uPAR is regulated via miR-561-3p and affects the progression and aggressiveness of CRC cells via the PI3K/AKT signaling pathway

Abstract

Urokinase plasminogen activator (uPA), the uPA receptor (uPAR) and plasminogen activator inhibitor type 1, constitute the plasminogen urokinase activation system that is hypothesized to be associated with the malignant biology of cancer cells. However, the regulation mechanism of uPAR expression in colorectal cancer (CRC) remains unclear. In the present study it was demonstrated that uPAR was upregulated in serum samples from patients with CRC and uPAR expression levels were associated with advanced stages of CRC and distant metastasis in patients. In CRC cells, uPAR was demonstrated to promote cell proliferation, migration and invasion, but inhibited cell apoptosis. Furthermore, the results demonstrated that uPAR expression was negatively regulated via miR-561-3p, via the binding of miR-561-3p to its 3’-untranslated region. Moreover, uPAR reduced the activation of the PI3K/AKT signaling pathway. In conclusion, uPAR was downregulated via miR-561-3p and potentially contributed to tumorigenesis via the PI3K/AKT signaling pathway in CRC.

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide and its incidence is increasing (Rawla et al., 2019). Advanced CRC has a high mortality rate and a poor prognosis, with the survival rate being higher when detected at an early stage (Keum and Giovannucci, 2019; Ladabaum et al., 2020). Therefore, it is important to investigate novel, more reliable biomarkers for the early diagnosis and prediction of CRC.

Urokinase plasminogen activator (uPA), the uPA receptor (uPAR) and plasminogen activator inhibitor type 1 constitute the plasminogen urokinase activation system, which is hypothesized to be associated with the malignant biology of cancer cells. uPAR, also known as CD87, is a glycosylphosphatidylinositol-anchored protein and a multifunctional cell surface receptor that is generally located in endothelial cells, fibroblasts and a variety of malignant cells (Dinesh and Rasool, 2018; Liu et al., 2018; Montuori et al., 2016; Simon et al., 2021). Increasing evidence suggests that uPAR is involved in the biology of cancer cells and therefore the expression of uPAR in numerous types of cancer is of clinical importance, including in colorectal cancer (CRC) (Illemann et al., 2014; Linders et al., 2021), gastric cancer (Alpizar-Alpizar et al., 2012) and breast cancer (Smaradhania et al., 2021). uPAR has been reported to be overexpressed in different types of cancer and can regulate a variety of tumorigenic processes, including cell proliferation, migration and invasion. Furthermore, uPAR has previously been investigated as a promising target for anti-cancer therapeutics (Fang et al., 2021; Kugaevskaya et al., 2018; Li Santi et al., 2021; Smith and Marshall, 2010). Recent studies have also demonstrated that uPAR acts as a tumor suppressor gene in the development of CRC (Biagioni et al., 2021; Biagioni et al., 2020). However, the underlying mechanism of uPAR expression regulation remains to be fully explored.

MicroRNAs (miRNAs/miRs) are evolutionally conserved noncoding RNAs ranging from 18–24 nucleotides. miRNAs are involved in gene expression regulation at the post-transcriptional level via binding to the 3’-untranslated region (3’UTR) of target mRNAs (Ambros, 2004; Bartel, 2004; Janiszewska, 2020; Lujambio and Lowe, 2012). miRNAs are also involved in tumor progression, as oncogenes or suppressor genes for tumors. It has previously been reported that miR-561-3p promotes the tumorigenesis of hepatocellular carcinoma (Chen et al., 2019), whereas miR-561-3p inhibits gastric cancer cell proliferation and invasion (Qian et al., 2016) and also inhibits cell proliferation in human non-small cell lung cancer (NSCLC) (Liao et al., 2020). Moreover, miR-561-3p has been reported to be involved in drug resistance in NSCLC (Hu et al., 2018). However, the role of miR-561-3p in CRC is unclear and remains to be elucidated.

The present study demonstrated that the axis of uPAR/miR-561-3p modulated the aggressiveness of CRC via the PI3K/AKT signaling pathway. Therefore, uPAR and/or miR-561-3p may be novel potential molecular biomarkers and therapeutic targets for the treatment of CRC.

Materials And Methods

Clinical specimens.

In total 30 samples from patients diagnosed with CRC and 30 samples from healthy individuals, who were identified via health checks and did not exhibit any health concerns during their physical examinations, were collected from Beijing Huairou Hospital (Beijing, China) from January 2018 to December 2020. Samples were snap-frozen and stored at -80˚C until further use. Informed consent was obtained from each patient and ethics approval was granted by the Ethics Committee of Beijing Huairou Hospital. The present study was performed in accordance with 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Cell culture and transfection.

The human colon cancer HCT116 and SW480 cell lines were purchased from the American Type Culture Collection and were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), HEPES (20 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml). Cells were incubated in a humidified atmosphere at 37˚C with 5% CO₂. All transfections were performed using lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol.

Plasmid construction.

The miR-561-3p mimic, the miR control, miR-561-3p inhibitor [miR-561-3p-antisense oligonucleotide (ASO)] and the normal scrambled negative control (NC; ASO-NC) were purchased from Shanghai GenePharma Co., Ltd. The uPAR overexpression vector was constructed using the coding sequence region of the uPAR gene, which was cloned into the pcDNA3.1 vector.

The small interfering RNA-uPAR was annealed and inserted into the plasmid of the pSilencer2.1-U6 neo vector. The uPAR 3’UTR containing the miR-561-3p wild-type (WT) or mutant (MUT) binding sites was annealed and cloned into the pmirGLO luciferase vector (Promega Corporation). All the constructed plasmids were confirmed via DNA sequencing.

Reverse transcription-quantitative PCR (RT-qPCR).

Total RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The experimental procedure of RNA extraction and RT-qPCR used in the present study was as previously described (Wan et al., 2014; Zhao et al., 2015). All primers are presented in Table I.

Dual-luciferase reporter assay. HCT116 cells were seeded into 48-well plates prior to transfection. Subsequently, the cells were co-transfected with the WT or MUT uPAR-3’UTR luciferase reporter and the miR-561-3p mimic, miR-561-3p-ASO or the corresponding control respectively. Luciferase activity was determined using the Dual-Glo Luciferase Assay System (Promega Corporation) according to the manufacturer’s protocol.

MTT and colony formation assays.

The MTT and colony formation assays were performed according to the manufacturer’s protocols, which were described previously (Wan et al., 2014; Zhao et al., 2015).

Migration and invasion assays.

Transfected HCT116 and SW480 cells (1x10⁵ cells) in serum-free medium were seeded into the upper chamber, either precoated with Matrigel for 2 h at 37˚C or uncoated. Medium with 20% FBS was added to the lower chamber. Following 24 h incubation, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Subsequently, cells were quantified in five random visual fields using a light microscope.

Cell apoptosis assay.

Transfected HCT116 and SW480 cells were seeded into six-well plates and treated for 24 h before being serum-starved for a further 24 h. The cells were then harvested. Apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit II (BD Biosciences) according to manufacturer’s protocol.

Bioinformatics.

miR-561-3p and target uPAR were identified using miRecords online software (http://miRecords.umn.edu/miRecords.)(Xiao et al., 2009).Among the potential miRNAs, miR-561-3p was chosen as the miRNA that markedly affected the growth phenotype of CRC cells based on previous data.

Western blotting.

Western blotting was performed to detect protein expression levels using previously described methods. The antibodies used were as follows: anti-GAPDH (Catalog No: SRP13406, 1:3,000 dilution), uPAR (Catalog No: SRP10752, 1:200 dilution), phosphorylated (p)-AKT (Catalog No: ab278559, Abcam, UK, 1:300 dilution), AKT (Catalog No: SRP00437, 1:300 dilution), p-PI3K (Catalog No: ab182651, Abcam, UK, 1:300 dilution) and PI3K (Catalog No: SRP01115, 1:300 dilution). The antibodies were used to incubate at 4°C overnight. Anti-GAPDH/uPAR/AKT were purchased from Tianjin Saierbio Technology, Inc.

Results

uPAR facilitates CRC cell proliferation, migration and invasion and inhibits cell apoptosis. To investigate the effect of uPAR on human CRC cells, the uPAR overexpression plasmid, pcDNA3.1/uPAR and three knockdown plasmid, pSilencer2.1/uPAR, were constructed. RT-qPCR and western blotting were performed to validate the efficiency of the plasmids in HCT116 cells. The results demonstrated that of the three knockdown plasmids, the inhibitory effect of short hairpin RNA (shR)3-uPAR on the mRNA and protein expression levels of uPAR was the most significant (Fig. 1a). Therefore, shR3-uPAR was used for the subsequent experiments. Moreover, to determine the effects of uPAR overexpression in HCT116 and SW480 cells the MTT, colony formation and Transwell migration/invasion assays were performed. The MTT and colony formation assays demonstrated that uPAR overexpression promoted cell proliferation compared with the cells transfected with pcDNA3.1 only. Furthermore, uPAR knockdown inhibited this effect (Fig. 1b-d). The results of the Transwell migration/invasion assays demonstrated that the migration and invasion abilities of uPAR-overexpressing CRC cells were markedly increased, whereas uPAR knockdown inhibited cell migration and invasion capacities (Fig. 1e and f). Moreover, flow cytometry was performed to explore whether uPAR regulated the process of cell apoptosis. The results demonstrated that uPAR overexpression inhibited the apoptosis of HCT116 and SW480 cells, whereas uPAR knockdown induced the opposite effect (Fig. 1g). These results suggested that uPAR potentially facilitates the proliferation, migration and invasion of CRC cells via the inhibition of cell apoptosis.

uPAR is the target of miR-561-3p.

To determine the miRNA that potentially regulated uPAR, bioinformatics analyses using miRecords combined with TargetScan6.2, RNA hybrid2.1.2 and PITA (Catalog version 5) was performed. Using the predicted scores and the function and expression levels of potential candidate targets in CRC, miR-561-3p was identified. It was demonstrated that the 3’UTR of uPAR contained a binding site for miR-561-3p. Therefore, to verify this predicted site, luciferase reporter vectors containing the WT or MUT 3’UTR of uPAR in the miR-561-3p binding region were produced (Fig. 2a). The results demonstrated that compared with the control group in HCT116 and SW480 cells, the ectopic expression of miR-561-3p group was reduced, whereas miR-561-3p knockdown group increased the luciferase fluorescence intensity of uPAR-3’UTR-WT. However, the fluorescence intensity was not altered in the uPAR-3’UTR-MUT group (Fig. 2b). Furthermore, miR-561-3p was overexpressed in HCT116 and SW480 cells and the results demonstrated a significant reduction in uPAR mRNA and protein expression levels. However, miR-561-3p knockdown increased the mRNA and protein expression levels of uPAR (Fig. 2c). These data indicated that uPAR may be a direct target of miR-561-3p and its expression may be downregulated by miR-561-3p overexpression in CRC cells.

miR-561-3p inhibits CRC cell aggressiveness via the regulation of uPAR expression.

To investigate the effect of miR-561-3p in CRC cells, the MTT and colony formation assays were performed to detect the effect of miR-561-3p on cell proliferation. The results demonstrated that miR-561-3p overexpression repressed cell proliferation and colony formation ability, whereas miR-561-3p inhibition facilitated the cell proliferation of HCT116 and SW480 cells (Fig. 3a and b). Furthermore, Transwell migration and invasion assays demonstrated that the migration and invasion abilities of miR-561-3p-overexpressing CRC cells were markedly decreased compared with the control group. However, miR-561-3p inhibition facilitated migration and invasion in CRC cells (Fig. 3c, d). Furthermore, flow cytometry was performed to determine whether miR-561-3p regulated cell apoptosis. The results demonstrated that miR-561-3p overexpression promoted apoptosis, whereas miR-561-3p inhibition decreased cell apoptosis in CRCs (Fig. 3e). These results indicated that miR-561-3p overexpression potentially inhibits CRC cell proliferation, migration and invasion via promoting cell apoptosis.

Moreover, to determine if the function of miR-561-3p in CRC cells was mediated via the regulation of uPAR, miR-561-3p mimics and uPAR overexpression vectors were co-transfected into CRC cells. The effects of the miR-561-3p inhibitor on cell proliferation, migration and invasion were significantly reversed following uPAR overexpression in CRC cells. Furthermore, the results also demonstrated that the miR-561-3p-mediated increase in cell apoptosis was significantly reduced by the ectopic expression of uPAR. These data suggested that miR-561-3p potentially inhibits CRC cell aggressiveness via the regulation of uPAR expression.

uPAR/miR-561-3p axis regulates the phosphorylation of the PI3K/AKT signaling pathway. Previous reports have demonstrated that uPAR can promote cancer cell tumorigenicity via the activation of the AKT/PI3K signaling pathway (Liu et al., 2018; Nowicki et al., 2011). In order to investigate whether the PI3K/AKT signaling pathway is responsible for the role of the uPAR/miR-561-3p axis in the aggressiveness of CRC, western blotting was performed to determine the protein expression levels of PI3K/AKT-associated proteins in HCT116 cells. The results demonstrated that miR-561-3p overexpression significantly reduced P13K/AKT-associated protein expression levels, whereas miR-561-3p inhibition increased the phosphorylation levels of PI3K/AKT-associated proteins in HCT116 cells. Furthermore, reintroduction of uPAR expression partially rescued the inhibitory function of miR-561-3p on the phosphorylation of PI3K/AKT-associated proteins in HCT116 cells (Fig. 4). These data demonstrated that miR-561-3p potentially inhibits the uPAR-mediated PI3K/AKT signaling pathway in CRC cells.

Serum uPAR expression levels are inversely associated with miR-561-3p and are related to the clinicopathological characteristics in patients with CRC.

To assess the importance of the uPAR/miR-561-3p axis in clinical samples, RT-qPCR was performed to determine uPAR and miR-561-3p expression levels in serum samples from 30 patients with CRC and 30 healthy individuals. The χ²-squared test demonstrated that there was no statistical significance in the age and gender of the two groups (data not shown). Moreover, uPAR was demonstrated to be upregulated, whereas miR-561-3p was demonstrated to be significantly downregulated in serum samples from patients with CRC compared with the healthy individuals (Fig. 5a). Furthermore, the association of uPAR expression levels with clinicopathological characteristics of patients with CRC was assessed. The results demonstrated that high uPAR expression levels were significantly associated with more advanced stages of CRC and distant metastasis compared with the healthy controls (Fig. 5b). The results indicated that uPAR expression levels were upregulated, whereas miR-561-3p expression levels were downregulated in serum samples from patients with CRC and were potentially associated with an aggressive phenotype.

Discussion

The uPA/uPAR system is recognized as a powerful driver of cancer development and progression (Mahmood et al., 2018; Ulisse et al., 2009). It has previously been reported that uPAR regulates cell proliferation, adhesion and migration. uPAR also serves a role in multiple signaling pathways and can therefore alter the biological properties of cells (Alfano et al., 2005; Smith and Marshall, 2010). The present study reviewed the results of these aforementioned reports and further demonstrated that uPAR was upregulated in the serum samples from patients with CRC compared with healthy individuals. Moreover, high expression levels of uPAR were significantly associated with the more advanced stages of CRC and distant metastases in patients. Furthermore, the results demonstrated that the upregulation of uPAR facilitated CRC cell proliferation, migration and invasion and inhibited apoptosis in CRC cells. It can therefore be hypothesized that uPAR acts as an oncogene in CRC.

To further explore the underlying molecular mechanism of uPAR dysregulation in CRC, the post-transcriptional regulation of uPAR expression via miRNAs was investigated. In the present study, uPAR was predicted to be a novel target of miR-561-3p via bioinformatics analysis. The dual-luciferase reporter assay demonstrated that miR-561-3p targets the 3’UTR of uPAR. Furthermore, miR-561-3p overexpression decreased the protein and mRNA expression levels of uPAR. However, the detailed underlying mechanism of uPAR involved in the regulation of CRC needs to be further elucidated. Moreover, previous studies have reported that the abnormal expression of miRNAs is associated with the pathogenesis of different types of cancer (Calin and Croce, 2006; Garo et al., 2021).

In the present study it was demonstrated that miR-561-3p was downregulated in the serum samples from patients with CRC compared with the healthy individuals. The high expression levels of miR-561-3p were demonstrated to inhibit CRC cell proliferation, migration and invasion and promote cell apoptosis. It can therefore be hypothesized that miR-561-3p may serve a role as a tumor suppressor via the downregulation of uPAR in CRC. Numerous studies have reported that the PI3K/AKT signaling pathway is abnormally activated in CRC and contributes towards cell proliferation, migration and invasion (Tan et al., 2018; Zhao and Qin, 2020). The present study demonstrated that miR-561-3p overexpression increased the phosphorylation levels of PI3K/AKT-associated proteins. Moreover, uPAR overexpression partially rescued the inhibitory function of miR-561-3p on the phosphorylation levels of PI3K/AKT-associated proteins in HCT116 cells. These data demonstrated that miR-561-3p potentially inhibited the uPAR-mediated PI3K/AKT signaling pathway in CRC cells.

The present study suggested that uPAR may be a novel target of miR-561-3p. Moreover, miR-561-3p may act as an inhibitor for cell proliferation, migration and invasion in CRC via inhibiting the expression of uPAR. The present study also indicated that the uPAR/miR-561-3p axis potentially regulated the development and progression of CRC via the PI3K/AKT signaling pathway. Moreover, high expression levels of uPAR were significantly associated with more advanced stages of CRC and distant metastasis in patients.

In conclusion, these results demonstrated that uPAR was downregulated via miR-561-3p and may potentially contribute to tumorigenesis via the PI3K/AKT signaling pathway. Furthermore, the uPAR/miR-561-3p axis may be a novel potential molecular biomarker and therapeutic target for patients with CRC.

Declarations

Funding

Scientific Research Backbone Fund of Beijing Huairou Hospital (HRYY-2019-03).

Acknowledgements

Not applicable.

Disclosure of interest

The Authors declare that there is no conflict of interest.

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Tables

Table 1. The primers and oligonucleotides used in this work. 

Primers used in the construction of plasmids and RT-qPCR.