Silencing of miR-17-5p suppresses cell proliferation and promotes cell apoptosis by directly targeting PIK3R1 in laryngeal squamous cell carcinoma
Increasing evidence has suggested that microRNAs (miRNAs) act as key post-transcriptional regulators in tumor progression. Previous studies have confirmed that miR-17-5p functions as an oncogene in multiple cancers and contributes to tumor progression. However, the role and biological functions of miR-17-5p in the development of laryngeal squamous cell carcinoma (LSCC) still remain unknown.
qRT-PCR was used to detect miRNA and mRNA expression levels in LSCC tissues and cell lines. CCK-8 assay was used to measure cell viability and flow cytometry was performed to evaluate cell apoptosis. Western blot analysis was used to detect the protein levels of BAX, BCL-2, cleaved Caspase-3, PIK3R1 and AKT. Luciferase reporter assay was used to detect the effect of miR-17-5p on PIK3R1 expression. Xenograft animal model was used to test the effect of miR-17-5p on LSCC cell in vivo.
In the present study, we found that miR-17-5p expression level was upregulated in LSCC tissues and cell lines. Depletion of miR-17-5p in LSCC cells significantly reduced cell proliferation and promoted cell apoptosis in vitro and in vivo. Mechanically, knockdown of miR-17-5p in LSCC cells inhibited BCL-2 expression while enhanced BAX and cleaved Caspase-3 protein expression. Moreover, depletion of miR-17-5p in LSCC cells suppressed AKT phosphorylation but did not influence PTEN expression. Importantly, miR-17-5p positively regulated PIK3R1 expression by directly binding to its 3′-untranslated region (UTR). Additionally, PIK3R1, which expression was downregulated in LSCC tissues and cell lines, was involved in LSCC cell survival by modulating the activation of AKT signal pathway. Dysregulation of miR-17-5p/PIK3R1 axis was participated in LSCC cell proliferation and apoptosis by inhibiting the activation of the PI3K/AKT signaling pathway.
In conclusion, our study indicates that the miR-17-5p/PIK3R1 axis plays an essential role in the development of LSCC and provides a potential therapeutic target for LSCC treatment.
KeywordsLaryngeal squamous cell carcinoma miR-17-5p PIK3R1 Proliferation Apoptosis
laryngeal squamous cell carcinoma
phosphoinositide-3-kinase, regulatory subunit 1
tension homology deleted on chromosome 10
reverse transcription-quantitative polymerase chain reaction
Laryngeal squamous cell carcinoma (LSCC) is the most common head and neck malignancy accounting for more than 95% of head and neck squamous cell carcinoma (HNSCC), with 177,422 new cases and 94,771 deaths worldwide in 2018 [1, 2]. Most of LSCC patients who are diagnosed at early stage may benefit from surgery, followed by radiotherapy and/or chemotherapy [3, 4]. However, the 5-year overall survival rate of patients with LSCC who are asymptomatic in the advanced stage remains lower than approximately 50% . Therefore, an understanding of the driven-element and molecular mechanisms of tumorigenesis in LSCC is crucial.
microRNAs (miRNAs) are the most important post-transcriptional regulators which suppress the expression of protein-coding genes by directly targeting mRNA at the 3′-untranslated region (UTR) for translational repression or degradation [6, 7]. Accumulating studies have shown that miRNAs are implicated in LSCC development, including proliferation, apoptosis, migration and invasion [8, 9, 10]. Our previous study has confirmed that miR-486 is involved in LSCC cell migration by targeting FLNA . Moreover, miR-370, which functions as a tumor suppressor, participates in LSCC cell growth by inducing FOXM1 expression . Overexpression of miR-613 reduces LSCC cell proliferation, invasion, and blocks G1/S phase transition by targeting the PDK1 gene . Furthermore, miR-1297, miR-143-3p, miR-503 and miR-205 also promote LSCC cell progression [14, 15, 16, 17]. Recent studies have confirmed that miR-17-5p plays critical roles in tumor progression, such as pancreatic cancer, breast cancer, hepatocellular carcinoma, gastric cancer and prostate cancer [18, 19, 20, 21, 22]. However, the expression and biological functions of miR-17-5p in LSCC remain unclear.
Increasing evidence has revealed that abnormal activation of PI3K/AKT pathway is associated with the generation of multiple tumors, including LSCC, via regulating cell survival, apoptosis, proliferation, migration, invasion and vesicle trafficking [23, 24, 25]. PIK3R1, which encodes the p85α protein, is best known as the regulatory subunit of class 1A PI3Ks through its interaction, stabilization and repression of PI3K-p110 catalytic subunits . PIK3R1 has been identified to be differentially expressed in many human cancers. For example, PIK3R1 functions as a tumor suppressor in hepatocellular carcinomas and renal cancer [27, 28], whereas acts as an oncogene in ovarian and colon tumors and plays a role in tumor progression and metastasis [29, 30]. However, the relationship between PIK3R1 and LSCC cell development has not been fully elucidated.
In the present study, we observed an increased level of miR-17-5p in LSCC tissues and cell lines. Knockdown of miR-17-5p reduced LSCC cell proliferation and induced apoptosis in vitro and in vivo by suppressing the activation of the PI3K/AKT pathway. Importantly, we demonstrated miR-17-5p positively regulated PIK3R1 mRNA and protein expression by targeting its 3′UTR. In addition, PIK3R1 may function as tumor suppressor in LSCC by promoting cell growth. Taken together, our findings indicate that the miR-17-5p/PIK3R1/AKT pathway plays a key role in LSCC proliferation and apoptosis, providing a potential therapeutic target for LSCC treatment.
Patients and samples
39 LSCC samples and non-cancerous adjacent normal tissues were obtained from the Department of Otolaryngology, Second Hospital of Hebei Medical University between September 2017 and July 2018. None of the LSCC patients were treated with radiotherapy or chemotherapy before surgery. All tissues were immediately frozen in liquid nitrogen after surgery and then later stored at − 80 °C for following use. This study was approved by Ethics Committee of Second Hospital of Hebei Medical University. The written informed consent was obtained from every patient. All the experiments in this paper obey World Medical Association Declaration of Helsinki.
Cell culture and transfection
The human LSCC cell lines (Hep2, SCC-2 and SCC-40) and the normal human oral keratinocyte cell line (HOK) were maintained in our lab. All cells were cultured in RPMI-1640 (Gibco, Beijing, China) with 10% fetal bovine serum (FBS) (Clark Bio, Claymont, DE), 100 units/ml penicillin and 100 μg/ml streptomycin and were incubated at 37 °C in a humidified incubator with 5% CO2. The miR-17-5p mimic, mimic-negative control (NC), miR-17-5p inhibitor and inhibitor-negative control (NC) were purchased from GenePharma Co., Ltd (Shanghai, China). Overexpression plasmid of PIK3R1 (pcDNA3.1-PIK3R1) and luciferase reporter vector were purchased from GENEWIZ Company (Suzhou, China). Cell transfections were carried out by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.
Cell proliferation assay
Cell proliferation assay was detected by using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratory, Kyushu, Japan) according to the manufacturer’s protocol. Briefly, LSCC cells were seeded in 96-well plates and then transfected with miR-17-5p inhibitor or pcDNA3.1-PIK3R1 respectively or co-transfected with them both for 72 h. At the indicated times, 10 μl CCK-8 solution was added into each well for another 3 h. Finally, the absorbance was measured at 450 nm by using a microplate reader (Thermo Fisher USA).
Cell apoptosis analysis
Cell apoptosis was detected by using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, USA) following the manufacturer’s instructions. Briefly, after transfection, cells were grown in 6-well plates for 48 h. Then cells were harvested and washed with ice-cold PBS thrice. Then, the cells were stained with Annexin V-fluorescein isothiocyanate and PI. The data analysis was performed using BD FACS Diva software (BD, USA).
Xenograft animal model
All animal studies were approved by the Institutional Animal Care Committee of Hebei Medical University. 6 weeks’ old BALB/C nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). 1 × 107 LV-anti-miR-17-5p or LV-anti-miR-NC-infected Hep2 cells were resuspended in 100 μl PBS mixed with 100 μl Matrigel (356234. BD, MA, USA); The suspension of cells was injected subcutaneously into the left dorsal flanks. The volume of xenograft was measured every 3 days. At the end of the experiment, the mice were euthanized by carbon dioxide asphyxiation. The tumor tissues were stored in liquid nitrogen immediately and stored at − 80 °C for western blot or qRT-PCR analysis.
RNA extraction and quantitative real-time PCR
Total RNA from tissues and cultured cells were extracted by using QIAzol Lysis Reagent (79306) according to the manufacturer’s protocol. For microRNA analysis, the miScripIIRT kit (QIAGEN GmbH, D-40724 Hilden, Germany) was used for reverse transcription, and the miScript SYBR® Green PCR kit was used for qRT-PCR with specific primers for microRNAs. RNU6b (U6) was used as internal control. For mRNA analysis, total cellular RNA was reverse-transcribed to first strand cDNA by using M-MLV First Strand Kit (Life Technologies). And Platinum SYBR Green qPCR Super Mix UDG Kit (Invitrogen) was used for the qRT-PCR of mRNAs. The mRNA expression was normalized to β-actin.
Luciferase assay was performed as previously described . Briefly, Hep2 cells were seeded into a 24-well plate, miR-17-5p mimic (or mimic-NC) was co-transfected with PIK3R1 reporter construct (wild-type or mutant) or the empty vector using Lipofectamine™ 2000 reagent for 48 h. Then the transfected cells were harvested in lysis buffer and detected by Dual-Glo Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s protocols. Firefly luciferase (FLuc) activity was measured and normalized against the Renilla luciferase (RLuc) activity.
Western blot analysis
The tissues and cultured cells were lysed with RIPA buffer. The protein was collected for western blot analysis. Equal amounts of protein were run on 10% SDS-PAGE, and electro-transferred to a polyvinylidene fluoride (PVDF) membranes (Millipore). The specific primary antibodies as follows: anti-PIK3R1 (1:1000, abcam, ERP18702), anti-PTEN (1:1000, abcam ab32199), anti-caspase 3 (1:1000, abcam, ab13847), anti-pan-AKT (1:1000, abcam, ab8805), anti-p-AKT (phospho T308) (1:1000, abcam, ab38449), anti-BCL-2 (1:1000, Proteintech, 12,789-1-AP), anti-BAX (1:1000, Proteintech, 50,599-2-Ig) or anti-β-actin (1:1000, Proteintech, 60,008-1-Ig). The memberanes were visualized with Immobilon ECL (Millipore). FusionCapt Advance Fx5 software (Vilber Lourmat) was used to capture the images.
Potential target genes of miR-17-5p were identified with following miRNA target prediction algorithms: Targetscan (http://www.targetcan.org/mmu_71/).
All of the data were represented as the mean ± S.E.M. Independent Student’s t-test was used for comparisons of differences between two groups. Results were considered statistically significant at P < 0.05. Graphpad Prism 7.0 software was using to perform the statistical analysis (GraphPad Software, San Diego, CA, USA).
miR-17-5p is upregulated in LSCC tissues and cell lines
Depletion of miR-17-5p inhibits cell proliferation and promotes cell apoptosis in LSCC in vitro
Depletion of miR-17-5p suppresses AKT phosphorylation
PIK3R1 is a direct target of miR-17-5p
PIK3R1 is downregulated in LSCC tissues and cell lines
miR-17-5p/PIK3R1 axis plays an essential role in LSCC cell proliferation
miR-17-5p is involved in LSCC proliferation in vivo
Dysregulation of signaling pathways has been identified to play a crucial role in initiating and maintaining malignant phenotypes of tumors . In the last decade, studies have demonstrated that uncontrolled activation of PI3K/AKT signaling pathway may be the most frequent driving events involved in development and progression of various cancers, including ovarian cancer, cervical cancer, gastric cancer, glioblastoma, hematological malignant and head and neck cancer . PI3K, containing a catalytic subunit (p110) and a regulatory subunit (p85), regulates the downstream phosphorylation of AKT gene, which is involved in promoting cell survival and proliferation by activating the downstream pathways . p85, which is encoded by PIK3R1, plays a critical role in the activation of PI3K/AKT pathway by binding and stabilizing PI3K p110 catalytic subunit . Therefore, aberrations of PIK3R1, such as gene mutations or downregulation, may play a role in different oncogenic mechanisms and influence distinct downstream signaling processes of AKT pathway. For example, PIK3R1 expression level is markedly downregulated in ovarian cancer, lung cancer, prostate cancer, liver, breast and kidney cancers . Moreover, the lower level of PIK3R1 is associated with a poor survival rate in patients with breast cancer . Further, lower mRNA level of PIK3R1 is a high-risk factor in stage I non-small cell lung cancers . Similarly, downregulation of PIK3R1 mRNA expression is involved in migration and invasion of breast cancer cell in vitro . Importantly, silencing of PIK3R1 in breast cancer could enhance the sensitivity to rapamycin . In the present study, we first confirmed that the levels of PKI3R1 mRNA and protein were significantly downregulated in LSCC tissues and cell lines. Functional experiments revealed that overexpression of PIK3R1 in LSCC cells obviously induced cell apoptosis and reduced cell proliferation. Moreover, overexpression of PIK3R1 in LSCC cells influenced AKT phosphorylation and the levels of downstream genes, such as BAX, BCL-2 and cleaved Caspases-3. These findings indicate that PIK3R1 can potentially be a biomarker and a target of treatment in LSCC. However, whether PIK3R1 level correlate with prognosis of patients with LSCC needs to be clarified with more clinical trials.
In addition to the effect of mutations in PIK3R1 that disrupt function, there are other different factors that affect the gene expression of PIK3R1. miRNAs, as a family of non-coding RNAs, have been widely accepted as regulators of PIK3R1 expression at the post-transcriptional level. For example, miR-479-5p directly targets PIK3R1 in gastric cancer cell and increases cell growth in vitro . miR-486-5p, suppresses cell proliferation in non-small cell lung cancer by regulating PIK3R1 expression and modulating AKT pathway activation . Furthermore, PIK3R1 level is reduced by miR-16-5p, which inhibits cell cycle and enhances cell apoptosis by modulating of the PI3K/AKT/NF-KB pathway . Besides, miR-495, miR-106a-5p, miR-455 and miR-15 were directly or indirectly contribute to the regulation of PIK3R1 expression [42, 43]. In the present study, we demonstrated that miR-17-5p could bind to PIK3R1 3′UTR directly. Overexpression of miR-17-5p could effectively decrease while knockdown of miR-17-5p increase the mRNA and protein levels of PIK3R1 and influence AKT phosphorylation. Moreover, protein levels of downstream targets known to play roles in cell proliferation and apoptosis including BCL-2, BAX, and cleaved Caspase-3 which were influenced by overexpression of miR-17-5p. Silencing of miR-17-5p enhanced the effect of PIK3R1 overexpression on cell proliferation and apoptosis. In addition, we found a negative relationship between miR-17-5p expression level and PIK3R1 mRNA level in LSCC tissues, indicating that dysregulation of the miR-17-5p/PIK3R1 regulatory pathway may be associated with LSCC cell survival.
It is well known that one miRNA may regulate different target genes, whereas one gene may be affected by multiple miRNAs. miR-17-5p acts as an oncogene in different cancers by specifically binding to and interfering with different targets. For example, miR-17-5p is overexpressed in gastric cancer tissues and cell lines. Knockdown of miR-17-5p reduces the proliferation and induces the apoptosis in SGC7901 cells by regulating the expression of Early Growth Response 2 (EGR2) . Similarly, miR-17-5p expression is reduced in breast cancer tissues. Overexpression of miR-17-5p inhibits cell proliferation, migration, and invasion by regulating AIB1 expression . Furthermore, miR-17-5p, which is upregulated in pancreatic cancer, functions as an oncogene by directly targeting RBL2 expression . Similar to the previous studies, we demonstrated that miR-17-5p was dramatically unregulated in LSCC tissues and LSCC cell lines. We also revealed that miR-17-5p level was correlated with LSCC T stage and TNM stage, indicating that higher expression may associate with the advanced tumor stage. Importantly, we observed that the higher level of miR-17-5p was associated with poor survival of patients with LSCC. Functionally, we further identified that miR-17-5p acted as an onco-miRNA in LSCC. Knockdown of miR-17-5p in LSCC cells reduced cell proliferation and promoted cell apoptosis in vitro and in vivo. These data suggest that miR-17-5p is a potential therapeutic target and a biomarker of LSCC.
In conclusion, our findings demonstrate that miR-17-5p is significantly increased in LSCC tissue and cell lines and may potentially be used as a diagnostic biomarker. Higher expression of miR-17-5p correlates with advanced stages of LSCC and a worse prognosis in LSCC. Dysregulation of miR-17-5p is involved in LSCC cell apoptosis and proliferation via directly targeting PIK3R1 3′UTR and modulation of AKT activation. These findings suggest that the miR-17-5p/PIK3R1/AKT axis can be a potential therapeutic target in LSCC treatment.
The authors thank Dr. Zi-Yuan Nie for technology support.
Conception and design: JW and CS; tissues collection: SL, JD and XR; development of methodology: JW, XJ and OX; acquisition of the data: HZ and JW; analysis of data: SL, XJ and SL; writing the manuscript: JW, XJ and CS. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study and was authorized Ethics Committee of Second Hospital of Hebei Medical University. All patients and volunteers were anonymous and provided written informed consent. All animal studies were approved by the Institutional Animal Care Committee of Hebei Medical University.
Consent for publication
Written consent was obtained from all participants.
The authors declare that they have no competing interests.
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