to cDNA with ExScript RT Reagent (Takara) according to the manufacturer’s protocol. Real-time RT-PCR was carried out using Platinum SYBR Green qPCR SuperMix-UDG reagents(Invitrogen) on the PRISM 7900HT system (Applied Biosystems). β-actin expression was used to normalize for variance. The expression levels of specific genes are reported as ratios of expression of β-actin in the same master reaction.
miRNA and shRNA transfection
The shRNA targeting the BTG2, miR-6875-3p mimic, inhibitor and the negative control were obtained from Ribobio (Guangzhou, China). Cells (5 × 105 cells/2 ml/well) were plated at 60% confluence in a six-well plate in DMEM without antibiotics. After 48 h, miR-6875-3p mimic, inhibitor or negative control oligonucleotide was transfected into cells with Lipofectamine 2000 at a final concentration of 50 nM according to the instructions of the manufacturer’s. After 4–6 h, the medium was replaced with fresh DMEM containing 10% FCS and the cells were cultured for further experiment. The transfection of shRNA was performed as described above. To inhibit FAK phosphorylation, dissociated cells were incubated with PF573228 (10 μM, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min at 37 °C before Western blot analysis.
Luciferase reporter assay
The 3’-UTR of BTG2 containing the predicted binding site of miR-6875-3p was amplified from normal fetal genomic DNA using PCR specific primers. The PCR product was restricted and inserted between the restriction sites SpeI and HindIII into pMIR-REPORT-basic vector (Applied Biosystems, USA). The consensus miR-6875-3p binding site was mutated via PCR using a QuikChange II XL site-directed mutagenesis kit (Stratagene, USA). All clones were verified by DNA sequencing.Then, miR-6875-3p mimic or negative control was co-transfected with luciferase reporter vectors into HCC cells as described above, followed by the detection of luciferase activity via a luminescence reporter gene assay system (PerkinElmer, Norwalk, CT, USA) according to the manufacturer’s instructions.
MTT assay
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution was used to detect the proliferative rate of indicated cell lines. MTT were added (final concentration 0.5 mg/ml, stock solution 5 mg/ml MTT in phosphate-buffered saline) in every group cells for 3 h. Cells were lysed in acidified 2-propanol and absorbance measured at 490 nm.
Animal experiment
The research was conducted in accordance with the declaration of Helsinki and with the guide for care and use of laboratory animals as adopted and promulgated by the United National Institutes of Health. Animal handling and research protocols were approved by the Animal Care and Use Ethnic Committee. Male BALB/c nude mice aged 4 to 6 weeks were purchased form Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). Cells were subcutaneously injected into the flank region of the mice. The animals were sacrificed 5 weeks post inoculation. Tumors were surgically removed and weighed. The tumor metastatic ability was determined by tail vein injection of the cells into male nude mice. After 6 weeks, the animals were ether-anesthetized, and their lungs were removed to determine the pulmonary metastatic foci.
Transwell and matrigel assay
The cell migration and invasion were assessed using the wound-healing and transwell assays as previously described [
15].
Magnetic cell sorting
CD31 and EPCAM positive cells were isolated from HL7702 and Huh7 cells using anti-CD31 and anti-EPCAM antibodies coupled to magnetic beads (Miltenyi Biotech) using the MACS system (Miltenyi Biotech).
The cells were plated in a six-well plate at 5 × 102 per well and incubated for 2 weeks for the colony formation assay. The cells were washed twice with PBS, fixed with methanol/acetic acid (3:1, v/v), and stained with 0.5% crystal violet (Sigma, China). The number of colonies was counted under a microscope (Olympus Vanox-T, Hamburg, Germany).
Statistical analysis
Data were recorded as the means ± standard deviation (SD). Survival analysis was analyzed using Kaplan-Meier method. Association between miR-6875-3p and BTG2 expression in HCC tissues was calculated using Spearman rank correlation test. The χ2 test was performed to analyze the relationship between miR-6875-3p expression and the clinicopathological characteristics. The differences between the groups were undertaken using the Student two-tailed t test. A p < 0.05 was considered statistically significant. The statistical analysis and figure generation were performed using SPSS version 23.0 (IBM).
Discussion
Increasing evidences have demonstrated that miRNAs play an important role in the occurrence and development of tumors. In this study, miR-6875-3p was found to be abnormally highly expressed in HCC tissues and cell lines, and negatively correlated with BTG2 expression while positively correlated with tumor staging, size, degree of differentiation, and vascular invasion of HCC. Moreover, in vitro and in vivo experiments have shown that miR-6875-3p regulates EMT and improve the proliferation, metastasis and stem cell-like properties of HCC cells. BTG2 was first identified as a direct and functional target of miR-6875-3p via the 3’-UTR of BTG2. We also confirmed that miR-6875-3p plays its biological functions via the BTG/FAK/Akt pathway (Fig.
7d).
So far, the study on the correlation between miR-6875-3p and tumors is limited. Metastatic colorectal cancer patients with high expression of miR-6875 and miR-6826 have been reported to have poor immunotherapy effect and poor prognosis [
5]. miR-6875 can be used in combination with four other miRNAs to detect early breast cancer [
4]. However, the biological functions and potential molecular mechanisms of miR-6875 in HCC remain unexplored. In our study, miR-6875-3p was found to be highly expressed in HCC tissues and cell lines, and positively correlated with the degree of malignancy and staging of tumors. Based on in vitro and in vivo experiments, it was determined that down-regulation of miR-6875-3p expression inhibited tumor proliferation, invasion and metastasis. Furthermore, we also found that the OS and RFS rates of HCC patients in the low miR-6875-3p expression group were significantly higher than those in the high expression group. These results suggested that miR-6875-3p may act as a cancer-promoting factor in HCC, and abnormally elevated miR-6875-3p may be a predictor of poor prognosis in HCC patients.
miRNAs perform their functions through inhibiting the expression of target mRNAs. Our study confirmed that the tumor promoter role of miR-6875-3p in HCC, but the underlying mechanism remains unclear. Therefore, we speculated that BTG2 may be a candidate target gene of miR-6875-3p using TargetScan, PicTar and miRanda databases. Then, the luciferase reporter assay results indicated that miR-6875-3p regulated BTG2 by directly acting on its 3’-UTR. To further verify whether the effect of miR-6875-3p on HCC cells was achieved by directly inhibiting the BTG2 expression, we down-regulated the expression of BTG2 in miR-6875-3p-silencing cells and detected the cell proliferation and metastasis. The results showed that the proliferation and metastasis of tumor cells were significantly increased, indicating that BTG2 is a mediator in which miR-6875-3p functions.
Studies have shown that BTG2 plays a crucial role in tumor occurrence and development as a tumor suppressor gene [
13]. BTG2 expression is decreased in various tumor tissues such as breast cancer, lung cancer, bladder cancer and HCC [
18‐
21], and the low expression of BTG2 is associated with the aggressive clinical manifestations and poor prognosis [
10,
11,
22]. The results of our study were consistent with these previous reports.
The molecular mechanism of miR-6875-3p-BTG2 axis promoting the proliferation, invasion and metastasis of HCC cells remains unclear. The results of microarray analysis suggested that the FAK pathway is the primary signal pathway with the changes of BTG2 expression in HCC cells. FAK, as a protein kinase, can promote tumor progression and metastasis, and its high expression is closely related to several progressive solid tumors [
23]. It has been reported that FAK/Akt signaling pathway is correlated with the invasion and metastasis of various tumors including HCC [
24‐
27]. Lim et al. [
28] discovered that BTG2 can inhibit tumor invasion and metastasis by inhibiting the activation of FAK pathway. In the present study, down-regulation of miR-6875-3p expression reduced the phosphorylation levels of FAK and Akt, whereas silencing BTG2 abolished the inhibitory effect of low-expressed miR-6875-3p on FAK/Akt phosphorylation. All three databases suggested that miR-6875-3p/BTG2 can promote FAK/Akt phosphorylation. Furthermore, PF573228 can counteract the promotion of proliferation, invasion and metastasis by miR-6875-3p, indicating that miR-6875-3p induces the proliferation and metastasis of HCC through the FAK/Akt signaling pathway. A recent study revealed that BTG2 inhibited the activation of FAK pathway by down-regulating reactive oxygen species (ROS) in tumor cell mitochondria [
26], but the specific mechanism of miR-6875-3p-BTG2 axis regulating FAK/Akt pathway in HCC cells remains unclear and needs to be further explored.
EMT and stem cell-like properties are critical for the distant metastasis of HCC cells and the formation of new tumors [
29,
30]. Our studies have shown that miR-6875-3p can regulate EMT, enhance the stem cell-like characteristics, and promote invasion and metastasis of HCC cells in vitro and in vivo; while down-regulation of miR-6875-3p has the opposite effect. Wang [
14] et al. also reported that the FAK/PI3K/AKT signaling pathway was involved in the regulation of EMT in HCC cells. Therefore, we concluded that miR-6875-3p may also regulate EMT of HCC via the FAK/Akt pathway. However, the specific mechanism of miR-6875-3p regulating EMT and stem cell-like characteristics of HCC cells still needs to be further explored.
In summary, we have first demonstrated the biological functions of miR-6875-3p in HCC in vitro and in vivo. We found that miR-6875-3p enhances EMT and stem cell-like properties, and promotes the proliferation, invasion, and metastasis of HCC cells via the BTG2/FAK/Akt pathway. Therefore, this study may provide new predictive indicators and therapeutic targets for HCC treatment strategies.
Conclusions
In the current study, our study demonstrates that miR-6875-3p is highly expressed in HCC tissue and cells, and its elevated expression is positively correlated with tumor staging, size, degree of differentiation, vascular invasion and poor prognosis in HCC patients. In addition, our study confirms that miR-6875-3p regulates EMT, enhances stem-like cell characteristics, and promotes the proliferation, invasion and metastasis of HCC cells. Furthermore, this study is the first report demonstrating that miR-6875-3p functions by regulating the BTG2/FAK/Akt signaling pathway to promote HCC cells proliferation, invasion and metastasis. Thus, our findings suggest that miR-6875-3p may be a new predictive indicator of survival and prognosis in patients with HCC and may serve as a target for the diagnosis and treatment of HCC.
Acknowledgments
We wish to particularly acknowledge the patients enrolled in this study for their participation, and the Department of Pathology and Physiopathology, Second Affiliated Hospital of Jilin University, for its collaboration in providing the human samples and the clinical information used in this project with appropriate ethics approval.