Abstract
The dysregulated lncRNA play essential roles in glioma development. This study aimed to investigate the role and mechanism of lncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/ antisense transcript 1 (KCNQ1OT1) in glioma progression. Tumor tissues and adjacent normal samples were collected from 30 glioma patients. The expression levels of lncRNA KCNQ1OT1, microRNA (miR)-338-3p and ribonucleotide reductase M2 (RRM2) were detected by quantitative real-time polymerase chain reaction or western blot analyses. Levels of cell viability, apoptosis, cell migration and invasion in glioma cell lines were determined using cell counting kit-8, flow cytometry with annexin V-FITC and trans-well assays, respectively. The role of KCNQ1OT1 in glioma development in vivo was investigated using a xenograft model. The target association between miR-338-3p and KCNQ1OT1 or RRM2 was validated by luciferase reporter assay. The results found that expression of KCNQ1OT1 was enhanced in glioma tissues and cells, and KCNQ1OT1 knockdown inhibited cell viability, migration and invasion, and xenograft tumor growth, but promoted apoptosis. miR-338-3p was targeted via KCNQ1OT1 and could reverse the effect of KCNQ1OT1 on glioma progression. RRM2 was targeted via miR-338-3p and attenuated the suppressive effect of miR-338-3p on glioma cell viability, migration and invasion. Besides, KCNQ1OT1 overexpression increased RRM2 expression, and this event was weakened via miR-338-3p up-regulation. In conclusion, the present finding suggest that silencing of KCNQ1OT1 can suppress the development and progression of glioma by up-regulating miR-338-3p and down-regulating RRM2.
1 Introduction
Glioma accounts for about 30% of primary brain tumors with high morbidity and mortality in adults and children [1, 2, 3]. Although many treatments including surgery, radiotherapy, chemotherapy and immunotherapy show great promise in glioma therapy [4, 5], the outcome for many patients remains poor. Hence, considerable hope is placed in exploring novel strategies to improve the outcomes for glioma patients.
Non-coding RNAs include long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), which do not have protein-coding ability [6]. LncRNAs with longer than 200 nucleotides play pivotal roles in many cellular processes in glioma [7]. For example, Liang et al. reported that knockdown of lncRNA paternally expressed gene 10 (PEG10) decreased proliferation, migration and invasion of glioma [8]. Moreover, Liu et al. suggested the involvement of small nucleolar RNA host gene 20 (SNHG20) as an oncogenic lncRNA in glioma that promotes cell proliferation [9]. Further it has been reported that lncRNA deleted in lymphocytic leukemia 1 (DLEU1) is involved in glioma development [10]. More recently, lncRNA potassium voltage-gated channel subfamily Q member 1 opposite strand/antisense transcript 1 (KCNQ1OT1) has been identified as an oncogenic lncRNA that plays a role in promoting progression of cancers, including hepatocellular carcinoma, breast cancer, cholangiocarcinoma and non-small-cell lung carcinoma [11, 12, 13, 14]. Additional studies have shown that knockdown of KCNQ1OT1 repressed proliferation, migration and invasion of glioma by activating the miR-370/CCNE2 axis [15]. However, the mechanism underpinning the role of KCNQ1OT1 in glioma progression appears to be complex and warrants further investigation.
miRNAs, as targets of lncRNAs, play important roles in regulation of glioma progression [16]. miR-338-3p has been reported to act as a tumor suppressor in many cancers, including bladder cancer, melanoma and oral squamous cell carcinoma [17, 18, 19]. Moreover, previous research has suggested that dysregulation of miR-338-3p is involved in glioma malignancy [20, 21, 22], and that Ribonucleotide reductase M2 (RRM2) may contribute to development of glioma [23, 24]. More importantly, predictions using starBase 2.0 indicate potential complementary sequences between miR-338-3p and KCNQ1OT1 or RRM2, however sequence interactions between these targets have not been validated previously. The present study advances the hypothesis that the progression of glioma is dependent on a competing endogenous RNA (ceRNA) network of KCNQ1OT1/miR-338-3p/RRM2. To investigate this hypothesis we measured the expression of KCNQ1OT1 in glioma tissues and cell lines and investigated the function of this lncRNA on glioma viability, apoptosis, migration and invasion in vitro. Furthermore, this study explored the functional roles of the ceRNA network involving KCNQ1OT1/miR-338-3p/RRM2 in glioma cells.
2 Materials and methods
2.1 Glioma tissues and cell lines
Thirty patients with glioma were recruited from Qianjiang Central Hospital of Hubei Province. The glioma patients were validated via histological examination and classified as grade I (n = 4), II (n = 6), III (n = 12) and IV (n = 8) according to the WHO classification system. When detecting gene expression levels, the samples were treated as tumor samples regardless of the classification. None of patients had received chemotherapy or radiotherapy prior to surgery. The tumor tissues and adjacent normal tissues were collected during surgery and stored at -80°C.
Human glioma cell lines (A172, U251 and LN229) and normal human astrocyte cells (NHAs) were acquired from BeNa Culture Collection (Beijing, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Solarbio, Beijing, China) containing 10% fetal bovine serum (Solarbio) at 37°C and 5% CO2 conditions, and culture medium was changed every 3 days.
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of Qianjiang Central Hospital of Hubei Province.
2.2 Construction of vectors or oligonucleotides and cell transfection
The KCNQ1OT1 overexpression vector (pcDNA3.1/ KCNQ1OT1) and RRM2 overexpression vector (pcDNA3.1/ RRM2) were generated by our laboratory, with pcDNA3.1 empty vector (Thermo Fisher, Wilmington, DE, USA) as control. The short hairpin RNA (shRNA) against KCNQ1OT1 (sh-KCNQ1OT1) and shRNA negative control (sh-NC) were designed and generated by Fulengen (Guangzhou, China). miR-338-3p mimic (miR-338-3p, 5’-CCAGCAUCAGUGAUU-3’) and miRNA negative control (miR-NC, 5’-UUCUCCGAACGUGUCACGUTT-3’) were generated by Hanbio (Shanghai, China). These constructed vectors or oligonucleotides were transfected into A172 and U251 cells with Lipofectamine 3000 (Thermo Fisher) according to the supplier’s instruction and the transfected cells were collected for further experiments after 24 h.
2.3 Quantitative real-time polymerase chain reaction (qRT-PCR) for RNA quantification
Tissues and cells were treated with Trizol reagent (Thermo Fisher) and total RNA was isolated. For the detection of levels of KCNQ1OT1 and RRM2, the complementary DNA (cDNA) was generated by high-Capacity cDNA Reverse Transcription kit (Thermo Fisher) and used for the qRT-PCR assay with SYBR mix (TaKaRa, Dalian, China) and the following specific primers (KCNQ1OT1: Forward, 5’-TGCAGAAGACAGGACACTGG-3’; Reverse, 5’-CTTTGGTGGGAAAGGACAGA-3’; RRM2: Forward, 5’-GCAAGCGATGGCATAGTA-3’; Reverse, 5’-TTCTATCCGAACAGCATTGA-3’). The internal control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Forward, 5’-AGACAGCCGCATCTTCTTGT-3’; Reverse, 5’-TGATGGCAACAATGTCCACT-3’). For the detection of levels of miR-338-3p, TaqMan microRNA Reverse Transcription kit (Thermo Fisher) was used for reverse transcription. The qRT-PCR response was performed with SYBR mix and specific primers (miR-338-3p: Forward, 5’-AACCGGTCCAGCATCAGTGATT-3’; Reverse, 5’-CAGTGCAGGGTCCGAGGT-3’), with U6 (Forward, 5’-ATTGGAACGATACAGAGAAGATT-3’; Reverse, 5’-GGAACGCTTCACGAATTTG-3’) as control. The relative gene expression was determined using the delta-delta cycle threshold method [25].
2.4 Cell counting kit-8 (CCK8) for cell viability
After the indicated transfection, A172 and U251 cells (1 x 104 per well) were collected and placed into 96-well plates in triplicates. At 0, 24, 48 and 72 h, each well was added with 10 μL CCK8 solution (Beyotime, Shanghai, China) and cells were continuously cultured for 2 h. The optical density at 450 nm was detected through a microplate reader (Bio-Rad, Hercules, CA, USA).
2.5 Flow cytometry for cell apoptosis
After the transfection, A172 and U251 cells were harvested and resuspended in DMEM at a density of 1 x 106 cells / mL. Aliquots of cell suspension (500 μL) were seeded into 24-well plates in triplicate and cultured for a further 72 h. After washing with phosphate buffer saline (PBS) twice, cells were resuspended in binding buffer and incubated with annexin V-fluorescein isothiocyante (FITC) and propidium iodide (PI) (Beyotime) for 10 min in the dark, followed by flow cytometric analysis (BD Biosciences, San Jose, CA, USA). The levels of apoptosis were expressed as % of cells at lower and upper right quadrants in the total number of cells.
2.6 Trans-well assay for cell migration and invasion
Transfected A172 and U251 cells were collected, washed with PBS and then resuspended in serum-free DMEM medium at a density of 1 x 105 cells / mL. Aliquots of cell suspension (200 μL) were added into the upper chambers of trans-well chambers precoated with or without Matrigel (BD Biosciences). The lower chambers contained 500 μL DMEM containing 10% serum. After 24 h incubation at 37°C, the cells in the upper chambers were removed and cells that had migrated across the membrane were stained with 0.5% crystal violet (Solarbio). Cell migration was quantified by counting 3 random fields of view using a microscope (200x magnification, Olympus, Tokyo, Japan).
2.7 Xenograft model of A172 cells
Ten male BALB/c nude mice (4-week-old) were purchased from Charles River (Beijing, China) and used for establishing a xenograft model. The mice were randomly divided into two groups (n = 5/group). A172 cells stably transfected with sh-KCNQ1OT1 or sh-NC were subcutaneously injected into nude mice at a dose of 2 x 106 cells / mouse). The tumor volume was monitored every 5 days and calculated using the following formula: length x width2 x 0.5. After 30 days, mice were sacrificed and tumor tissues were weighed.
Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. Animal experiments were approved by the Animal Ethic Committee of Qianjiang Central Hospital of Hubei Province.
2.8 Luciferase reporter vector construction and luciferase activity assay
The starBase 2.0 database predicted potential complementary sequences between miR-338-3p and KCNQ1OT1 or RRM2. The sequences of KCNQ1OT1 and 3’ untranslated regions (UTR) sequences of RRM2 containing miR-338-3p binding sites were obtained and inserted downstream of psiCHECK-2 (Promega, Madison, WI, USA) to generate wild-type (wt) luciferase reporter vectors for KCNQ1OT1-wt and RRM2-wt. The corresponding mutant (mut) luciferase reporter vectors KCNQ1OT1-mut and RRM2-mut were constructed by mutating the seed sites of miR-338-3p. These constructed vectors together with miR-338-3p or miR-NC were transfected into A172 and U251 cells for 24 h and analysed for luciferase activity with a dual-luciferase reporter assay system (Promega).
2.9 Western blot for protein expression assay
Tissue homogenates and cell suspensions were lysed in radio-immunoprecipitation assay buffer (Beyotime) at 4°C and centrifuged. Supernatants were retained and protein concentration were determined using the bicinchoninic acid protein assays kit (Thermo Fisher). After denaturization (100°C), proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electro-transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). After the membrane transfer, the membranes were blocked using western blocking buffer (Beyotime) and then incubated with the following primary antibodies against RRM2 (ab57653, 1:100 dilution, Abcam, Cambridge, MA, USA), PCNA (ab152112, 1:3000 dilution, Abcam), Bcl-2 (ab196495, 1:1000 dilution, Abcam), Bax (ab199677, 1:1000 dilution, Abcam), MMP-9 (ab73734, 1:1000 dilution, Abcam) or GAPDH (ab9484, 1:2000 dilution, Abcam) as a loading control, and corresponding secondary antibody. Enhanced chemiluminescence reagent (Solarbio) was used for determining protein expression levels.
2.10 Statistical analysis
GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA, USA) was used for graphics and statistical analyses. The data of 3 independent experiments were presented as mean ± standard deviation (S.D.). The linear correlation between levels of miR-338-3p and KCNQ1OT1 in glioma tissues was measured using the Pearson correlation coefficient test. Student’s t-test was used to compare the differences between two groups and ANOVA analysis followed by Tukey’s test was used for the comparison between multiple groups. The significant difference was considered at p value < 0.05.
3 Results
3.1 KCNQ1OT1 expression is enhanced in glioma tissues and glioma cell lines
To measure the lncRNA expression levels in glioma, 30 paired tumor and adjacent samples were collected from glioma patients. As shown in Figure 1A, the level of KCNQ1OT1 was significantly increased in glioma tissues compared with that in normal tissues. Moreover, expression of KCNQ1OT1 was also determined in glioma cell lines. Results showed high levels of KCNQ1OT1 expression in glioma cell lines (A172, U251 and LN229) when compared to expression levels in NHAs cells (Figure 1B). Since A172 and U251 cells expressed relatively higher levels of this lncRNA, these cell lines were used for further experiments.
The expression of KCNQ1OT1 is increased in glioma tissues and cell lines. (A) qRT-PCR assays was performed to detect the levels of KCNQ1OT1 in glioma tissues (n=30) and normal samples (n=30). (B) The expression of KCNQ1OT1 was quantified in glioma cell lines (A172, U251 and LN229) and normal human astrocyte cells (NHAs) by qRT-PCR. *p<0.05.
3.2 Silencing of KCNQ1OT1 inhibits cell viability, migration and invasion, and tumor growth, but induces apoptosis in glioma cell lines
To investigate the effect of KCNQ1OT1 on glioma progression in vitro, A172 and U251 cells were transfected with sh-KCNQ1OT1 or sh-NC. As displayed in Figure 2A and 2B, the transfection efficacy was confirmed with the results that KCNQ1OT1 abundance was markedly reduced in A172 and U251 cells after transfection with sh-KCNQ1OT1 compared with cells transfected with sh-NC. Moreover, cell viability was measured via CCK8 assay, and the data of CCK8 assay suggested that knockdown of KCNQ1OT1 evidently decreased the viability of A172 and U251 cells at 72 h (Figure 2C and 2D). Meanwhile, down-regulation of KCNQ1OT1 in these two cell lines induced high levels of apoptosis at 72 h (Figure 2E and 2F). Stably transfected A172 cells were used to establish a xenograft model to investigate the role of KCNQ1OT1 in glioma development in vivo. In the xenograft model mice were observed for a period of 30 days and, xenograft tumor volume and weight were significantly decreased in the sh-KCNQ1OT1 group when compared with animals in the sh-NC group (Figure 2G and 2H). In addition, glioma cell migration and invasion was investigated at 24 h and the results showed that silencing of KCNQ1OT1 led to decreased levels of migration and invasion in both A172 and U251 cells (Figure 3). Moreover, knockdown of KCNQ1OT1 led to a reduction in the expression of PCNA (pro-proliferation), Bax (pro-apoptotic) and MMP-9 (pro-metastatic), but an elevation in the expression of Bcl-2 (anti-apoptotic) in A172 and U251 cells (Supplementary Figure 1A and 1B).
Knockdown of KCNQ1OT1 suppresses cell viability and tumor growth but promotes apoptosis in glioma cell lines. A172 and U251 cells were transfected with sh-KCNQ1OT1 or sh-NC for 24 h and then relative levels of KCNQ1OT1 expression were determined (A and B). Levels of cell viability at 0, 24, 48 and 72 h (C and D) and apoptosis at 72 h (E and F) were detected by qRT-PCR, CCK8 assay and flow cytometry with FITC-conjugated annexin-V. In addition, a xenograft model using stably transfected A172 cells was established in nude mice and tumor volume (G) and weight (H) were determined in in groups treated with sh-KCNQ1OT1 and sh-NC (n=5). *p<0.05.
Knockdown of KCNQ1OT1 suppresses migration and invasion in glioma cell lines. Trans-well assays were used to determine levels of migration and invasion by A172 and U251 cells transfected with sh-KCNQ1OT1 or sh-NC at 24 h (Bar: 100 μm). *p<0.05.
3.3 Overexpression of KCNQ1OT1 increases cell viability, migration and invasion, but inhibits apoptosis by decreasing miR-338-3p in glioma cell lines
To explore the ceRNA network of KCNQ1OT1 in glioma development, the potential targets of KCNQ1OT1 were searched using starBase v2.0, and the tumor suppressor miR-338-3p was a candidate target. The predicted complementary sequences of KCNQ1OT1 and miR-338-3p are displayed in Figure 4A. To validate their association, KCNQ1OT1-wt and KCNQ1OT1-mut vectors were generated and transfected into A172 and U251 cells. The results of luciferase reporter assay described that overexpression of miR-338-3p led to more than 45% reduction in luciferase activity of KCNQ1OT1-wt in the two cell lines, while it showed little effect on luciferase activity of KCNQ1OT1-mut (Figure 4B). Furthermore, miR-338-3p expression was obviously increased via knockdown of KCNQ1OT1 (Figure 4C). In addition, miR-338-3p level was markedly decreased in glioma tissues and cells (Figure 4D and 4E). Meanwhile, miR-338-3p level in glioma tissues was negatively correlated with KCNQ1OT1 expression (Figure 4F).
KCNQ1OT1 is a decoy of miR-338-3p in glioma cell lines. (A) The complementary sequences of KCNQ1OT1 and miR-338-3p were searched using starBase v2.0. (B) Luciferase reporter assays were performed in A172 and U251 cells transfected with KCNQ1OT1-wt or KCNQ1OT1-mut and miR-338-3p or miR-NC. (C) The expression of miR-338-3p was measured in A172 and U251 cells transfected with sh-KCNQ1OT1 or sh-NC by qRT-PCR. (D and E) The expression of miR-338-3p was detected in glioma tissues (n=30) and cell lines. (F) The linear association between levels of miR-338-3p and KCNQ1OT1 in glioma tissues was analyzed. *p<0.05.
In order to further explore whether KCNQ1OT1-mediated regulation of glioma progression involved miR-338-3p, A172 and U251 cells were transfected with pcDNA3.1, pcDNA3.1/KCNQ1OT1, pcDNA3.1/KCNQ1OT1 and miR-NC or miR-338-3p. As shown in Figure 5A, the expression of miR-338-3p was effectively elevated in A172 and U251 cells after the transfection with miR-338-3p mimic. Additionally, overexpression of KCNQ1OT1 led to increased levels of cell viability, migration and invasion, as well as a decrease in the levels of apoptosis in the two cell lines, while these events were attenuated by addition of miR-338-3p (Figure 5B-5E and Supplementary Figure 2A and 2B).
Overexpression of KCNQ1OT1 increases cell viability, migration and invasion, but inhibits apoptosis by decreasing levels of miR-338-3p in glioma cell lines. (A) The expression of miR-338-3p was measured in A172 and U251 cells transfected with miR-338-3p or miR-NC for 24 h. Cell viability at 0, 24, 48 and 72 h (B), apoptosis at 72 h (C), migration (D) and invasion (E) at 24 h were detected in A172 and U251 cells transfected with pcDNA3.1, pcDNA3.1/KCNQ1OT1, pcDNA3.1/KCNQ1OT1 and miR-NC or miR-338-3p by CCK8, flow cytometry and transwell assay. *p<0.05.
3.4 Overexpression of miR-338-3p represses cell viability, migration and invasion, but induces apoptosis, by decreasing RRM2 in glioma cell lines
To further explore the ceRNA network in this study, the targets of miR-338-3p were analyzed. The binding sites of miR-338-3p and RRM2 were predicted by starBase v2.0 (Figure 6A). For validation of this prediction, luciferase reporter assays was performed in A172 and U251 cells. The luciferase activity in RRM2-wt group was notably reduced by overexpression of miR-338-3p, whereas it was not affected in RRM2-mut group (Figure 6B). Moreover, the mRNA and protein expression levels of RRM2 were significantly enhanced in glioma tissues and cell lines in comparison to their matched controls (Figure 6C-6E). In addition, the abundance of RRM2 at mRNA and protein levels were remarkably decreased by overexpression of miR-338-3p in A172 and U251 cells (Figure 6F).
RRM2 is a target of miR-338-3p in glioma cell lines. (A) The binding sites of RRM2 and miR-338-3p were predicted using starBase v2.0. (B) Luciferase reporter assays were performed in A172 and U251 cells transfected with RRM2-wt or RRM2-mut and miR-338-3p or miR-NC. (C) The mRNA and protein levels of RRM2 were measured in glioma tissues (n = 30). (D and E) The mRNA and protein levels of RRM2 were detected in glioma cell lines. (F) The abundances of RRM2 at mRNA and protein levels were examined in glioma cell lines transfected with miR-338-3p or miR-NC. *p<0.05.
To evaluate the function of miR-338-3p and whether it was associated with RRM2, A172 and U251 cells were transfected with miR-NC, miR-338-3p, miR-338-3p and pcDNA3.1 or pcDNA3.1/RRM2. The mRNA and protein levels of RRM2 were significantly inhibited by miR-338-3p overexpression in A172 and U251 cells, which was restored by introduction of a RRM2 overexpression vector (Figure7A and 7B). Furthermore, overexpression of miR-338-3p remarkably suppressed the viability and migration and invasion of these two cell lines, but promoted apoptosis, which was abrogated by restoration of RRM2 (Figure 7C-7F and Supplementary Figure 3A and 3B).
Overexpression of miR-338-3p represses cell viability, migration and invasion, but induces apoptosis by decreasing RRM2 in glioma cell lines. RRM2 mRNA and protein levels (A and B), cell viability at 0 h, 24 h, 48 h and 72 h (C), apoptosis at 72 h (D), migration (E) and invasion (F) at 24 h were determined in A172 and U251 cells transfected with miR-NC, miR-338-3p, miR-338-3p and pcDNA3.1 or pcDNA3.1/RRM2 by qRT-PCR, western blot, CCK8, flow cytometry and trans-well assay. *P<0.05.
3.5 Overexpression of KCNQ1OT1 up-regulates RRM2 expression by down-regulating miR-338-3p in glioma cell lines
To explore whether KCNQ1OT1 could regulate RRM2 via miR-338-3p in glioma, A172 and U251 cells were transfected with pcDNA3.1, pcDNA3.1/KCNQ1OT1, pcDNA3.1/ KCNQ1OT1 and miR-NC or miR-338-3p. The data of qRT-PCR revealed that RRM2 mRNA levels were significantly up-regulated by KCNQ1OT1 overexpression in A172 and U251 cells, which was weakened by the addition of miR-338-3p (Figure 8A). Similarly, the protein expression levels of RRM2 in the two cell lines was also increased by the presence of KCNQ1OT1 and decreased by introduction of miR-338-3p (Figure 8B).
Overexpression of KCNQ1OT1 up-regulates RRM2 expression by down-regulating miR-338-3p in glioma cell lines. (A and B) The RRM2 abundances at mRNA (A) and protein (B) levels were measured in A172 and U251 cells transfected with pcDNA3.1, pcDNA3.1/ KCNQ1OT1, pcDNA3.1/KCNQ1OT1 and miR-NC or miR-338-3p by qRT-PCR and western blot analysis. *p<0.05.
4 Discussion
It has been identified that lncRNAs play important roles in the diagnosis, prognosis and treatment of glioma [26]. However, the mechanisms underpinning the involvement of lncRNA KCNQ1OT1 in glioma progression remain to be elucidated. In the present work, we investigated the inhibitory effect of silencing KCNQ1OT1 in the progression of glioma, as defined by the inhibition of cell viability, migration and invasion, as well as promotion of glioma cell apoptosis. Furthermore, our study has for the first time identified an association with the miR-338-3p/RRM2 axis.
Previous studies have suggested that KCNQ1OT1 plays an oncogenic role in many human cancers [13, 27]. This present study showed high expression of KCNQ1OT1 in glioma tissues and glioma cell lines, suggesting its oncogenic role in glioma development. To investigate the potential effects of this lncRNA in glioma, loss-of-function experiments were performed. We found that KCNQ1OT1 knockdown promoted a decrease in cell viability and increased levels of apoptosis in glioma cells. The mechanism involved in KCNQ1OT1-induced apoptosis is currently unknown and could be further investigated in the future. Moreover, tumor migration and invasion are major risk factors for malignant gliomas [28]. By using trans-well migration and invasion assays, we confirmed the anti-migratory and anti-invasive roles of KCNQ1OT1 knockdown in glioma cells. These results indicated that knockdown of KCNQ1OT1 played an anti-cancer role in glioma, which was also consistent with previous study [15]. Furthermore, along with the in vivo xenograft model, this study uncovered an anti-cancer role of KCNQ1OT1 silencing in glioma development, indicating that this lncRNA may be a promising target for therapeutics in glioma.
LncRNAs may be involved in targeting miRNAs in glioma [29]. Previous research suggests that KCNQ1OT1 can promote cancer progression by acting as a sponge or ceRNA of miRNAs, such as miR-27b-3p and miR-140-5p [13, 14]. To find out whether KCNQ1OT1 serves as a ceRNA in glioma progression, we explored the targets of KCNQ1OT1 and confirmed an interaction between miR-338-3p and this lncRNA. In this study, the expression of miR-338-3p was decreased in glioma, suggesting the potential inhibitory role of this miRNA in glioma, which is supported by previous studies [20]. Moreover, it was reported that miR-338-3p suppressed cell proliferation and metastasis in glioma [21, 22]. Similarly, we also confirmed the anti-cancer role of miR-338-3p in glioma in vitro. In addition, overexpression of miR-338-3p abrogated the carcinogenic effect of KCNQ1OT1 on glioma development, indicating that KCNQ1OT1 regulated progression of glioma by decreasing miR-338-3p. Furthermore, we explored the functional target of miR-338-3p in glioma. RRM2 has been revealed as an oncogene that promotes glioma cell proliferation and migration [24]. Here we validated RRM2 as a functional target of miR-338-3p in the developments of glioma. Meanwhile, we found that KCNQ1OT1 could induce RRM2 expression by competitively binding with miR-338-3p because of the similar binding sites. Further, this study investigated the effects of KCNQ1OT1 knockdown using an in vivo xenograft model, which further confirmed the anti-cancer role of KCNQ1OT1 silencing in glioma in vivo. Although the current study implicates KCNQ1OT1 in the progression of glioma, it is important to emphasise that certain limitations also exist. Firstly, the clinical glioma sample size is too small to investigate the prognostic role for KCNQ1OT1 in patients. Secondly, the regulatory network involving KCNQ1OT1/miR-338-3p/RRM2 has not been validated in vivo. Hence, more comprehensive and detailed in vivo studies need to be performed in the future.
In conclusion, our study has showed the suppressive effect of KCNQ1OT1 inhibition on glioma progression by decreasing cell viability, migration and invasion, as well as increasing apoptosis, possibly via up-regulation of miR-338-3p and down-regulation of RRM2, thus elucidating a novel mechanism underlying the pathogenesis of glioma.
Conflict of interest: Authors state no conflict of interest.
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Supplementary Figures
KCNQ1OT1 regulates the levels of protein associated with cell viability, apoptosis, migration and invasion. (A and B) The protein levels of PCNA, Bcl-2, Bax and MPP-9 were detected in A172 and U251 cells transfected with sh-NC or sh-KCNQ1OT1. *P<0.05.
KCNQ1OT1 regulates the levels of protein associated with cell viability, apoptosis, migration and invasion by miR-338-3p. (A and B) The protein levels of PCNA, Bcl-2, Bax and MPP-9 were detected in A172 and U251 cells transfected with pcDNA3.1, pcDNA3.1/KCNQ1OT1, pcDNA3.1/KCNQ1OT1 + miR-NC or miR-338-3p. *p<0.05.
miR-338-3p regulates the levels of protein associated with cell viability, apoptosis, migration and invasion by RRM2. (A and B) The protein levels of PCNA, Bcl-2, Bax and MPP-9 were detected in A172 and U251 cells transfected with miR-NC, miR-338-3p, miR-338-3p + pcDNA3.1 or pcDNA3.1/RRM2. *p<0.05.
© 2020 Zhangxing Yin et al. published by De Gruyter
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