Knockdown of long non‑coding RNA HCP5 suppresses the malignant behavior of retinoblastoma by sponging miR‑3619‑5p to target HDAC9
- Authors:
- Published online on: March 3, 2021 https://doi.org/10.3892/ijmm.2021.4907
- Article Number: 74
Abstract
Introduction
Retinoblastoma (RB) is an eye malignancy that affects children primarily triggered by mutations in RB genes, the aberrant expression of intracellular molecules and by the activation of oncogenic pathways during the development of the retina (1-3). Approximately 8,000 children are diagnosed with RB each year (4). The clinical symptoms of RB include white pupillary reflex, conjunctival congestion, vitreous opacity, corneal edema, strabismus and increasing intraocular pressure (5,6). Currently, focal therapy (laser and cryotherapy), surgery and chemotherapy are the major treatment strategies for RB (7,8). RB can be cured over time with a graduated-intensity approach based on pathology in developed or even developing countries (9-11). However, the therapeutic effect remains unsatisfactory for children with metastatic and/or advanced RB (9). Therefore, the development of novel targets at the molecular level for RB therapy is crucial.
Long non-coding RNAs (lncRNAs), a type of non-coding RNA composed of >200 nucleotides, regulate the expression of genes by regulating transcription, post-transcription and chromatin modifications (12,13). lncRNAs function as important regulators in the progression of RB (14-16). For instance, the overexpression of lncRNA RB-associated transcript-1 can accelerate the tumorigenesis of RB (14). The downregulation of lncRNA ILF3 antisense RNA 1 and lncRNA LINC00324 has been shown to suppress the proliferation and invasion of RB cells, as well as tumor growth in mice (15,16). lncRNA HLA complex P5 (HCP5), a member of the new pseudogene family P5 (17), is mainly expressed in the immune system, such as in the spleen, blood and thymus (18). HCP5 plays a role in the progression of several types of human cancers (19-21). The study by Bai et al indicated that the downregulation of HCP5 suppressed the viability, migration and invasion of colorectal cancer (CRC) cells and impeded the malignant behavior of CRC in vivo (19). Wang et al revealed that the knockdown of HCP5 exerted an inhibitory effect on the growth and metastasis of ovarian cancer (OC) in mice (20). Similarly, Yuan et al suggested that the progression of pancreatic cancer was significantly suppressed by the silencing of HCP5 (21). Nevertheless, the specific role and potential mechanisms of HCP5 in RB are relatively unknown.
The participation of microRNAs (miRNAs or miRs) in the pathogenesis of RB has been uncovered (22,23). Wan et al reported that miR-25-3p exertd a protective effect against RB tumorigenesis by dampening the proliferation, migration and invasion of RB (22). Li and You demonstrated that miR-758 functioned as a suppressor of the metastasis of RB (23). Recent studies demonstrated that miR-3619-5p played an anti-tumor role in liver cancer and RB (24,25). More importantly, the interaction of miR-3619-5p with lncRNA LINC00202 has been shown to considerably contribute to the suppression of the progression of RB (25). It remains unclear however, as to whether the inhibitory effect of miR-3619-5p in RB tumorigenesis is modulated by HCP5.
Histone deacetylase 9 (HDAC9), a member of class II HDACs, possesses a conserved domain of HDAC and can interact with tissue-specific transcription factors and co-repressors (26). In recent years, the carcinogenesis of HDAC9 in RB has attracted increasing attention (27-29). Both Zhang et al (27) and Xu et al (29) suggested that the downregulation of HDAC9 suppressed the proliferation of RB cells, thus, attenuating RB in vitro (27,29). Jin et al revealed that the overexpression of HDAC9 reversed the anti-proliferative effect of miR-101-3p on RB cells (28). Nevertheless, the interactions between miR-3619-5p and HDAC9, as well as the HCP5/miR-3619-5p/HDAC9 axis in the progression of RB remain relatively unknown.
The present study thus focused on investigating the expression and roles of HCP5 and miR-3619-5p in RB, and on exploring the association between HCP5, miR-3619-5p and HDAC9 in RB cells. Collectively, the findings of the present study may provide a novel target for the treatment of RB.
Materials and methods
Collection of samples
RB tissues and adjacent normal tissues in pairs (n=71) were obtained from patients who were diagnosed with RB (unilateral, 51; bilateral, 20) from June, 2017 to July, 2019 at the Affiliated Hospital of Weifang Medical University. Among the patients, 41 lymph node metastases and 2 distant metastasis cases (1 cerebral spinal fluid and 1 brain) were included. The tumor node metastasis (TNM) stage (30), intraocular international RB classification (IIRC) stage (31) and Reese Ellsworth (RE) stage (32) were used to classify RB. The present study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Affiliated Hospital of Weifang Medical University [no. 2020(12)]. Written informed consent was obtained from all patients, and parental consent was obtained in cases where the patient was <18 years old.
Cells and cell culture
A human normal retinal pigmented epithelium cell line (APRE-19) and 4 human RB cell lines (Y79, HXO-RB44, WERI-Rb-1 and SO-RB50) were purchased from the American Type Culture Collection (ATCC). The ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The Y79, Weri-RB1, SO-RB50 and HXO-RB44 cells were cultured in modified Roswell Park Memorial Institute-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS. All cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA isolated from the tissues or cells was analyzed using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The extracted RNA was reverse transcribed into complementary DNA (cDNA) using a Prime-Script RT reagent kit (Takara Biotechnology Co., Ltd.). Subsequently, SYBR® Premix Ex Taq™ II (Takara Biotechnology Co., Ltd.) was utilized for the amplification of cDNA. The reaction conditions were 95°C for 5 min, 40 cycles of 95°C for 15 sec, 60°C for 20 sec, and 70°C for 15 sec. The primer sequences used in the present study are listed in Table I. The relative expression of HCP5, miR-3619-5p and HDAC9 was calculated using the 2−ΔΔCq method (33). The endogenous controls were U6 and GAPDH.
Cell transfection
Short hairpin (sh) RNA against HCP5 (sh-HCP5), sh-negative control (sh-NC), miR-NC and miR-3619-5p mimics, as well as miR-3619-5p inhibitor, pcDNA-HDAC9, pcDNA-MS2, pBobi-MS2-GFP and pcDNA-HCP5-MS2 were purchased from Guangzhou RiboBio Co., Ltd. sh-HCP5 or sh-NC were firstly cloned into a lentiviral vector. These factors (all, 50 nM) were then transfected into Y79 and/or HXO-RB44 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 48 h after transfection, the cells were harvested for further experiments.
Target prediction
The target miRNAs of HCP5 were predicted using StarBase v2.0 software (http://starbase.sysu.edu.cn). A total of 8 miRNAs (miR-3681-3p, miR-29b-3p, miR-520a-5p, miR-3619-5p, miR-216a-3p, miR-20b-5p, miR-128-3p, and miR-4701-5p) were selected for verification via RNA immunoprecipitation (RIP) assay. Similarly, the target genes of miR-3619-5p were also predicted using StarBase v2.0 software. HDAC9 was selected due to its important role in RB.
RIP assay
pcDNA-MS2, pBobi-MS2-GFP and pcDNAHCP5-MS2 were transfected into the Y79 or HXO-RB44 cells. The RIP assay was performed with anti-GFP antibody (1:5,000; ab6673, Abcam), as well as a MagnaRIP RNA-Binding Protein Immunoprecipitation kit (EMD Millipore). The expression of the aforementioned miRNAs was detected by RT-qPCR.
Dual luciferase reporter (DLR) assay
The 3′-UTR fragment of HCP5, including the predicted or mutated binding site for miR-3619-5p, was introduced into psiCHECK2 (Promega Corporation) to establish HCP5 wild-type (wt) or HCP5 mutant-type (mut). Similarly, the 3′-UTR sequence of HDAC9 containing the predicted or mutated binding site for miR-3619-5p was inserted into psiCHECK2 (Promega Corporation) to construct HDAC9 wt or mut. For reporter assays, one of the above-mentioned vectors (80 ng) along with miR-3619-5p mimics or miR-NC (50 nM) were co-transfected into the Y79 and HXO-RB44 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h, relative luciferase activity was examined using a DLR assay System (Promega Corporation). The activity of Firefly luciferase was normalized to that of Renilla luciferase.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay
The transfected cells were plated into 96-well plates at a density of 5,000 cells/well and cultured for 48 h at 37°C. MTT reagent (20 µl; Sigma-Aldrich; Merck KGaA) was then added to each well at different time points (24, 48, 72 and 96 h) followed by incubation for 4 h at 37°C. After discarding the medium, 150 µl of dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) were added to each well. The optical density was measured at 490 nM using an enzyme immunoassay instrument (Bio-Tek Instruments, Inc.).
Cell migration and invasion assays
The 24-well Transwell chambers (8 µM pore size; BD Biosciences) coated with Matrigel (BD Biosciences) were used to evaluate cell invasion. Cells (1×105) were resuspended in 200 µl of serum-free medium and then plated into the upper chambers of each Transwell apparatus. A total of 600 µl of medium containing 10% FBS was added to the lower chambers followed by incubation at 37°C for 48 h. Subsequently, the cells in the upper chambers were wiped off using a cotton swab, and those adhering to the lower chambers were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (TCI chemicals) at 37°C for 30 min. Stained cells were imaged using an inverted light microscope (Olympus Corporation) and analyzed using ImageJ software [version 1.46, National Institutes of Health (NIH)].
For the cell migration assay, the procedure was similar to the cell invasion assay, except that the 24-well Transwell chambers were not pre-coated with Matrigel.
Western blot analysis
Total protein was extracted from the transfected Y9 and/or HXO-RB44 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was detected using the BCA Protein Assay kit (Abcam). Proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were then blocked with 5% non-fat milk for 2 h followed by incubation with the primary antibodies anti-HDAC9 (1:1,000; ab109446, Abcam) and anti-GAPDH (1:1,000; ab9485, Abcam) overnight at 4°C. Tris-buffered saline Tween-20 was then used to wash the membranes 3 times. Subsequently, HRP-conjugated anti-rabbit IgG secondary antibody (1:5,000; ab205718, Abcam) was added followed by incubation for 1 h at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.). The relative protein expression over GAPDH was quantified using Alpha Innotech imaging software (ProteinSimple).
Xenograft tumor model
Female BALB/c nude mice (4-5 weeks of age, weighing 20-25 g) were obtained from the Shanghai Laboratory Animal Centre (Shanghai, China). All mice were housed under controlled conditions (25°C, 50% humidity, 12 h light/dark cycle) and were provided with free access to food and water. Subsequently, the mice were randomly divided into the 2 following groups: The sh-NC group (n=5) and the sh-HCP5 group (n=5). sh-HCP5 or sh-NC were integrated into a lentiviral vector and transfected into the Y79 cells. Furthermore, the transfected Y79 cells (2×106 cells/100 µl, s.c.) were injected into the right flanks of the nude mice. Tumor volumes were measured every other week using the following formula: (A × B2)/2, (A, the longest diameter; B, the shortest diameter). After 4 weeks, the mice were anaesthetized with pentobarbital sodium (50 mg/kg) and euthanized by cervical dislocation. The tumor xenografts were separated from the mice and weighed. The animal experiments were approved by the Ethics Committee of the Affiliated Hospital of Weifang Medical University [no. 2020(12)] and were performed in accordance with the institutional guide for the care and use of laboratory animals (National Institutes of Health).
Statistical analysis
In vitro experiments were performed in triplicate, and each experiment was repeated 3 times. In vivo experiments were performed using 5 mice per group. Statistical analyses were performed using SPSS Statistics 22.0. The Student's t-test was used for comparisons between 2 groups (paired t-test for the data in Figs. 1A, 4E, and 6C; unpaired t-test for the data in Figs. 1B, C, D, 2C, D, 3A, C, 4D, 5C, D and 6F). One-way ANOVA was used to assess differences among multiple groups, followed by Tukey's post hoc test. Pearson's correlation analysis was used to determine the correlation between HCP5 and miR-3619-5p expression, as well as that between miR-3619-5p and HDAC9, and HDAC9 and HCP5 expression in RB tissues. Data are presented as the means ± standard deviation (SD). The Chi-squared test was used to analyze the categorical data presented in Table II. Statistically significant differences were regarded as those with values of P<0.05.
Results
lncRNA HCP5 is highly expressed in RB tissues and cells
First, the expression of HCP5 was determined by RT-qPCR and it was found that the expression of HCP5 in the tumor tissues was distinctly increased compared to that in adjacent tissues (P<0.01; Fig. 1A). Subsequently, the association between HCP5 expression and the pathological features of RB cases was analyzed. It was found that a high and low expression of HCP5 exhibited significant differences in TNM stage (P=0.003), lymph node metastasis (P=0.043), IIRC stage (P=0.009) and RE stage (P=0.024) (Table II). As illustrated in Fig. 1B-D, an increased expression of HCP5 was observed in TNM stage III/IV, IIRC stage D/E and RE stage IV/V (P<0.01), compared to TNM stage I/II, IIRC stage A/C and RE stage I/II/III, respectively. The expression of HCP5 was also determined in RB cells and an elevated expression of HCP5 was found in the Rb cell lines (Y79, HXO-RB44, WERI-Rb-1 and SO-RB50) (P<0.01; Fig. 1E). In addition, a relatively higher expression of HCP5 was found in the HOX-RB44 and Y79 cell lines among the 4 RB cell lines. Therefore, the HOX-RB44 and Y79 cells were used in the following experiments.
Silencing of lncRNA HCP5 suppresses the viability, migration and invasion of RB cells
To explore the function of HCP5 in RB cells, the transfection efficiency of sh-HCP5 in both the HXO-RB44 and Y79 cell lines was initially detected. Transfection with sh-HCP5 markedly reduced the expression of HCP5 (all P<0.01; Fig. 2A). Subsequently, functional experiments were conducted on HCP5. MTT assay revealed that transfection with sh-HCP5 led to a notable decrease in the viability of the HXO-RB44 and Y79 cells (P<0.05; Fig. 2B). Additionally, the relative migration and invasion of the Y79 and HXO-RB44 cells were reduced by the knockdown of HCP5 (all P<0.01; Fig. 2C and D).
Silencing of lncRNA HCP5 suppresses the growth of tumor xenografts in vivo
The expression of HCP5 in mouse tissues was also determined. The results of RT-qPCR demonstrated that HCP5 expression was decreased by the injection of sh-HCP5 (P<0.01, Fig. 3A). The effect of sh-HCP5 on tumor xenografts was then examined. As illustrated in Fig. 3B-D, it was found that tumor weight in the sh-HCP5 group was lower than that in the sh-NC group, and the tumor volume was markedly decreased by sh-HCP5 on the 28th day after the injection in mice (P<0.01).
HCP5 serves as a competing endogenous RNA for miR-3619-5p
To explore the regulatory mechanisms of HCP5 in RB, the target miRNAs of HCP5 were predicated using StarBase v2.0 software. RIP assay was performed to verify 8 selected miRNAs. As shown in Fig. 4A, the Y79 and HXO-RB44 cells in the MS2-HCP5 group were enriched with miR-29b-3p, miR-3619-5p and miR-128-3p compared with the MS2 group. Among these 3 miRNAs, miR-3619-5p was selected due to its relatively high enrichment with HCP5. The predicted binding site of HCP5 and miR-3619-5p is shown in Fig. 4B. DLR assay further revealed that the relative luciferase activity of the Y79 and HXO-RB44 cells co-transfected with HCP5 wt and miR-3619-5p mimics was conspicuously lower than that of the Y79 and HXO-RB44 cells co-transfected with HCP5 wt and miR-NC (all P<0.01; Fig. 4C). Subsequently, the effect of HCP5 on miR-3619-5p expression was examined and it was observed that the expression of miR-3619-5p increased following the downregulation of HCP5 in the Y79 and HXO-RB44 cells (all P<0.01; Fig. 4D). Moreover, miR-3619-5p expression in tumor tissues was diminished in comparison with adjacent normal tissues (P<0.01; Fig. 4E). A negative correlation was validated between HCP5 and miR-3619-5p in RB tissues using Pearson's correlation analysis (P=0.004, r=-0.3372; Fig. 4F).
Overexpression of miR-3619-5p inhibits the viability, migration and invasion of RB cells
Accordingly, the role of miR-3619-5p in RB cells was explored; miR-3619-5p mimics and inhibitors were transfected into RB cells. As was expected, miR-3619-5p expression increased by the addition of miR-3619-5p mimics, whereas it was decreased by transfection with miR-3619-5p inhibitor in the HXO-RB44 and Y79 cells (all P<0.01; Fig. 5A). Importantly, it was discovered that the overexpression of miR-3619-5p caused a decrease in the viability of the Y79 and HXO-RB44 cells (all P<0.01; Fig. 5B). Moreover, the overexpression of miR-3619-5p suppressed the migration and invasion of the Y79 and HXO-RB44 cells (all P<0.01; Fig. 5C and D).
Identification of HDAC9 as a target gene of miR-3619-5p
To elucidate the mechanisms of miR-3619-5p in the progression of RB, the target gene of miR-3619-5p was predicted using StarBase v2.0 software and it was found that miR-3619-5p shared a complementary binding sequence at the 3-UTR with HDAC9 (Fig. 6A). Furthermore, the DLR assay verified that transfection with miR-3619-5p mimics markedly reduced the luciferase activity of the HDAC9 wt vector compared with that of miR-NC, whereas it did not exert any significant effect on the luciferase activity of the HDAC9 mut vector in the Y79 and HXO-RB44 cells (all P<0.01; Fig. 6B). Simultaneously, it was discovered that HDAC9 expression was markedly increased in tumor tissues in contrast to adjacent normal tissues (P<0.01; Fig. 6C). Pearson's correlation analysis demonstrated that HDAC9 expression negatively correlated with miR-3619-5p expression (r=-0.3909), and HDAC9 expression positively correlated with HCP5 expression in RB tissues (r=0.3376; all P<0.01; Fig. 6D and E). The regulatory association between miR-3619-5p and HDAC9 was then examined, and it was observed that the relative protein expression of HDAC9 was reduced by the upregulation of miR-3619-5p in the Y79 and HXO-RB44 cells (all P<0.01; Fig. 6F).
lncRNA HCP5 suppresses RB cell viability, migration and invasion by sponging miR-3619-5p to target HDAC9
Subsequently, Y79 cells with a relatively high expression of HCP5 were used to perform rescue experiments. To investigate whether HCP5 affects RB cells by regulating the miR-3619-5p/HDAC9 axis, HDAC9 was first overexpressed in Y79 cells. As shown in Fig. 7A, HDAC9 expression was markedly upregulated by transfection with pcDNA-HDAC9 in the Y79 cells (P<0.01). Furthermore, rescue assays were performed. MTT assay revealed that cell viability was reduced by transfection with sh-HCP5, while the addition of miR-3619-5p inhibitor or pcDNA-HDAC9 reversed the sh-HCP5-mediated decrease in the viability of Y79 cells (all P<0.01; Fig. 7B). Transwell assays indicated that sh-HCP5 suppressed the migration and invasion of Y79 cells, whereas the suppressive effects of sh-HCP5 on the migratory and invasive abilities were partially eliminated by miR-3619-5p inhibitor or pcDNA-HDAC9 in the Y79 cells (all P<0.01; Fig. 7C and D).
Discussion
RB is an eye malignant tumor that mainly affects children (8). The abnormal expression of lncRNA HCP5 plays a clinically predictive role in tumorigenesis in a number of types of cancer (34-36). For example, HCP5 expression is higher in cervical cancer tissues than in paracancerous tissues, which is negatively associated with the survival rate of patients with cervical cancer (34). The upregulation of HCP5 has been observed in prostate cancer, eventually resulting in tumor metastasis (35). Increased HCP5 levels have also been found in oral squamous cell carcinoma, and it has been shown to be significantly associated with lymph node metastasis and an advanced TNM stage (36). In the present study, it was discovered that HCP5 was upregulated in RB tissues and cells compared to the controls. Concurrently, it was found that a high expression of HCP5 was closely associated with TNM stage and lymph node metastasis. In addition, strong associations were identified between enhanced HCP5 and IIRC stage/RE stage, suggesting that HCP5 may be an onco-lncRNA in RB.
HCP5 plays a vital role in the cellular processes of several human cancers (19,20,37). Wang et al revealed that the downregulation of HCP5 suppressed the proliferation of triple-negative breast cancer (TNBC) cells in vitro and the tumor growth of TNBC in vivo (37). Bai et al indicated that HCP5 silencing suppressed the progression of CRC in vivo (19). Wang et al demonstrated that the knockdown of HCP5 greatly contributed to the inhibition of the proliferation, migration and invasion of OC cells (20). In the present study, the proliferation, migration and invasion of RB cells, as well as the growth of tumor xenografts were suppressed by HCP5 downregulation. Based on these results, it can be conjectured that the silencing of HCP5 acts as a suppressor in the progression of Rb.
miR-3619-5p plays an anti-tumor role in the malignancy of several cancers (38,39). For instance, miR-3619-5p is minimally expressed in non-small cell lung cancer (NSCLC) tissues, whereas the overexpression of miR-3619-5p suppresses the growth and invasion of NSCLC cells in vitro (38). A high expression of miR-3619-5p suppresses the proliferation, migration and invasion of breast cancer cells (39). In the present study, a decreased miR-3619-5p expression was identified in RB tissues, and the upregulation of miR-3619-5p exerted suppressive effects on the proliferation and metastasis of RB cells. A recent study by Yan et al revealed that the proliferation, migration and invasion of RB cells were impeded by transfection with miR-3619-5p mimics (25). These results suggest that miR-3619-5p may also be function as an anti-tumor miRNA in RB. In addition, miR-3619-5p interacts with lncRNAs and is involved in cancer progression, such as LINC00202-miR-3619-5p in RB (25), DGCR5-miR-3619-5p in gallbladder cancer (40) and PVT1-miR-3619-5p in gastric cancer (41). We hypothesised that the anti-tumour function of miR-3619-5p in RB may also be regulated by HCP5. Herein, miR-3619-5p was initially identified as a target of and was negatively modulated by HCP5. The results of the present study demonstrated that the reduction of the proliferation and metastasis of Y79 cells transfected with sh-HCP5 was reversed by transfection with miR-3619-5p inhibitor. These results suggest that the silencing of HCP5 attenuates RB carcinogenicity by interacting with miR-3619-5p.
In recent years, increasing attention has been paid to the involvement of HDAC9 in the pathological process of RB (27,29). Xu et al suggested that HDAC9 expression was increased in RB tissues, and its downregulation inhibited the proliferation, migration and invasion of RB cells (29). In addition, Zhang et al confirmed that the silencing of HDAC9 suppressed the proliferation of RB cells in vitro (27). Consistent with these studies, an increased expression of HDAC9 was identified in the present study, indicating the oncogenicity of HDAC9 in RB. Simultaneously, it was found that HDAC9 was a target gene of and was negatively regulated by miR-3619-5p, and a positive correlation was observed between HDAC9 and HCP5 expression in RB tissues. Therefore, it was demonstrated miR-3619-5p is involved in the tumorigenesis of Rb through regulation by HCP5. It was further hypothesized that HDAC9 may also be modulated by HCP5 to play a role in the progression of Rb. Feedback verification experiments revealed that the inhibitory effects of HCP5 knockdown on the proliferation, migration and invasion of Y79 cells were reversed by HDAC9 overexpression, which confirmed this hypothesis. It was concluded that the downregulation of HCP5 suppressed the progression of Rb by sponging miR-3619-5p and regulating HDAC9. However, there may be a limitation in to the presents tudy. Only one Rb cell line (Y79) was used to perform the feedback verification experiments, and further experiments using other cell lines are required to validate this conclusion.
In conclusion, the present study demonstrates that HCP5 acts as an endogenous sponge of miR-3619-5p to regulate HDAC9. The knockdown of HCP5 mitigates the progression of RB by regulating the miR-3619-5p/HDAC9 axis. Overall, the present study provides perspectives for novel therapeutic targets for RB.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
YZ made substantial contributions to the conception and design of the study, data acquisition, the drafting of the article, and gave final approval of the version to be published. FH made substantial contributions to the conception and design of the study, data analysis and interpretation, and in the drafting of the article. Both authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University [No. 2020(12)]. Written informed consent was obtained from all the patients. The study was conducted in accordance with the Declaration of Helsinki. The animal experiments were approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University, and were performed in accordance with the institutional guide for the care and use of laboratory animals (National Institutes of Health).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
Funding
The present study was funded by the Shandong Medical and Health Science and Technology Development Project (Project name: Study on the mechanism of mirNA-21 on retinal microvascular endothelial cells of RF/6A in diabetic rats; no. 2019WS599).
Abbreviations:
|
lncRNAs |
long non-coding RNAs |
|
RB |
retinoblastoma |
|
RT-qPCR |
reverse transcription-quantitative polymerase chain reaction |
|
DLR |
dual-luciferase reporter |
References
|
Villegas VM, Hess DJ, Wildner A, Gold AS and Murray TG: Retinoblastoma. Curr Opin Ophthalmol. 24:581–588. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Cimino PJ, Robirds DH, Tripp SR, Pfeifer JD, Abel HJ and Duncavage EJ: Retinoblastoma gene mutations detected by whole exome sequencing of merkel cell carcinoma. Mod Pathol. 27:1073–1087. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Venkatesan N, Deepa PR, Khetan V and Krishnakumar S: Computational and in vitro investigation of miRNA-Gene regulations in retinoblastoma pathogenesis: miRNA mimics strategy. Bioinform Biol Insights. 9:89–101. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Dimaras H, Corson TW, Cobrinik D, White A, Zhao J, Munier FL, Abramson DH, Shields CL, Chantada GL, Njuguna F and Gallie BL: Retinoblastoma. Nat Rev Dis Primers. 1:150212015. View Article : Google Scholar : PubMed/NCBI | |
|
Okimoto S and Nomura K: Clinical manifestations and treatment of retinoblastoma in Kobe children's hospital for 16 years. J Pediatr Ophthalmol Strabismus. 51:222–229. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Abu-Ain MS, Shatnawi R, Yousef YA and Watts P: Heterochromia irides and mistaken identity of retinoblastoma. BMJ Case Rep. 12:e2310912019. View Article : Google Scholar : PubMed/NCBI | |
|
Jenkinson H: Retinoblastoma: Diagnosis and management-the UK perspective. Arch Dis Child. 100:1070–1075. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Rao R and Honavar SG: Retinoblastoma. Indian J Pediatr. 84:937–944. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Sullivan EM, Wilson MW, Billups CA, Wu J, Merchant TE, Brennan RC, Haik BG, Shulkin B, Free TM, Given V, et al: Pathologic risk-based adjuvant chemotherapy for unilateral retinoblastoma following enucleation. J Pediatr Hematol Oncol. 36:e335–e340. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Qaddoumi I, Nawaiseh I, Mehyar M, Razzouk B, Haik BG, Kharma S, Jaradat I, Rodriguez-Galindo C and Wilson MW: Team management, twinning, and telemedicine in retinoblastoma: A 3-tier approach implemented in the first eye salvage program in Jordan. Pediatr Blood Cancer. 51:241–244. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Chantada GL, Dunkel IJ, Schaiquevich PS, Grynszpancholc EL, Francis J, Ceciliano A, Zubizarreta PA, Fandiño AC and Abramson DH: Twenty-Year collaboration between north American and South American retinoblastoma programs. J Glob Oncol. 2:347–352. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Wang X, Arai S, Song X, Reichart D, Du K, Pascual G, Tempst P, Rosenfeld MG, Glass CK and Kurokawa R: Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature. 454:126–130. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Fatica A and Bozzoni I: Long non-coding RNAs: New players in cell differentiation and development. Nat Rev Genet. 15:7–21. 2014. View Article : Google Scholar | |
|
He X, Chai P, Li F, Zhang L, Zhou C, Yuan X, Li Y, Yang J, Luo Y, Ge S, et al: A novel LncRNA transcript, RBAT1, accelerates tumorigenesis through interacting with HNRNPL and cis-activating E2F3. Mol Cancer. 19:1152020. View Article : Google Scholar : PubMed/NCBI | |
|
Han S, Song L, Chen Y, Hou M, Wei X and Fan D: The long non-coding RNA ILF3-AS1 increases the proliferation and invasion of retinoblastoma through the miR-132-3p/SMAD2 axis. Exp Cell Res. 393:1120872020. View Article : Google Scholar : PubMed/NCBI | |
|
Dong Y, Wan G, Yan P, Qian C, Li F and Peng G: Long noncoding RNA LINC00324 promotes retinoblastoma progression by acting as a competing endogenous RNA for microRNA-769-5p, thereby increasing STAT3 expression. Aging (Albany NY). 12:7729–7746. 2020. View Article : Google Scholar | |
|
Vernet C, Ribouchon MT, Chimini G, Jouanolle AM, Sidibe I and Pontarotti P: A novel coding sequence belonging to a new multicopy gene family mapping within the human MHC class I region. Immunogenetics. 38:47–53. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Liu Y, Helms C, Liao W, Zaba LC, Duan S, Gardner J, Wise C, Miner A, Malloy MJ, Pullinger CR, et al: A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci. PLoS Genet. 4:e10000412008. View Article : Google Scholar : PubMed/NCBI | |
|
Bai N, Ma Y, Zhao J and Li B: Knockdown of lncRNA HCP5 suppresses the progression of colorectal cancer by miR-299-3p/PFN1/AKT axis. Cancer Manag Res. 12:4747–4758. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Wang L, He M, Fu L and Jin Y: Role of lncRNAHCP5/microRNA-525-5p/PRC1 crosstalk in the malignant behaviors of ovarian cancer cells. Exp Cell Res. 394:1121292020. View Article : Google Scholar : PubMed/NCBI | |
|
Yuan B, Guan Q, Yan T, Zhang X, Xu W and Li J: LncRNA HCP5 regulates pancreatic cancer progression by miR-140-5p/CDK8 axis. Cancer Biother Radiopharm. 35:711–719. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Wan W, Long Y, Li Q, Jin X, Wan G, Zhang F, Lv Y, Zheng G, Li Z and Zhu Y: MiR-25-3p promotes malignant phenotypes of retinoblastoma by regulating PTEN/Akt pathway. Biomed Pharmacother. 118:1091112019. View Article : Google Scholar : PubMed/NCBI | |
|
Li J and You X: MicroRNA758 inhibits malignant progression of retinoblastoma by directly targeting PAX6. Oncol Rep. 40:1777–1786. 2018.PubMed/NCBI | |
|
Hao P, Yue F, Xian X, Ren Q, Cui H and Wang Y: Inhibiting effect of MicroRNA-3619-5p/PSMD10 axis on liver cancer cell growth in vivo and in vitro. Life Sci. 254:1176322020. View Article : Google Scholar : PubMed/NCBI | |
|
Yan G, Su Y, Ma Z, Yu L and Chen N: Long noncoding RNA LINC00202 promotes tumor progression by sponging miR-3619-5p in retinoblastoma. Cell Struct Funct. 44:51–60. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Parra M and Verdin E: Regulatory signal transduction pathways for class IIa histone deacetylases. Curr Opin Pharmacol. 10:454–460. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang Y, Wu D, Xia F, Xian H, Zhu X, Cui H and Huang Z: Downregulation of HDAC9 inhibits cell proliferation and tumor formation by inducing cell cycle arrest in retinoblastoma. Biochem Biophys Res Commun. 473:600–606. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Jin Q, He W, Chen L, Yang Y, Shi K and You Z: MicroRNA-101-3p inhibits proliferation in retinoblastoma cells by targeting EZH2 and HDAC9. Exp Ther Med. 16:1663–1670. 2018.PubMed/NCBI | |
|
Xu L, Li W, Shi Q, Wang M, Li H, Yang X and Zhang J: MicroRNA936 inhibits the malignant phenotype of retinoblastoma by directly targeting HDAC9 and deactivating the PI3K/AKT pathway. Oncol Rep. 43:635–645. 2020.PubMed/NCBI | |
|
TNM8: The updated TNM classification for retinoblastoma. Community Eye Health. 31:342018. | |
|
Shields CL and Shields JA: Basic understanding of current classification and management of retinoblastoma. Curr Opin Ophthalmol. 17:228–234. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Yousef YA, Al-Hussaini M, Mehyar M, Sultan I, Jaradat I, AlRawashdeh K, Khurma S, Deebajah R and Nawaiseh I: Predictive value of tnm classification, international classification, and reese-ellsworth staging of retinoblastoma for the likelihood of high-risk pathologic features. Retina. 35:1883–1889. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar | |
|
Yu Y, Shen HM, Fang DM, Meng QJ and Xin YH: LncRNA HCP5 promotes the development of cervical cancer by regulating MACC1 via suppression of microRNA-15a. Eur Rev Med Pharmacol Sci. 22:4812–4819. 2018.PubMed/NCBI | |
|
Hu R and Lu Z: Long noncoding RNA HCP5 promotes prostate cancer cell proliferation by acting as the sponge of miR4656 to modulate CEMIP expression. Oncol Rep. 43:328–336. 2020. | |
|
Zhao J, Bai X, Feng C, Shang X and Xi Y: Long non-coding RNA HCP5 facilitates cell invasion and epithelial-mesenchymal transition in oral squamous cell carcinoma by miR-140-5p/SOX4 axis. Cancer Manag Res. 11:10455–10462. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Wang L, Luan T, Zhou S, Lin J, Yang Y, Liu W, Tong X and Jiang W: LncRNA HCP5 promotes triple negative breast cancer progression as a ceRNA to regulate BIRC3 by sponging miR-219a-5p. Cancer Med. 8:4389–4403. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Niu X, Liu S, Jia L and Chen J: Role of MiR-3619-5p in β-catenin-mediated non-small cell lung cancer growth and invasion. Cell Physiol Biochem. 37:1527–1536. 2015. View Article : Google Scholar | |
|
Lv MH, Mao QX, Li JT, Qiao J, Chen X and Luo S: Knockdown of LINC00665 inhibits proliferation and invasion of breast cancer via competitive binding of miR-3619-5p and inhibition of catenin beta 1. Cell Mol Biol Lett. 25:432020. View Article : Google Scholar : PubMed/NCBI | |
|
Liu S, Chu B, Cai C, Wu X, Yao W, Wu Z, Yang Z, Li F, Liu Y, Dong P and Gong W: DGCR5 promotes gallbladder cancer by sponging MiR-3619-5p via MEK/ERK1/2 and JNK/p38 MAPK pathways. J Cancer. 11:5466–5477. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Wu C, Hu Y, Ning Y, Zhao A, Zhang G and Yan L: Long noncoding RNA plasmacytoma variant translocation 1 regulates cisplatin resistance via miR-3619-5p/TBL1XR1 axis in gastric cancer. Cancer Biother Radiopharm. 35:741–752. 2020. View Article : Google Scholar : PubMed/NCBI |
