Background
Glioblastoma has been regarded as one of the most common brain malignancy with high mortality [
1]. In spite of advances in modern therapies like chemotherapy, radiotherapy and surgery, glioblastoma patients remained to display the poor prognosis with low 5-year survival rate [
2‐
4]. Worse still, the pathogenesis and development of glioblastoma were extremely complicated [
5]. Thereby, gene-targeted therapy was supposed to be a relatively effective therapeutic tactic for patients with glioblastoma.
Genome‑wide sequencing has disclosed that less than 2% of genes were capable to encode proteins and more than 98% of the genome was non-coding genes. Long non-coding RNA (lncRNA) was one of non-coding RNAs (ncRNAs) with more than 200 nucleotides in length, which has been identified to play a critical role in diverse biological processes to stimulate diseases or tumors initiation and progression. SNHG8 was reported to directly sponge with miR-663 to regulate the growth, migration, and invasion of colorectal cancer cells [
6]. MALAT1 has been confirmed to improve cell cycle progression of pulmonary artery hypertension [
7]. Small nucleolar RNA host gene 29 (SNHG29) was also known as LRRC75A-AS1, which has been identified to regulate the development osteosarcoma [
8]. Additionally, SNHG29 was proved to be involved in the modulation of vascular calcification [
9]. Nevertheless, the biological roles of SNHG29 in glioblastoma remained unknown.
LncRNAs were supposed to regulate gene expression in several mechanisms such as gene imprinting, epigenetic modification and degrading miRNAs. Except those regulatory mechanisms, emerging researches proposed that lncRNAs functioned as a competitive endogenous RNA (ceRNA) by sponging microRNAs (miRNAs) to regulate the expression of target genes. As examples, the regulatory mechanism of HOTAIR in prostate cancer was to sponge miR-152 to increase the expression of FOXR2, modulating proliferation and apoptosis of prostate cancer cells [
10]. Long non-coding RNA PVT1 regulate miR-143/HK2 axis to drive tumor progression in gallbladder cancer [
11]. However, the regulatory mechanism of SNHG29 in glioblastoma has not been explored yet.
This research was aimed to explore the biological function and regulatory mechanism of SNHG29 in glioblastoma. We confirmed that SNHG29 regulates miR-223-3p/CTNND1 axis to promote glioblastoma progression via Wnt/β-catenin signaling pathway, implying a potential tactic for the treatment of glioblastoma patients.
Materials and methods
Tissue samples and cell lines
The glioblastoma samples (n = 33) and normal brain tissues (n = 33) were obtained from Qilu Hospital of Shandong University. All the specimens were rapidly maintained at − 80 °C. All of these patients have not received other anticancer treatment prior to operation. Informed consents of this research were signed by patients before surgery, and this exploration was approved by the Ethics Committee of normal human astrocyte (NHA) was purchased from Sciencell Research Laboratories (Carlsbad, CA, USA). In addition, human glioblastoma cells including U87, U251, Hs683 and CCD-25Lu were acquired from Chinese Academy of Science Cell Bank (shanghai, China). NHA cells were grown in astrocyte medium, while glioblastoma cells were cultivated in the Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) added with 10% fetal bovine serum (FBS). A damp atmosphere containing 5% CO2 at 37 °C was suitable for incubation of cell lines.
Cell transfection
The short hairpin RNA (shRNA) targeting SNHG29 or CTNND1 (sh-SNHG29#1/2/3 or sh-CTNND1#1/2/3) with negative control (sh-NC) was employed to knockdown SNHG29 or CTNND1 expression. Overexpressed miR-233-3p was obtained by transfected with miR-223-3p mimics with negative control (NC mimics). Overexpression vector for CTNND1 was constructed by cloning the full length of CTNND1 into pcDNA3.1 vector, and the empty pcDNA3.1 was taken as control. All the plasmids were synthesized by GenePharma (Shanghai, China) and transfected into glioblastoma cells through using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction.
Real-time reverse-transcription polymerase chain reaction (RT-qPCR)
Total RNA extraction from the tissues and cells was made with Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). The expression of SNHG29, CTNND1, FOXO1, CTNNB1, cyclin D1, c-myc and their internal control GAPDH in glioblastoma tissues, cells as well as corresponding controls was detected by SYBR Prime-Script RT-PCR Kit (TakaraBio, Japan). The expression of miR-223-3p and its endogenous control U6 was examined by TaqMan MicroRNA Reverse Transcription kit and TaqMan Universal Master Mix II (Applied Biosystems, Foster City, CA, USA). Relative expression values were measured and calculated through using 2−∆∆Ct method.
Cell proliferation assay
The Cell Counting Kit-8 (CCK-8) assay and colony formation assay were adopted to assess cell proliferation. For CCK-8 assay, U87 and U251 cells (1000 cells/well) transfected with sh-NC or SNHG29-specific shRNAs were cultivated in 96-well plates. 10 µl of CCK-8 solvent was supplemented to each well at five time-points. The absorbance at the wavelength of 450 nm was assessed using a microplate reader.
For colony formation assays, U87 and U251cells (250) were cultured for 14 days after being seeded in each well of six-well plates. To calculate colonies, 4% paraformaldehyde and crystal violet were separately used for fixation and staining.
Nuclear-cytoplasmic fractionation
The cytoplasmic or nuclear SNHG29 was segregated with the application of a PARIS kit (Life Technologies, MA, USA). Briefly, collected U87 and U251 cells were lysed on ice. The supernatant was harvested after centrifugation. The extracted RNAs and two controls (GAPDH for cytoplasm, U6 for nucleus) were tested by RT-qPCR, respectively.
Transwell assay
SNHG29-downregulated U87 and U251 cells were placed in the upper chamber. Afterwards, the lower chamber was supplemented with 600 µl of DMEM containing 10% FBS (Hyclone, Shanghai, China). After incubated for 24 h, cells on the lower chamber were subjected to methanol and crystal violet for fixation and staining. The number of migrated cells was counted under a microscope (IX71, Olympus, Tokyo, Japan).
Western blot
U87 and U251 cells were lysed with the help of RIPA lysis buffer (Beyotime Biotechnology, China). Primary antibodies, including E-cadherin (ab1416), N-cadherin (ab18203), Vimentin (ab92547), β-catenin (ab32572), cyclin D1 (ab16663), c-myc (ab32072) and GAPDH (ab8245) were purchased from Abcam company (Abcam, Cambridge, UK). Blots were imaged by ECL detection reagents (Amersham Biosciences, Sweden).
Microarray and KEGG pathway analysis
Total RNA extracted from glioblastoma tissues was washed by RNeasy mini kit (QIAGEN, Germany) under the instructions by manufacturers. RNA quality was thoroughly assessed by Qubit Fluorometer (Thermo Fisher Scientific, USA). Raw data were processed and subjected to KEGG pathway analysis.
RNA immunoprecipitation (RIP) assay
Magna RNA immunoprecipitation (RIP) kit (Millipore, Billerica, USA) was used for conducting RIP assay in U87 and U251 cells. Magnetic beads containing Ago2 or IgG (negative control) antibodies were added into cell lysate which was preserved in RIP buffer before. Relative expression of SNHG29, miR-223-3p and CTNND1 was detected by RT-qPCR.
Luciferase reporter assay
SNHG29-WT (or CTNND1-WT) and SNHG29-Mut (or CTNND1-Mut) were constructed into pmirGLO plasmids (Promega, Madison, USA). The constructed plasmids were co-transfected with miR-223-3p mimics or NC mimics into U87 and U251 cells for 48 h. Luciferase reporter assay system (Promega, Madison WI) was adopted to examine the relative luciferase activities.
Statistical analysis
SPSS 17.0 statistics software (SPSS, Inc., Chicago, IL, USA) was responsible to statistical analysis. Data in graphs were shown as the mean ± standard deviation (SD) from experiments that were repeated at least 3 times. The group differences were separately analyzed by Student’s t test and one-way ANOVA test. A value of P less than 0.05 was regarded as statistically significant.
Discussion
Glioblastoma is one of the most lethal types of tumors in the central nervous system [
12]. Surgery section, chemotherapy and radiotherapy were widely adopted to treat patients with malignant brain tumor whereas the result of these methods did not improve patient’s condition [
13]. The pathology and progression of glioblastoma was associated with both genetic and epigenetic changes [
14]. Alterations of molecules also have been reported to be associated with the recurrence of patients in primary glioblastoma [
15]. Thus, better understanding and further exploration of underlying mechanism of glioblastoma was urgently needed.
LncRNAs is a category of non-coding RNAs, playing an important part in pathological and physiological aspects [
16,
17]. The dysregulation of lncRNAs was closely related to cellular processes of tumors. Up-egulation of lncRNA LINC00174 promotes cell proliferation to facilitate colorectal carcinoma progression via miR-1910-3p/TAZ axis [
18]. Additionally, up-regulation of SNHG14 boosts cell migration and invasion in renal cell carcinoma [
19]. Likewise, glioblastoma tissues and cells also displayed higher SMHG29 expression than normal tissues and cells. Moreover, knockdown of SNHG29 limited glioblastoma cell proliferation, migration and EMT process.
Mechanistically, LncRNAs have been proved by abundant explorations to serve as a ceRNA to regulate tumor progression [
20‐
22]. Based on the theory of ceRNA pattern, we speculated that SNHG29 also functioned in this pattern. In our research, miR-223-3p expression was validated to combine with SHNG29 after prediction and screening. The expression of miR-223-3p was negatively correlated with SNHG29 expression. Furthermore, CTNND1 was then proved to serve as a target gene of miR-223-3p after the prediction of starBase and screening. Additionally, CTNND1 was negatively correlated with miR-223-3p. The rescue assays suggested that CTNND1 overexpression restored the inhibitory influence of SNHG29 knockdown on cell proliferation, migration and EMT process.
The Wnt/β-catenin signaling pathway has been identified to be closely associated with the regulation of many cellular events (proliferation, differentiation, migration, or EMT process) through modulating the ability of β-catenin protein [
23‐
25]. Recent studies demonstrated that some lncRNAs could affect the Wnt/β-catenin signaling pathway in multiple cancers [
26‐
28] In the current research, the markers expression of Wnt/β-catenin signaling pathway including β-catenin, c-myc and cyclin D1 was respectively decreased by CTNND1 suppression and increased by addition of LiCl. At last, the retraining effect on cell proliferation, migration and EMT process of Wnt/β-catenin signaling pathway inactivation caused by CTNND1 repression was abolished by LiCl addition. Collectively, this study analyzed the association between ANHG29 expression and the overall survival of glioblastoma patients, indicating the prognostic potential of SNHG29 in glioblastoma patients. Further clinical study will be made in our future study. Lack of animal study and absent of mechanism investigation on SNHG29 upstream are pitfalls of our current study, we will investigate more deep mechanism of this molecular pathway in future study.
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