CircLASP1 silence strengthens the therapeutic effects of MK-2206 on nasopharyngeal cancer through upregulating miR-625

Abstract

Therapeutic effects of MK-2206 are largely limited due to the complexity of the pathogenesis of nasopharyngeal cancer (NPC). Here, we aimed to investigate whether and how circLASP1 is involved in the therapeutic effects of MK-2206 on NPC. We showed circLASP1 was increased while miR-625 was decreased in NPC tissues and cell lines. CircLASP1 silence strengthened the therapeutic effects of MK-2206 via suppressing NPC cell proliferation and inducing autophagy and apoptosis in vitro. In mechanism analyses, we found that circLASP1 indirectly released AKT by directly binding to miR-625 in NPC cells, and miR-625 acted as a tumor suppressor in NPC and activated cell autophagy through inhibiting the AKT/mTOR pathway. Most importantly, knockdown of circLASP1 was revealed to enhance the therapeutic effects of MK-2206 on NPC in vivo. Our results suggest that the circLASP1/miR-625 axis is involved the therapeutic effects of MK-2206 on NPC by regulating autophagy, proliferation, and apoptosis through the AKT/mTOR pathway. miR-625 is involved in NPC tumorigenesis.

Abbreviations
• ANOVA
• analysis of variance
• CCK-8
• cell-counting kit-8
• circRNAs
• circular RNAs
• FISH
• fluorescence in situ hybridization
• LASP1
• LIM and SH3 protein 1
• miRNAs
• microRNAs
• MUT
• mutant
• NC
• negative control
• NPC
• nasopharyngeal cancer
• OE-AKT
• AKT overexpression vector
• OE-circLASP1
• circLASP1 overexpression vector
• qRT-PCR
• quantitative real-time PCR
• RIP
• RNA immunoprecipitation
• SD
• standard deviation
• WT
• wild type

INTRODUCTION

Nasopharyngeal cancer (NPC) arises from the nasopharyngeal mucosal lining.¹ It was estimated that approximately 66,098 new NPC cases will occur in China and the United States in 2022.² Although NPC appears to be sensitive to chemo- and radiotherapy, the prognosis of NPC patients with metastasis is far from satisfactory, with an extremely high recurrence rate of 82%.^3,4 MK-2206 is a selective AKT inhibitor with broad preclinical antitumor activity. Both in vitro and in vivo evidence has indicated that MK-2206 could repress the NPC tumor growth by repressing the AKT signaling pathway,⁵ yet its therapeutic effects are largely limited due to the complexity of the pathogenesis of NPC. Thus, it is urgent to develop strategies for reinforcing the inhibitory activities of MK-2206 on NPC.

In recent years, circular RNAs (circRNAs) and microRNAs (miRNAs) have been reported to play key roles in NPC tumorigenesis.^6,7 Moreover, the interaction between the circRNA/miRNA axis and AKT1 (also called AKT in human) has been increasingly identified to be linked with the pathogenesis of cancers.^8,9 As a key structural scaffolding protein, LIM and SH3 protein 1 (LASP1) was revealed to be associated with the growth of many metastatic cancers, including NPC.¹⁰ Nevertheless, whether its circular transcript (circLASP1) plays a role in NPC pathogenesis remains undetermined. As LASP1 possesses the ability to regulate the PI3K/AKT pathway,¹¹ we explored the interaction between circLASP1 and AKT using bioinformatics analysis (Starbase) and found that miR-625 might be a bridge between circLASP1 and AKT. miR-625 was reported to play a role in the tumorigenesis of NPC¹²; however, the interaction between miR-625 and circLASP1 or AKT in NPC has not been investigated.

As an important phagocytosis process, the dysfunction of autophagy has been correlated with multiple human diseases, especially cancers.¹³ Induction of autophagy was revealed to repress the invasion and migration but promote the apoptosis of NPC cells.^14,15 Moreover, evidence has shown that the inhibition of AKT/mTOR signaling could induce autophagy in NPC cells.¹⁶

Here, we attempt to examine the potential roles of circLASP1 in reinforcing the therapeutic effects of MK-2206 through the AKT/mTOR signaling pathway on NPC. Our study can help to further understand the pathogenesis of NPC and provides several novel effective targets for NPC treatment.

MATERIALS AND METHODS NPC tissue collection and cell line culture

A total of 46 pairs of NPC and adjacent normal tissues were obtained from NPC patients during surgery from The Second Xiangya Hospital, Central South University. All tumor samples were stored at −80°C before use. Research protocols were approved by the Institutional Ethical Review Board of The Second Xiangya Hospital, Central South University, and written informed consent was supplied by each subject involved in this study. Four NPC cell lines, 5-8F, 6-10B, SUNE1, and C666, as well as a normal human nasopharyngeal epithelial cell line, NP69, were all supplied by the Cell Bank of the Chinese Academy of Sciences. NPC cell lines CNE-3 and HNE-3 were obtained from the Experimental Center of Scientific Research, People's Hospital of Guangxi Zhuang Autonomous Region. NPC cell lines were cultured in RPMI-1640 medium (Invitrogen) containing 10% FBS (Gibco), and the NP69 cell line was maintained in keratinocyte/serum-free medium (Invitrogen) containing bovine pituitary extract (BD Biosciences). All cells were maintained at 37°C in an incubator with 5% CO₂. For the MK-2206 (Merck & Co., Inc.) treatment, 6-10B cells were treated with 1 μM MK-2206 and SUNE1 cells were treated with 0.5 μM MK-2206.

Cell transfection

CircLASP1 overexpression vector (OE-circLASP1) was established by cloning the human circLASP1 fragment into the pLCDH-ciR vector (Geenseed Biotech). To knockdown circLASP1, siRNA targeting the back splice junction of circLASP1 was generated by Geenseed Biotech. The siRNA was subcloned into the lentivirus vector (pHBLV-U6-MCS-CMV-ZsGreen-PGK-Puro) to construct the sh-circLASP1 vector. The lentiviral vector was transfected into HEK293T cells with two assistant vectors. Stably infected NPC cells were then screened in the presence of polybrene (Sigma-Aldrich). miR-625 inhibitor, miR-625 mimics, and their negative controls including mimics NC and inhibitor NC were all obtained from GenePharma. The AKT overexpression vector (OE-AKT) and its negative control (OE-NC) were purchased from Addgene. Cell transfection was executed by Lipofectamine 3000 (Invitrogen) for 48 hours.

Sanger sequencing

CircLASP1 was amplified and cloned into a T-vector (Takara). Sanger sequencing (Sangon Biotech) was adopted to validate the head-to-tail spliced structure of circLASP1.

Dual-luciferase reporter assay

The wild type (WT) and mutant (MUT) miR-625 binding sequences of AKT or circLASP1 were, respectively, inserted into pmirGLO vector (Promega), referred to as AKT-WT and AKT-MUT or circLASP1-WT and circLASP1-MUT. To examine the interaction between miR-625 and AKT, 6-10B and SUNE1 cells were cotransfected with AKT-WT or AKT-MUT and miR-625 mimics or miR-625 inhibitor using Lipofectamine 3000 reagent. After 48 hours of cotransfection, the intensities of firefly and renilla luciferase were detected by the Dual-Luciferase Reporter Assay Kit (Promega). The interaction between circLASP1 and miR-625 was examined as described above.

RNA immunoprecipitation (RIP)

RNA immunoprecipitation was conducted using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). In brief, treated 6-10B and SUNE1 cells were lysed by RIP buffer, and then the cell lysate was collected and incubated with magnetic beads conjugated with Ago2 (Millipore) or IgG (Millipore). After purification, immunoprecipitated RNA was examined by qRT-PCR.

Fluorescence in situ hybridization (FISH)

The colocalization of circLASP1 and miR-625 in 6-10B and SUNE1 cells was examined using Cy3-labeled circLASP1 and FITC-labeled miR-625 probes (GenePharma). FISH analysis was carried out by a Fluorescent In Situ Hybridization Kit (Geenseed Biotech). In brief, cells mixed with sh-NC and sh-circLASP1 were fixed with 4% paraformaldehyde at room temperature for 30 minutes. Cells were then permeabilized with 0.1% Triton X-100 in PBS for approximately 10 minutes and incubated with circLASP1 and miR-625 RNA oligonucleotide probes in a hybridization buffer at 37°C overnight. DAPI was used to stain cell nuclei, and images were photographed using a fluorescence microscope (Olympus).

Immunofluorescence analysis

6-10B and SUNE1 cells were seeded on 24-well plates and fixed with 4% paraformaldehyde for 30 minutes, followed by 20 minutes incubation with 0.1% Triton X-100. After being blocked with 5% BSA in PBS, cells were incubated with anti-LAMP2 (1:1000, ab25631; Abcam) and anti-LC3 (1:2000, #12741; Cell Signaling Technology) antibodies at 4°C overnight. Cells were then incubated with secondary antibodies (1:2000, ab150077 and ab150115; Abcam) for 2 hours at room temperature. Finally, signals were viewed with a fluorescence microscope (Olympus).

Cell proliferation and apoptosis assessment

The proliferation of treated NPC cells was tested by Cell-Counting Kit-8 (CCK-8; Dojindo Laboratories). In brief, treated NPC cells (5 × 10³) were cultured in 96-well plates and incubated with CCK-8 solution for 2 hours. Afterward, the optical density of each well was measured at 490 nm by the FLUOstar OPTIMA microplate reader (BMG Labtech GmbH). The apoptosis of treated NPC cells was evaluated by flow cytometry analysis. After staining with the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences), cell apoptosis was examined using flow cytometry (Thermo Fisher Scientific).

Xenograft tumor assay

To evaluate the roles of circLASP1 in the therapeutic effects of MK-2206 on NPC, 6-10B and SUNE1 cells stably expressing sh-circLASP1 were inoculated into the left flank of BALB/c nude (nu/nu) mice (6-week-old, male). Animals were supplied by the SJA Laboratory Animal Company and randomized into four groups (n = 6 per group): the sh-NC group, the sh-circLASP1 group, the MK-2206 + sh-NC group, and the MK-2206 + sh-circLASP1 group. MK-2206 (240 mg/kg, three times per week) was administered by oral gavage from day 1 after inoculation until day 35. After 35 days of inoculation, tumors were harvested and their volume and weight were measured. Tumor volume calculation formula: (length × width²/2). All animal manipulations were approved by the Medical Ethics Committee of The Second Xiangya Hospital, Central South University.

For Ki-67 staining, tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and then sliced into 5-μm sections. After dewaxing, rehydration, and blocking the endogenous peroxidase activity, tumor sections were incubated overnight with primary antibody against Ki-67 (1:500, ab15580; Abcam) and then probed by secondary antibody biotinylated goat anti-rabbit immunoglobulin (Abcam) for 2 hours. Next, tumor sections were incubated with streptavidin-peroxidase and DAB solution (Solarbio) to visualize the expression.

Western blot

Proteins from NPC cells and tumor tissues were isolated using RIPA buffer (Beyotime); the concentration of protein samples was tested using a BCA Protein Assay Kit (Solarbio). Afterward, 50-μg protein samples were loaded and separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% BSA for 2 hours, the membranes were incubated overnight with indicated primary antibodies against LC3B (1:1000, #43566), p62 (1:1000, #39749), Beclin 1 (1:1500, #3495), uncleaved caspase-3 (1:2000, #14220), cleaved caspase-3 (1:2000, #9664), uncleaved PARP (1:1500, #9532), cleaved PARP (1:1500, #5625), Bax (1:1000, #2772), Bcl-2 (1:2000, #3498), p-AKT (1:1500, ab38449; Abcam), AKT (1:1000, ab8805, Abcam), p-mTOR (1:2000, ab109268; Abcam), mTOR (1:2000, ab32028, Abcam), p-p70S6K (1:1000, #97596), p70S6K (1:2000, #2708), p-ULK1 (1:1000, #5869), ULK1 (1:2000, #8054), and GAPDH (1:5000, ab8245; Abcam). After three washes with PBS, the membranes were probed by corresponding HRP-conjugated secondary antibody for 2 hours. Enhanced chemiluminescence reagent (EMD Millipore) was employed to visualize signal, and Image J software was used to analyze signal. Antibodies were all obtained from Cell Signaling Technology unless indicated otherwise.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNAs of NPC tissues and cell lines were extracted using TRIzol Reagent (Thermo Fisher Scientific). RNA samples (5 μg) were reversely transcribed into cDNA by using the TaqMan® microRNA reverse transcription kit (Thermo Fisher Scientific) or PrimeScript RT Kit with gDNA Eraser (Takara), and the cDNA was subjected to qPCR in the presence of specific primers using the TaqMan® microRNA assay kit (Thermo Fisher Scientific) or TaqMan® universal PCR mixture kit (Thermo Fisher Scientific). The PCR process was conducted on the ABI7500 Fast Real-Time PCR System (Applied Biosystems). The relative expression levels were quantified using the 2^−△△Ct method and normalized to GPDAH or U6. Sequences of primers used in this study were purchased from Sangon Biotech.

Statistical analysis

All data were analyzed by GraphPad Prism 6.0 and presented as the mean ± standard deviation (SD). All experiments were performed in at least three biological replicates, and each biological replicate contained three technical replicates. Statistical differences were performed using Student's t test (two tailed) between two groups or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparison. p < 0.05 was considered statistically significant.

RESULTS miR-625 exhibited a low expression and was involved in the regulation of autophagy in NPC

Compared with normal tissues, miR-625 level was dramatically decreased in NPC tissues (Figure 1A). Moreover, miR-625 expression was found decreased in metastatic NPC tissues (n = 20) compared with nonmetastatic tissues (n = 26) (Figure 1B). Consistently, we found a remarkable downregulation of the miR-625 level in NPC cell lines including CNE-3, HNE-3, 5-8F, 6-10B, SUNE1, and C666 (Figure 1C). Then, transfection of 6-10B and SUNE1 cells with miR-625 mimics caused an upregulation of miR-625, and miR-625 inhibitor caused a downregulation of miR-625 (Figure 1D). Overexpression of miR-625 resulted in a dramatic upregulation of immunofluorescent LC3 puncta, while knockdown of miR-625 exhibited opposite effects (Figure 1E). Moreover, miR-625 overexpression triggered colocalization of LC3 puncta and LAMP2, which indicated the formation of autolysosomes, while miR-625 silencing exhibited opposite effects (Figure 1F). Additionally, miR-625 overexpression resulted in an upregulation of the LC3 II to LC3 I ratio and Beclin 1 expression and a downregulation of p62 in both 6-10B and SUNE1 cells, while miR-625 silence exhibited opposite effects (Figure 1G). These results suggested that miR-625 was decreased in NPC and miR-625 overexpression induced autophagy in NPC cells.

FIGURE 1. miR-625 exhibited a low expression and was involved in the regulation of autophagy in nasopharyngeal cancer (NPC). qRT-PCR analysis of miR-625 relative expression in (A) NPC and adjacent normal tissues (n = 46), in (B) metastatic (n = 26) and nonmetastatic (n = 20) NPC tissues, and in (C) NPC cell lines CNE-3, HNE-3, 5-8F, 6-10B, SUNE1, and C666 (NP69 was adopted as normal control cell line). (D) The overexpression and knockdown efficiency of miR-625 mimics and inhibitor in 6-10B and SUNE1 cells were measured via qRT-PCR. 6-10B and SUNE1 cells were transfected with miR-625 mimics or inhibitor and subjected to immunofluorescence microscopy for (E) LC3 or for (F) LC3 and LAMP2 colocalization. (G) Treated 6-10B and SUNE1 cells were subjected to Western blot detection of LC3 (LC3 II/LC3 I), p62, and Beclin 1. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 repressed NPC cell proliferation and facilitated cell apoptosis depending on the caspase-3 pathway

Next, we estimated the roles of miR-625 in NPC cell growth in vitro by a series of assays. The results indicated that miR-625 overexpression caused a remarkable reduction of proliferation in both 6-10B and SUNE1 cells, while miR-625 silencing enhanced their proliferation (Figure 2A). An increased apoptosis rate was observed in miR-625–overexpressed 6-10B and SUNE1 cells, while a reduced apoptosis rate was observed in miR-625–silenced ones (Figure 2B). In addition, in 6-10B and SUNE1 cells, miR-625 overexpression elevated the expression levels of cleaved caspase-3, cleaved PARP, and Bax and reduced the expression levels of uncleaved caspase-3, uncleaved PARP, and Bcl-2, while miR-625 knockdown exhibited opposite effects (Figure 2C). These findings implied that miR-625 served as a tumor repressor of NPC.

FIGURE 2. miR-625 repressed nasopharyngeal cancer (NPC) cell proliferation and facilitated cell apoptosis. After 48 h of transfection with miR-625 mimics or miR-625 inhibitor, 6-10B and SUNE1 cells were subjected to (A) proliferation analysis using CCK-8 assay, (B) apoptosis analysis using flow cytometry, and (C) apoptosis-related protein (cleaved caspase-3, uncleaved caspase-3, cleaved PARP, uncleaved PARP, Bax, and Bcl-2) expression analysis using Western blot. Mimics NC and inhibitor NC were used as negative controls. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 interacted with AKT

As AKT activation has been found during NPC pathogenesis and recent publications have found an interaction between miRNAs and AKT in various cancers,^17,18 we were wondering if miR-625 could interact with AKT in NPC. Starbase database analysis showed that the AKT 3′–UTR region contains miR-625 binding sites (Figure 3A). miR-625 overexpression caused a significant AKT downregulation, while miR-625 knockdown caused a significant AKT upregulation (Figure 3B,C). Transfection of miR-625 mimics inhibited relative luciferase activity of 6-10B and SUNE1 cells driven by AKT-WT, while transfection of miR-625 inhibitor increased the relative luciferase activity (Figure 3D). Transfection of miR-625 mimics and inhibitor had no effects on the luciferase activity of 6-10B and SUNE1 cells driven by AKT-MUT (Figure 3D). We also examined the expression of AKT in NPC tissues and found that AKT was dramatically increased in NPC tissues compared with normal ones (Figure 3E). Moreover, AKT level was negatively correlated with the miR-625 level in NPC tissues (Figure 3F). These results indicated that miR-625 directly targeted and interacted with AKT.

FIGURE 3. miR-625 interacted with AKT. (A) Starbase analysis predicting AKT as a target of miR-625. B, Western blot and (C) qRT-PCR analyses of the expression of AKT protein and mRNA in 6-10B and SUNE1 cells transfected with miR-625 mimics or miR-625 inhibitor. D, Interplay between miR-625 and AKT was validated by dual-luciferase reporter assay. E, qRT-PCR showing the AKT mRNA expression in 46 pairs of nasopharyngeal cancer (NPC) and adjacent normal tissues. F, Pearson's analysis showing the correlation between the expression of miR-625 and AKT in NPC tissues. The sample correlation coefficient (R) is a measure of the closeness of association of the points in a scatter plot to a linear regression line based on those points. The coefficient of determination (R2) is a number between 0 and 1 that measures how well a statistical model predicts an outcome. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 facilitated NPC cell autophagy by directly targeting and inhibiting AKT

Additionally, AKT overexpression had no effect on the miR-625 level in miR-625–overexpressed 6-10B and SUNE1 cells but reversed the downregulation of AKT induced by miR-625 (Figure 4A). AKT overexpression abolished the repressive effects of miR-625 overexpression on p-AKT, AKT, p-mTOR, p-p70S6K, and p-ULK1 in 6-10B and SUNE1 cells (Figure 4B). Thus, miR-625 could inhibit the AKT/mTOR pathway by directly targeting AKT in NPC. Results from Western blot and immunofluorescence assays showed that AKT overexpression abrogated the promotive influences of miR-625 on autophagy (Figure 4C,D). Taken together, miR-625 promoted NPC cell autophagy via the repression of the AKT/mTOR pathway by targeting and repressing AKT.

FIGURE 4. miR-625 facilitated nasopharyngeal cancer (NPC) cell autophagy by directly targeting and inhibiting AKT. After 48 h of transfection with miR-625 mimics plus OE-AKT, 6-10B and SUNE1 cells were subjected to qRT-PCR analysis of (A) miR-625 and AKT. The miR-625 mimics– and OE-AKT– transfected 6-10B and SUNE1 cells were also subjected to Western blot analysis of (B) p-AKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, p-ULK1, and ULK1 and (C) LC3 II/LC3 I, p62, and Beclin 1, and to (D) immunofluorescence microscopy of LC3. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 repressed NPC cell proliferation and facilitated cell apoptosis by directly targeting and inhibiting AKT

Next, we examined whether the regulatory influences of miR-625 on NPC cells are mediated by AKT. AKT overexpression reversed the repressive impacts of miR-625 on the proliferation of 6-10B and SUNE1 cells (Figure 5A). The apoptosis of 6-10B and SUNE1 cells induced by miR-625 overexpression was abolished by the transfection of OE-AKT, as demonstrated by flow cytometry analysis (Figure 5B) and Western blot analysis of apoptosis-related proteins (Figure 5C). These results indicated that miR-625 repressed NPC cell proliferation and facilitated NPC cell apoptosis by directly targeting and inhibiting AKT.

FIGURE 5. miR-625 repressed nasopharyngeal cancer (NPC) cell proliferation and facilitated cell apoptosis by directly targeting and inhibiting AKT. After 48 h of transfection with miR-625 mimics and OE-AKT, 6-10B and SUNE1 cells were subjected to (A) proliferation analysis using CCK-8 assay, (B) apoptosis analysis using flow cytometry, and (C) apoptosis-related protein (cleaved caspase-3, uncleaved caspase-3, cleaved PARP, uncleaved PARP, Bax, and Bcl-2) expression analysis using Western blot. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 enhanced the effects of MK-2206 on autophagy through suppressing the AKT/mTOR pathway in NPC cells

Based on the profound effects of miR-625 on autophagy, we evaluated the influences of miR-625 on a most common inhibitor, MK-2206, targeting AKT in NPC. miR-625 was only increased in the miR-625 mimics and MK-2206 combined treatment group; treatment with MK-2206 alone did not affect the miR-625 expression of 6-10B and SUNE1 cells (Figure 6A). MK-2206 treatment repressed p-AKT, p-mTOR, p-p70S6K, and p-ULK1 in 6-10B and SUNE1 cells, and the combination therapy of miR-625 and MK-2206 enhanced the inhibitory effects of MK-2206 (Figure 6B). Moreover, the combination therapy of miR-625 and MK-2206 was found to enhance the promotive effects of MK-2206 on the autophagy of 6-10B and SUNE1 cells, as shown by the analysis of LC3 and p62 using Western blot or/and immunofluorescence analyses (Figure 6C,D). In addition, MK-2206 treatment triggered colocalization of LC3 puncta and LAMP2, and the combination therapy of miR-625 and MK-2206 enhanced this phenomenon (Figure 6E). These results indicated that miR-625 enhanced the effects of MK-2206 on promoting autophagy via suppressing the AKT/mTOR pathway in NPC cells.

FIGURE 6. miR-625 enhanced the effects of MK-2206 on the AKT/mTOR pathway and autophagy in nasopharyngeal cancer (NPC) cells. (A) qRT-PCR analysis showing the expression of miR-625 in 6-10B and SUNE1 cells treated with MK-2206 alone or in combination with miR-625 mimics. Protein expression of (B) p-AKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, p-ULK1, and ULK1 and (C) LC3 II/LC3 I and p62 were detected with Western blot in 6-10B and SUNE1 cells treated with MK-2206 alone or in combination with miR-625 mimics. 6-10B and SUNE1 cells were treated with MK-2206 alone or in combination with miR-625 mimics and subjected to immunofluorescence microscopy for (D) LC3 or for (E) LC3 and LAMP2 colocalization. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

miR-625 enhanced the effects of MK-2206 on NPC proliferation and apoptosis

Treatment with MK-2206 alone could repress the proliferation of 6-10B and SUNE1 cells, and the combination therapy of miR-625 and MK-2206 enhanced the repressive effects of MK-2206 on cell proliferation (Figure 7A). MK-2206 treatment–induced apoptosis of 6-10B and SUNE1 cells was increased by the combination therapy of miR-625 and MK-2206, as demonstrated by flow cytometry (Figure 7B) and Western blot analysis (Figure 7C). These findings indicated that miR-625 enhanced the effects of MK-2206 on NPC proliferation and apoptosis.

FIGURE 7. miR-625 enhanced the effects of MK-2206 on nasopharyngeal cancer (NPC) proliferation and apoptosis. After 48 h of treatment with MK-2206 alone or in combination with miR-625 mimics, 6-10B and SUNE1 cells were subjected to (A) proliferation analysis using CCK-8 assay, (B) apoptosis analysis using flow cytometry, and (C) apoptosis-related protein (cleaved caspase-3, uncleaved PARP, Bax, and Bcl-2) expression analysis using Western blot. *p [less than] 0.05 and **p [less than] 0.01

CircLASP1 was increased in NPC and acted as a sponge of miR-625

Although LASP1 was reported to be involved in NPC tumorigenesis,¹⁰ whether its circular transcript circLASP1 plays a role in NPC remains undetermined. To address this question, Starbase database analysis revealed miR-625 binding sites in circLASP1 (Figure 8A). Compared with normal tissues or the NP69 cell line, circLASP1 was dramatically increased in tumor tissues or NPC cell lines (Figure 8B,C). CircLASP1 (hsa_circ_0043387) is derived from exon 5 and 6 of the LASP1 gene, and the junction site was verified by Sanger sequencing (Figure 8D). We also demonstrated that circLASP1 was more stable than its linear mRNA in 6-10B and SUNE1 cells after treatment with RNase R (Figure 8E) and actinomycin D (Figure 8F). Moreover, a negative correlation was observed between the expression of circLASP1 and miR-625 in NPC tissues (Figure 8G). The overexpression and knockdown efficiency of circLASP1 were confirmed by qRT-PCR (Figure 8H); moreover, LASP1 mRNA expression was not affected by circLASP1 overexpression and knockdown (Figure 8H). Overexpression or knockdown of circLASP1 was found to decrease or increase the miR-625 level in both 6-10B and SUNE1 cells (Figure 8I), indicating that circLASP1 could negatively regulate the expression of miR-625. Transfection with miR-625 mimics or inhibitor repressed or enhanced, respectively, the luciferase activity of 6-10B and SUNE1 cells driven by circLASP1-WT (Figure 8J). Moreover, RIP assay indicated that circLASP1 could be effectively pulled down by the anti-Ago2 antibody in the presence of miR-625 mimics (Figure 8K). Additionally, we mixed the cells of sh-NC and sh-circLASP1 and then performed FISH assay to detect the localization of circLASP1 and miR-625. The results indicated that circLASP1 and miR-625 exhibited colocalization in cytoplasm (Figure 8L). These findings suggested that circLASP1 was increased in NPC and negatively regulated the expression of miR-625.

FIGURE 8. CircLASP1 was increased in nasopharyngeal cancer (NPC) and acted as a sponge of miR-625. A, Starbase analysis predicting miR-625 as a target of circLASP1. B, qRT-PCR was performed to show the circLASP1 relative expression in (B) NPC and adjacent normal tissues (n = 46), as well as in (C) NPC cell lines CNE-3, HNE-3, 5-8F, 6-10B, SUNE1, and C666 (NP69 was adopted as normal control). D, Schematic illustration of formation of circLASP1 (hsa_circ_0043387). The junction site was verified by Sanger sequencing. E, F, Relative expression of circLASP1 and its linear mRNA were detected after treatment with RNase R (E) and actinomycin D (F). G, Pearson's analysis showing the correlation between the expression of miR-625 and circLASP1 in NPC tissues. The sample correlation coefficient (R) is a measure of the closeness of association of the points in a scatter plot to a linear regression line based on those points. The coefficient of determination (R2) is a number between 0 and 1 that measures how well a statistical model predicts an outcome. After 48 h of transfection with OE-circLASP1 or sh-circLASP1, the relative expression levels of (H) circLASP1 and LASP1 and (I) miR-625 in 6-10B and SUNE1 cells were examined with qRT-PCR. Interplay between miR-625 and circLASP1 in 6-10B and SUNE1 cells was validated by (J) dual-luciferase reporter assay, (K) RIP assay, and (L) FISH assay. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

CircLASP1 regulated NPC cell autophagy, proliferation, and apoptosis through directly targeting miR-625

As miR-625 was demonstrated to be a downstream agent of circLASP1, we then investigated whether circLASP1 is involved in the influence of miR-625 on NPC cells. By transfecting 6-10B and SUNE1 cells with sh-circLASP1, we found that miR-625 level was markedly increased, while cotransfection with sh-circLASP1 and miR-625 inhibitor abolished the upregulation of miR-625 (Figure 9A). Knockdown of circLASP1 repressed p-AKT, AKT, p-mTOR, p-p70S6K, and p-ULK1 of 6-10B and SUNE1 cells, while miR-625 inhibition abrogated the repressive effects of circLASP1 knockdown on the AKT/mTOR pathway (Figure 9B). By analyzing the expression of LC3, Beclin 1, and p62 using Western blot and immunofluorescence, we found that cotransfection with sh-circLASP1 and miR-625 inhibitor abolished the promotive influences of circLASP1 knockdown on the autophagy of 6-10B and SUNE1 cells (Figure 9C,D). The inhibitory effects of circLASP1 knockdown on proliferation (Figure 10A), as well as the promotive effects of circLASP1 knockdown on apoptosis (Figure 10B,C), were all reversed by cotransfection with sh-circLASP1 and miR-625 inhibitor. Additionally, Western blot was performed to test the effects of circLASP1 overexpression on AKT/mTOR signaling and autophagy. Results indicated that circLASP1 overexpression significantly increased the expression levels of p-AKT, AKT p-mTOR, p-p70S6K, and p-ULK1 in 6-10B and SUNE1 cells (Figure S1A). CircLASP1 overexpression was also found to increase the expression of p62 while decrease the ratio of LC3 II/LC3 I in 6-10B and SUNE1 cells (Figure S1B). These results implied that circLASP1 overexpression could repress autophagy by activating the AKT/mTOR signaling pathway. Flow cytometry analysis and Western blot assay were performed in circLASP1-overexpressed 6-10B and SUNE1 cells. Results from flow cytometry indicated that circLASP1 overexpression slightly repressed the apoptosis of 6-10B and SUNE1 cells (Figure S2A). Moreover, expression levels of cleaved caspase-3 and Bax were found to be decreased while uncleaved PARP and Blc-2 were found to be increased in 6-10B and SUNE1 cells after circLASP1 overexpression (Figure S2B). These results indicated that circLASP1 overexpression inhibited apoptosis in 6-10B and SUNE1 cells. These results indicated that circLASP1 is involved in regulating NPC cell autophagy, proliferation, and apoptosis by the miR-625/AKT axis.

FIGURE 9. CircLASP1 regulated nasopharyngeal cancer (NPC) cell autophagy through the miR-625/AKT axis. A, qRT-PCR was performed to show the relative expression of miR-625 in 6-10B and SUNE1 cells transfected with sh-circLASP1 alone or in combination with miR-625 inhibitor. B, C, Protein expressions of p-AKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, p-ULK1, ULK1, LC3 II/LC3 I, Beclin 1, and p62 were examined by Western blot in 6-10B and SUNE1 cells transfected with sh-circLASP1 and miR-625 inhibitor. D, After transfection with sh-circLASP1 and miR-625 inhibitor, 6-10B and SUNE1 cells were subjected for immunofluorescence microscopy for LC3. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

FIGURE 10. CircLASP1 regulated nasopharyngeal cancer (NPC) cell proliferation and apoptosis through the miR-625/AKT axis. After 48 h of transfection with sh-circLASP1 alone or in combination with miR-625 inhibitor, 6-10B and SUNE1 cells were subjected to (A) CCK-8 and (B) flow cytometry assays. C, Western blot analysis of apoptosis-related proteins (cleaved caspase-3, uncleaved PARP, Bax, and Bcl-2) in 6-10B and SUNE1 cells transfected with sh-circLASP1 and miR-625 inhibitor. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

Knockdown of circLASP1 enhanced the therapeutic effects of MK-2206 on NPC in vivo

We next investigated whether circLASP1 is involved in the therapeutic effects of MK-2206 on NPC in vivo. After 35 days of inoculation, tumors were harvested and their volumes and weights were measured (Figure 11A). Results indicated that sh-circLASP1 treatment alone was able to reduce tumor volume and weight, and the combination treatment of sh-circLASP1 and MK-2206 caused a greater inhibition on tumor volume and weight (Figure 11B,C). Immunohistochemical results indicated that the expression of Ki-67 in tumors of mice treated with either sh-circLASP1 or MK-2206 alone was decreased, and the combination treatment of sh-circLASP1 and MK-2206 led to a greater inhibition of the Ki-67 expression level (Figure 11D). Treatment with MK-2206 alone did not affect the levels of circLASP1 and miR-625, while sh-circLASP1 treatment alone or in combination with MK-2206 was found to reduce circLASP1 level and increase miR-625 level in tumors derived from 6-10B and SUNCE1 cells (Figure 11E,F). Additionally, MK-2206 treatment repressed the AKT pathway while promoted autophagy in the tumors derived from 6-10B and SUNE1 cells, and the combination therapy of sh-circLASP1 and MK-2206 enhanced the impacts of MK-2206 on the AKT pathway and autophagy (Figure 11G). These findings suggested that knockdown of circLASP1 enhanced the therapeutic effects of MK-2206 on NPC in vivo.

FIGURE 11. Knockdown of circLASP1 enhanced the therapeutic effects of MK-2206 on nasopharyngeal cancer (NPC) in vivo. A, Representative images showing the tumors derived from 6-10B and SUNE1 cells with different treatments: sh-NC, sh-circLASP1, sh-NC + MK-2206, and sh-circLASP1 + MK-2206. B, C, Volume and weight of tumors from different groups. D, Proliferative activity evaluated by Ki-67 staining in the tumor slices from different groups. qRT-PCR was performed to examine the relative expression of (E) circLASP1 and (F) miR-625 of tumors derived from treated 6-10B and SUNE1 cells. G, Western blot analysis of p-AKT, AKT, LC3 II/LC3 I, Beclin 1, and p62 of tumors derived from treated 6-10B and SUNE1 cells. *p [less than] 0.05, **p [less than] 0.01, and ***p [less than] 0.001

DISCUSSION

Overwhelming evidence has pointed out the importance of circRNAs in the initiation and progression of NPC.^19,20 Thus, a thorough understanding of the biological roles of NPC-specific circRNA in its malignant characteristics is critical for screening potential targets for NPC therapy. Herein, we revealed that circLASP1 was dramatically elevated and negatively correlated with miR-625 expression in NPC. In functional analyses, we found that circLASP1 was involved in the regulation of NPC cell autophagy, proliferation, and apoptosis in vitro. In mechanism analyses, we demonstrated that circLASP1 indirectly released AKT by directly binding to miR-625 in NPC cells. In addition, we demonstrated that the combination therapy of circLASP1 silence and MK-2206 showed stronger antitumor effects on NPC than treatment with MK-2206 alone.

Only one study showed that miR-625 could inhibit NPC cell growth and metastasis.¹² Consistently, miR-625 overexpression was revealed to repress NPC cell proliferation and promote NPC cell apoptosis in this study. Besides, accumulating evidence proved that miRNAs functioned in NPC by regulating autophagy. For example, miR-185 was proved to repress NPC cell proliferation while promote cell apoptosis and autophagy by inhibiting the TGF-β1/mTOR pathway.²¹ Recently, miR-106a-5p was reported to facilitate NPC malignant phenotype by repressing autophagy.²² Our study was the first to show the promotive roles of miR-625 in NPC cell autophagy, implying that the repressive impacts of miR-625 on NPC cells may be mediated by autophagy regulation.

It is well known that the modulatory ability of cancer progression of miRNAs is dependent on its regulatory effects on corresponding tumor-related mRNAs.²³ miRNAs affected the expression of target genes in two ways: leading to mRNA degradation directly and preventing translation. In recent years, the AKT signaling pathway has been found to be targeted and modulated by numerous miRNAs, thus affecting the progression of NPC.^18,24 Similarly, for the first time, we showed that miR-625 binds to the 3′UTR of the AKT gene for suppressing gene expression via degrading AKT mRNA and inhibiting AKT protein synthesis in NPC. Moreover, functional analyses suggested that the regulatory impacts of miR-625 on NPC cell autophagy, proliferation, and apoptosis were mediated by AKT pathway inhibition.

As a common AKT inhibitor, MK-2206 has been strongly supported by a body of evidence to show repressive effects on NPC progression.^5,25 A previous study suggested that MK-2206 could inhibit NPC cell proliferation by reducing the expression of cyclin D1 (a cyclin required for the transition from G1 to S phase), yet it rarely caused NPC cell apoptosis.⁵ Thus, strategies for enhancing the cytotoxicity of MK-2206 are essential to make it more valuable. As miRNAs are involved in the regulation of various tumor-related signaling pathways, including the AKT pathway, the combination of miRNA and antitumor agents has been considered to be a promising way to increase the efficacy of cancer treatment.²⁶ In view of the repressive effects of miR-625 on the AKT pathway, we tested the effects of combination therapy of miR-625 and MK-2206 on NPC. Our study was the first to show that miR-625 dramatically enhanced the repressive effects of MK-2206 on NPC progression by activating autophagy and apoptosis.

Numerous miRNAs have been found to twist with circRNAs to modulate the biological activities of NPC cells.^27,28 Likewise, we also found that circLASP1 targeting miR-625 influenced NPC progression. The upregulation of the parental gene of circLASP1, LASP1, has been observed in multiple malignant tumors, indicating its oncological and clinical significance.^10,29,30 Nevertheless, the role of circLASP1 in cancer progression has not been investigated yet. Sanger sequencing results indicated circLASP1 is derived from exon 5 and 6 of the LASP1 gene. Our study, for the first time, also revealed that circLASP1 regulated NPC cell proliferation, apoptosis, and autophagy through targeting the miR-625/AKT axis. Moreover, circLASP1 knockdown was also found to enhance the therapeutic effects of MK-2206 on NPC progression.

In summary, our data suggested that the circLASP1/miR-625 axis regulated NPC progression by modulating proliferation, apoptosis, and autophagy through the AKT/mTOR pathway, and circLASP1 and miR-625 could be two promising targets for enhancing the therapeutic effects of MK-2206 on NPC. Our findings not only contribute to understanding the molecular mechanism of NPC but also provide a new effective way to enhance the efficacy of MK-2206 on NPC.

ACKNOWLEDGMENTS

We would like to give our sincere gratitude to the reviewers for their constructive comments.

FUNDING INFORMATION

None.

DISCLOSURE

The authors have no commercial or other associations that might pose a conflict of interests.

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ETHICS STATEMENT

Research protocols were approved by the Institutional Ethical Review Board of The Second Xiangya Hospital, Central South University.

INFORMED CONSENT

Informed consent was obtained from study participants. All animal manipulations were approved by the Medical Ethics Committee of The Second Xiangya Hospital, Central South University.