Medulloblastoma is the most common malignant brain tumor of childhood, with great potential to metastasize. However, the mechanisms of how medulloblastoma develops and progresses remain to be elucidated. The present study assessed the role of long noncoding RNA LOXL1-AS1 (lncRNA LOXL1-AS1) in the cell proliferation and metastasis in human medulloblastoma. It was initially found that LOXL1-AS1 was significantly overexpressed in clinical medulloblastoma tissues compared with the adjacent noncancerous tissues. LOXL1-AS1 was also highly expressed in medulloblastoma at advanced stages and differentially expressed in a series of medulloblastoma cell lines. Knockdown of LOXL1-AS1 using shRNAs significantly inhibited cell viability and colony formation capacities in D283 and D341 cells. Moreover, the cell proportion in the S phase was significantly increased, while the cell proportion in the G2/M phase was decreased after knockdown of LOXL1-AS1 in D283 cells and D341 cells. Cell cycle arrest led to eventual cell apoptosis by LOXL1-AS1 knockdown. Moreover, in a xenograft model of human medulloblastoma, knockdown of LOXL1-AS1 significantly inhibited tumor growth and promoted tumor cell apoptosis. In addition, knockdown of LOXL1-AS1 inhibited cell migration and reversed epithelial-to-mesenchymal transition (EMT). Western blot analysis further revealed that knockdown of LOXL1-AS1 decreased the phosphorylated levels of PI3K and AKT without affecting their total protein levels. These results suggest that LncRNA LOXL1-AS1 promoted the proliferation and metastasis of medulloblastoma by activating the PI3K-AKT pathway, providing evidence that knockdown of LncRNA LOXL1-AS1 might be a potential therapeutic strategy against medulloblastoma.
Medulloblastoma is the most common malignant brain tumor of childhood characterized with frequent extraneural metastasis [
Currently, the origin of cancer is considered as a step-by-step accumulation of alterations in cell function and molecular expression, which are widely reported to relate with mechanisms involving transcriptional regulation [
Emerging data has shown the critical role of lncRNAs in the development and progression of medulloblastoma. Tumor growth and metastasis of medulloblastoma have been reported to be strictly controlled by lncRNAs such as CCAT1 [
lncRNA LOXL1-antisense RNA (LOXL1-AS1) is encoded on the opposite strand of LOXL1. It is a novel lncRNA that has recently been identified using sequencing and genetic analysis [
The role of LOXL1-AS1 in human tumorigenesis remains unknown, so the present study aimed to investigate the expression profile and functional role of LOXL1-AS1 in medulloblastoma. To this end, the LOXL1-AS1 level was initially evaluated in clinical medulloblastoma tissues and in a series of medulloblastoma cell lines. Specific shRNAs targeting LOXL1-AS1 were then synthesized to modulate the expression of LOXL1-AS1. Cell viability, colony formation, and cell migration capacities were examined
A total of 50 cases that were clinically diagnosed with medulloblastoma at Jining No. 1 People’s Hospital and Sishui People’s Hospital were included in the present study. For each case, its cancerous tissues and the matched adjacent noncancerous tissues were obtained. All patients showed their full consent to participate in our study, and a written consent form was obtained from each patient. Protocols for using human tissues were approved by the ethical committee board at Jining No. 1 People’s Hospital and Sishui People’s Hospital University.
Human medulloblastoma cell lines Daoy, D283, D425, D341, and D458 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Los Angeles, CA, USA) supplied with 10% fetal bovine serum (FBS) (Gibco). Culture medium was refreshed every two days unless otherwise stated. Primary antibodies were commercially purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) except for the phosphorylation detection antibodies which were obtained from Cell Signaling Technology (Boston, MA, USA). For knockdown of lncRNA LOXL1-AS1, two specific shRNAs were chemically synthesized by GenePharma (Shanghai, China). A scramble shRNA was also synthesized serving as control shRNA.
Total RNAs of human tissues and cultured cells were isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. The quality and concentration of extracted RNAs were determined by collecting the absorbance at 260 nm with Nanodrop 2000. The RNAs were immediately transcribed into cDNAs using the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan). All real-time PCRs were performed with the SYBR Premix Ex Taq Kit (TaKaRa, Japan) in an ABI PRISM 7500 Real-Time System.
Total proteins were extracted using a RIPA lysis buffer (pH = 7.5, Beyotime Biotechnology, Nantong, China) to generate the whole protein lysate. An equal amount of 40
D283 and D341 cells with indicated treatments were seeded on sterile coverslips in a 24-well plate in DMEM with 10% FBS. After 24 h, cells were rinsed with PBS and fixed for 30 min in 4% paraformaldehyde. Subsequently, cells were penetrated with 2% Triton X-100 for 20 min and then blocked in 5% FBS for 1 h. After that, cells were incubated with primary antibodies at 4°C overnight. After washing by PBS, cells were incubated with corresponding secondary antibody in the dark for 1 h and counterstained for 30 min with 4
D283 and D341 cell viability were assessed using a cell counting kit-8 (CCK-8) assay (Beyotime, Nantong, China) according to the manufacturer’s protocol. Briefly, cells were seeded into 6 cm dishes and transfected with specific shRNAs against LOXL1-AS1 (shRNA1 or shRNA2 group) or with a scramble shRNA (control group). Twenty-four hours later, each group of cells were trypsinized and resuspended. Cells were then seeded into a 96-well plate at an initial concentration of 8000 cells per well. Cell proliferative rates were monitored for the following 5 days. On each monitored time point, an aliquot of 10
Human medulloblastoma cell lines D283 and D341 were pretransfected with specific shRNA against LOXL1-AS1 and spread into 12-well plates. Then, all plates were incubated for 2 weeks to allow colony formation. After that, colonies were stained with crystal violet (0.1%) for 30 min. A colony is defined as cell accumulation with over 50 cells. The total number of colonies was manually counted and averaged for each group.
For the wound-healing assay, cells were plated on 6-well plates to form a confluent monolayer. Wounds were made with sterile pipette tips. Wound recovery was observed every 6 hours. The wound recovery rate was then calculated after each monitored time point. A migration assay was carried out using Boyden chambers (tissue culture-treated, 6.5 mm diameter, 8
Flow cytometry analysis was performed to analyze cell cycle progression. Briefly, control or LOXL1-AS1-depleted D283 cells and D341 cells were washed with PBS twice and fixed with 70% ethanol for 30 min on ice. To degrade RNAs, 20 mg/ml of RNase (Sigma-Aldrich, NY, USA) was added for 1 h at 37°C. After RNA degradation, samples were then stained with 20 mg/ml propidium iodide (PI, Sigma-Aldrich), and cell proportion at each phase was assessed by FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) equipped with the ModiFit LT v2.0 software.
Cell apoptosis was analyzed using the Annexin V/PI apoptosis kit (Invitrogen, Shanghai, China) according to the manufacturer’s instruction. Briefly, D283 and D341 cells were seeded in a 6-well plate (2 × 106 cells/well) and transfected with a scramble shRNA (control group) or with the specific shRNA against LOXL1-AS1 (shRNA group). Both cell lines were then cultured with complete medium for 48 h. After that, cells were washed with iced PBS and resuspended with 100
Male athymic BALB/c nude mice that were 5 weeks old were maintained in a special pathogen-free (SPF) condition. A total of 10 mice were fed and randomly divided into the control or shRNA group (
Tumor tissues from the mouse model were paraffin embedded and cut into 4
Data were expressed as means ± standard deviation (SD). Comparisons between groups were analyzed using Student’s
Initially, the expression of LOXL1-AS1 was examined in clinical medulloblastoma tissues. In the 50 cases, the mean level of LOXL1-AS1 in medulloblastoma tissues was approximately 1.5-fold that in the adjacent noncancerous tissues (Figure
lncRNA LOXL1-AS1 was highly expressed in medulloblastoma. (a) qRT-PCR analysis of LncRNA LOXL1-AS1 in 50 cases of clinical medulloblastoma as well as their adjacent normal tissues. (b) The paired expression of LOXL1-AS1 was shown in the 50 cases. (c) All the 50 cases were subgrouped as T1, T2, T3, and T4 based on the tumor size. Relative level of LOXL1-AS1 in T3 and T4 cancerous tissues was significantly higher than that in T1 and T2 tissues. (d) qRT-PCR analysis of the relative level of LOXL1-AS1 in 5 medulloblastoma cell lines. The level of LOXL1-AS1 in the D458 cells was set as 1. LOXL1-AS1 levels in other cell lines were normalized to those in D458 cells. The
In view of the highest expression of LOXL1-AS1 in D283 and D341 cells, these two cell lines were selected as optimal to investigate the functional roles of LOXL1-AS1 in medulloblastoma. Specific shRNAs against LOXL1-AS1 were synthesized. It was shown that specific shRNAs depleted the expression of LOXL1-AS1 in D283 cells (Figure
Knockdown of LOXL1-AS1 inhibited cell viability and colony formation capacity in D283 and D341 cells. (a, b) Knockdown efficiency of two synthesized shRNAs against LOXL1-AS1 (termed as shRNA1 and shRNA2) was assessed in D283 cells and D341 cells, respectively. (c, d) After knockdown of LOXL1-AS1 in D283 and D341 cells, cell proliferative rates were monitored in a consecutive of 5 days. The absorbance on day 1 was set as 1 for each group of cells. (e, f, g) Control and LOXL1-AS1-depleted cells were subject to colony formation assay in D283 cells and D341 cells. Formed colonies were stained with crystal violet, and all colonies in each group were manually counted and averaged from three independent assays.
The effects of LOXL1-AS1 knockdown were then assessed on cell survival (Figure
Knockdown of LOXL1-AS1 arrested cell cycle at the S phase in medulloblastoma. (a) Cell cycle progression was analyzed in both D283 cells and D341 cells that were pretreated with or without specific shRNAs against LOXL1-AS1. (b, c) Cell proportions in the G0/G1, S, and G2/M phases were calculated in D283 cells and D341 cells, respectively. It was found that cells were proportionally accumulated in the S phase while cells in the G2/M phase were significantly decreased in both cell lines.
Subsequently, cell apoptosis was analyzed (Figure
Knockdown of LOXL1-AS1 promoted cell apoptosis in D283 and D341 cells. (a) Cell survival was determined in both cell lines with or without LOXL1-AS1 depletion. (b, c) The percentage of cell apoptosis was shown for D283 cells and D341 cells. It was found that the cell apoptosis was significantly promoted by either shRNA in both cell lines.
A xenograft model of human medulloblastoma was established by inoculating D283 cells into nude mice. D283 cells were pretreated with a scrambled shRNA or the shRNA1 prior to inoculation. QRT-PCR analysis was performed to confirm the efficiency of the shRNA1 to knockdown LOXL1-AS1 (Figure
Depletion of LOXL1-AS1 inhibited tumor growth in medulloblastoma
Cell migration capacity was then assessed using a wound-healing assay. It was observed that both D283 and 341 cells recovered the artificial wound, regardless of what treatments they received. However, the shRNA-treated cells showed slower recovery ability at 18 h in comparison with control cells (Figure
Knockdown of LOXL1-AS1 inhibited cell metastasis and reversed EMT processes in medulloblastoma. (a, b) Control or shRNA-treated D283 and D341 cells were subject to wound-healing assay. Representative images showing the wound recovery were shown at 0 h and 18 h for both cell lines. The wound-recovered area which represented the cell migration capacity was calculated for each group of cells. (c, d) Both D283 and D341 cells were subject to Transwell migration assay. Cells that migrated to the lower surface were stained with crystal violet. Transmigrated cells were counted and averaged from 5 randomly selected fields. (e) Immunofluorescent analysis of vimentin (mesenchymal marker) and E-cadherin (epithelial marker). (f) Western blot analysis of vimentin and E-cadherin in D283 and D341 cells. Expression of vimentin was less detected, whilst that of E-cadherin was largely detected after knockdown of LOXL1-AS1.
In addition, the epithelial marker, E-cadherin, and mesenchymal marker, vimentin, were detected, with an increase in E-cadherin immunofluorescence and a decrease in vimentin immunofluorescence observed after knockdown of LOXL1-AS1 (Figure
The molecular mechanisms that contributed to the LOXL1-AS1-mediated phenotype were explored. It was detected that the phosphorylated levels of PI3K (p-PI3K) and AKT (p-AKT) were remarkably decreased in LOXL1-AS1-depleted cells, whereas the total protein levels of PI3K and AKT remained unaffected by LOXL1-AS1 knockdown, indicating that LOXL1-AS1 activated the PI3K/AKT pathway in medulloblastoma (Figure
LOXL1-AS1 positively regulated the PI3K/AKT pathway in medulloblastoma cell lines. D283 cells and D341 cells were pretreated with scramble shRNA or specific shRNA1 or shRNA2 before collection of cell lysates. PI3K and AKT were detected at both the dephosphorylated and phosphorylated forms using Western blot analysis.
Medulloblastoma remains a major health problem threating children’s lives worldwide. Forty percent of patients suffering from medulloblastoma were found to have distant metastasis at diagnosis [
The present study provided
In addition, it was also found that knockdown of LOXL1-AS1 impaired cell migration capacities as evidenced by the wound-healing and Transwell migration assays. Cell migration was inhibited by approximately 50% in LOXL1-AS1-depleted D283 cells and D341 cells. EMT is a common manifestation of tumor metastasis and reflects epithelial cell plasticity, in which multiple regulatory molecules are involved, including the Zeb and Snail families [
Phosphorylation of PI3K and AKT is pivotal for their activation. Upon phosphorylation, PI3K activates and phosphorylates the downstream AKT to cause a cascade reaction [
In summary, the present study identified a novel lncRNA, LOXL1-AS1, as a critical mediator of cell proliferation and migration in medulloblastoma. LOXL1-AS1 is significantly overexpressed in clinical medulloblastoma tissues, while knockdown of LOXL1-AS1 expression impairs tumor cell growth and migration, as well as inactivation of PI3K/AKT. This is the first report of the expression profile and functional role of LOXL1-AS1 in human tumorigenesis, providing strong evidence that a synthetic compound or reagent targeting LOXL1-AS1 or the PI3K/AKT pathway might serve as promising clinical therapeutics against medulloblastoma.
The authors declare that they have no conflicts of interest.
Ran Gao and Rui Zhang contributed equally to this study.