Long non-coding RNAs (lncRNAs) play vital roles in human cancers. It has been reported that lncRNA SNHG17 expression is dysregulated in different types of cancer and involved in cancer progression. However, the role of SNHG17 in lung adenocarcinoma (LUAD) remains unclear. The present study aimed to investigate the role of SNHG17 in LUAD. Reverse transcription-quantitative (RT-q) PCR analysis was performed to detect SNHG17 expression in LUAD tissues and cells. The effects of SNHG17 on cancer cell migration, invasion, proliferation and epithelial-to-mesenchymal transition (EMT) were assessed via Transwell, MTT and western blot assays, respectively. The interactions between SNHG17 and microRNA (miRNA/miR)-193a-5p, miR-193a-5p and neuropilin and tolloid-like 2 (NETO2) were assessed via the dual-luciferase reporter assay. NETO2 expression and its potential role in LUAD were analyzed via RT-qPCR analysis and the UALCAN database. The results demonstrated that SNHG17 expression was significantly upregulated in LUAD tissues and cells, and high SNHG17 expression was associated with tumor-node-metastasis stage and poor prognosis of patients with LUAD. SNHG17 knockdown inhibited cell migration, invasion, proliferation and the EMT process. In addition, the results revealed that SNHG17 functions as a competing endogenous RNA of miR-193a-5p. The results of the dual-luciferase reporter assay confirmed that miR-193a-5p can directly target SNHG17. NETO2 was also predicted as a target protein of miR-193a-5p, which was confirmed via the dual-luciferase reporter assay. The roles of NETO2 knockdown in cancer cells were rescued following transfection with miR-193a-5p inhibitor or overexpression of SNHG17. Notably, high NETO2 expression was associated with poor prognosis of patients with LUAD. Bioinformatics analysis demonstrated that the promoter methylation level of NETO2 decreased in LUAD. Taken together, the results of the present study suggest that SNHG17 expression is upregulated in LUAD tissues and cells, and SNHG17 exerts tumor promoting effect by targeting the miR-193a-5p/NETO2 axis.
Lung cancer is one of the most common malignancies and one of the leading causes of cancer-associated mortality worldwide (
Long non-coding RNAs (lncRNAs) play pivotal roles in various biological events (
Recent studies have reported that lncRNAs play roles in diseases by acting as ceRNAs to directly sponge miRNAs to upregulate mRNAs expression. For example, lncRNA TTN-AS1 is negatively associated with miR-142-5p expression in LUAD, and accelerates LUAD progression by promoting cell migration, invasion and epithelial-to-mesenchymal transition (EMT) by regulating the miR-142-5p/CDK5 axis (
The aim of the present study was to investigate the potential mechanism and roles of the SNHG17/miR-193a-5p/NETO2 axis in LUAD.
A total of 50 pairs of LUAD tumor tissues and corresponding adjacent normal tissues (4–5 cm from tumor tissue) were collected from patients with LUAD at the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) between June 2016 and June 2019. The tissue samples were stored at −80°C. Among them, there were 32 males and 18 females (age, <60 (n=26), 52±8; ≥60 (n=24), 69±9. No patients received radiotherapy and/or chemotherapy prior to surgery. The inclusion criteria were as follows: i) patients diagnosed with LUAD; ii) had no other diseases, and iii) provided informed consent. Patients not meeting these criteria were excluded. Patient characteristics are listed in
The normal lung cell line, BEAS-2B and the cancer cell lines, A549, H1299, H1650, H1975 and CALU-3 were purchased from the American Type Culture Collection. All cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc.). The 293T cells were purchased from Chinese Academy of Sciences and maintained in DMEM medium (Gibco; Thermo Fisher scientific, Inc.) supplemented with 10% FBS. All cells were maintained at 37°C with 5% CO2.
Total RNA was extracted from tumor tissues or cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed into cDNA using the cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). The following temperature protocol for RT was used: 85°C for 5 sec, 37°C for 15 min, then the samples were stored at 4°C. qPCR was subsequently performed on a Real-Time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBRGreen RT-PCR reagents kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The following thermocycling conditions were used: Initial denaturation at 95°C for 5 min, then 95°C for 5 sec, 60°C for 30 sec for 40 cycles. U6 was the internal control of miR-193a-5p, while GAPDH was the internal control for SNHG17 and NETO2. Relative expression level of gene was calculated using the 2−ΔΔCq method (
To obtain the SNHG17 overexpression vector, the full-length sequence of SNHG17 was cloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The short hairpin RNAs (shRNAs) of SNHG17 and negative control shRNA (sh-NC), small inhibiting (si) RNA NC and siRNA targeting NETO2 (50 nM) were synthesized by Shanghai GenePharma Co., Ltd. The miR-193a-5p mimics (5′-UGGGUCUUUGCGGGCGAGAUGA-3′) and miR-193a-5p inhibitor (5′-UCAUCUCGCCCGCAAAGACCCA-3′) were designed and synthesized by Guangzhou RiboBio Co., Ltd. The sequence of the NC mimics is 5′-UCACAACCUCCUAGAAAGAGUAGA-3′. The sequence of the NC inhibitor is 5′-UCUACUCUUUCUAGGAGGUUGUGA-3′. The concentration of mimics and inhibitor was 50 nM. The concentration of shRNA was 0.5 µg. Transfection was performed at 37°C for 6 h, then the medium was changed. Subsequent experiments were performed after 48 h of transfection. All transfections were performed using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The following sequences were used: sh-NC, 5′-UCUCCGAACGUGUCACGUU-3′; sh-SNHG17#1, 5′-GGATTGTCAGCTGACCTCTGT-3′ and sh-SNHG17#2, 5′-GGTGACGTGTCTTCAAGAAGA-3′; si-NETO2#1, 5′-GAUGGAAGUUGAUAAGGAAAU-3′; si-NETO2#2, 5′-GAAUAGAUGAGACGGGUACAUAGG-3′.
The MTT assay was performed to assess cell proliferation. Transfected A549 or H1299 cells were seeded into 96-well plates at a density of 2×103 cells/well. Cells were cultured for 24, 48 and 72 h at 37°C. MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA) were added into each well. Following incubation for 4 h at 37°C, absorbance was measured at a wavelength of 490 nm, using a microplate reader (BioTek Epoch; Agilent).
RIP was performed using the EZ-Magna RIP kit (MilliporeSigma), according to the manufacturer's instructions. A549 and H1299 cells were collected and lysed using RIP lysis buffer (MilliporeSigma). Cell lysis solutions were incubated with anti-Ago2 (cat. no. 2897; 1:50) or anti-IgG (cat. no. 6990S; 1:50) (both from Cell Signaling Technology, Inc.) conjugated with magnetic beads for 4 h at 4°C. The immunoprecipitated RNA was harvested and detected via RT-qPCR analysis.
The biotin-labeled SNHG17 and no-biotin-labeled NC were synthesized by Shanghai GenePharma Co., Ltd., and cultured with 50 µl M-280 Streptavidin magnetic beads (Invitrogen; Thermo Fisher Scientific, Inc.). The pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) was used to construct SNHG17 overexpression plasmids and transfected as aforementioned. The A549 or H1299 cells were lyzed with IP Lysis Buffer (Thermo Fisher Scientific, Inc.). Lysates (50 µg) were incubated with probe-coated beads (Thermo Fisher Scientific, Inc.). The RNA-bound complex was subsequently eluted with Elution Buffer (Thermo Fisher Scientific, Inc.) and the isolation of completes was performed using a magnet. The contents of target RNA were detected using RT-qPCR.
The cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured using a BCA kit (Beyotime Institute of Biotechnology). Proteins (35 µg per lane) were separated using 10% SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma). The membranes were blocked with 5% skimmed milk at room temperature for 2 h, then incubated with the following primary antibodies overnight at 4°C: E-cadherin [cat. no. 3195S; 1:1,000; Cell Signaling Technology, Inc. (CST)], β-catenin (cat. no. 8480S; 1:1,000; CST), vimentin (cat. no. 5741S; 1:1,000; CST), snail (cat. no. 3879S; 1:1,000; CST), twist (cat. no. 69366S; 1:1,000; CST), NETO2 (cat. no. ab109288; 1:1,000; Abcam) and β-actin (cat. no. 3700S; 1:10,000; CST). Following which, the membranes were incubated with the following HRP-conjugated secondary antibodies at room temperature for 1 h: Goat anti-rabbit (cat. no. ab205718; 1:2,000) and goat anti-mouse (cat. no. ab205719; 1:2,000) (both from Abcam). Protein bands were detected using ECL substrate (Thermo Fisher Scientific, Inc.).
Transfected cells (5×104 cells) were seeded into the upper chambers of Transwell plates (Corning, Inc.) in culture medium without FBS. RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS was plated in the lower chambers. The upper chambers of Transwell plates were coated with and without Matrigel (37°C for 8 h) for the invasion and migration assays, respectively. After incubation for 24 h at 37°C, the invasive cells were fixed with 4% paraformaldehyde for 10 min and subsequently stained with 0.1% crystal violet for 10 min, both at room temperature. Images of the stained cells were captured from five randomly selected fields using an inverted fluorescence microscope (magnification ×400).
The target proteins of miR-193a-5p were predicted using the online databases, TargetScan (
The wild-type (WT) or mutant (MUT) reporter plasmids of SNHG17 and NETO2 were constructed by cloning WT or MUT sequences into pGL3 luciferase reporter plasmids (Promega Corporation). The 293T cells (1×105/well) were seeded into a 24-well plate and co-transfected with SNHG17-WT, SNHG17-MUT, NETO2-WT or NETO2-MUT reporter plasmids, and the aforementioned miR-193a-5p mimics or mimics NC, using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activities were detected using the Dual-Luciferase Reporter Assay System (Promega Corporation) and normalized to
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc.). Data are presented as the mean ± SD. All the experiments were repeated three times. Student's t-test was used to compare differences between two groups, while one-way ANOVA followed by Tukey's post hoc test were used to compare differences between multiple groups. Kaplan-Meier survival analysis was used to analyze the effect of SNHG17 expression on survival time in patients with LUAD and the log-rank test was used to determine statistical significance. According to the median of SNHG17 expression level, the patients were divided into the high and low expression level groups. Association between SNHG17 expression and the clinicopathological characteristics of patients with lung adenocarcinoma was analyzed using a χ2 test. The correlation of gene expression was analyzed using linear regression analysis. P<0.05 was considered to indicate a statistically significant difference.
To determine the role of SNHG17 in LUAD progression, RT-qPCR analysis was performed to detect SNHG17 expression in LUAD cancer tissues and adjacent normal tissues. The results demonstrated that SNHG17 expression was significantly upregulated in cancer tissues compared with that in normal tissues (
In the migration and invasion assays, transfection with shRNAs significantly inhibited the migratory and invasive abilities of A549 cells (
It is well-known that lncRNAs can sponge miRNAs and inhibit their expression and functions (
The luciferase reporter vectors, including WT or MUT binding sequences were constructed and co-transfected with miR-193a-5p mimics into 293T cells. The luciferase activity of WT reporter vectors was significantly inhibited following transfection with miR-193a-5p mimics, while no differences were observed between the MUT groups (
To investigate the effects of miR-193a-5p on LUAD progression
The downstream target proteins of miR-193a-5p were predicted via bioinformatics analysis. Among the 33 potential target genes (
The role of NETO2 in LUAD cells was investigated. The binding sequence between miR-193a-5p and NETO2 was predicted (
Increasing evidence suggest that lncRNAs are aberrantly expressed in different types of human cancer (
Previous studies have reported the carcinogenic role of SNHG17. For example, SNHG17 contributes to the progression of pancreatic cancer by sponging miR-942 (
It has been reported that lncRNAs affect cancer progression by regulating the expression of miRNAs (
To further investigate the role of miR-193a-5p in inhibiting LUAD progression, bioinformatics analysis and the dual-luciferase report assay were performed, which confirmed that NETO2 is a direct target of miR-193a-5p. Previous studies have reported that NETO2 plays a critical role in cancer progression. For example, NETO2 expression was significantly upregulated in gastric cancer (GC) tissues compared with that in normal tissues, and positively associated with cancer stage and lymph node metastasis (
In conclusion, the results of the present study demonstrated that SNHG17 expression is upregulated in LUAD. SNHG17 knockdown inhibited cell proliferation, migration, invasion and EMT, suggesting that SNHG17 contributes to the progression of LUAD by acting as an oncogene. The regulation of SNHG17 in LUAD was realized by targeting the miR-193a-5p/NETO2 axis. Thus, SNHG17 may be used as a novel diagnostic marker or therapeutic target of LUAD. However, further studies are required to determine how the SNHG17/miR-193a-5p/NETO2 axis regulates LUAD progression. Thus, prospective studies will focus on investigating the molecular mechanism and effects of SNHG17 in LUAD tumor growth and tumor immune microenvironment
Not applicable.
No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
ZWZ analyzed the patient data. ZWZ and CLW designed the experiments and confirmed the authenticity of all the raw data. ZWZ, YLY, BZ, YCM and CC performed the experiments and analyzed the data. ZWZ and CLW drafted the original manuscript. All authors have read and approved the final manuscript.
The present study was approved by the Ethics Committees of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China; approval no. TJWZXYXEC-). Written informed consent was provided by all patients prior to the study start.
Not applicable.
The authors declare that they have no competing interests.
SNHG17 expression is upregulated in LUAD. (A) SNHG17 expression in normal lung tissues (n=50) and lung cancer tissues (n=50). ***P<0.001. (B) SNHG17 expression at different stages of LUAD; I/II (n=23), III/IV (n=27). ***P<0.001. (C) Kaplan-Meier survival analysis was performed to detect the survival time of patients with LUAD, based on SNHG17 expression. (D) Reverse transcription-quantitative PCR analysis was performed to detect SNHG17 expression in normal BEAS-2B lung cells and five LUAD cell lines. *P<0.05, **P<0.01 vs. BEAS-ZB. (E) A549 and H1299 cells were transfected with sh-NC, sh-SNHG17#1 or sh-SNHG17#2, and SNHG17 expression was detected. **P<0.01 vs. sh-NC. (F) SNHG17 expression increased in A549 and H1299 cells following transfection with pcDNA-SNHG17. *P<0.05, **P<0.01. LUAD, lung adenocarcinoma; sh, short hairpin; NC, negative control.
Regulation of SNHG17 on the migration, invasion, proliferation and EMT of A549 and H1299 cells. (A) Migration and invasion analyses of A549 and H1299 cells transfected with sh-NC, sh-SNHG17#1 or sh-SNHG17#2. *P<0.05, **P<0.01 vs. sh-NC; #P<0.05, ##P<0.01 vs. sh-NC. (B) The MTT assay was performed to assess cell proliferation in the indicated groups. **P<0.01 vs sh-NC; ##P<0.01 vs. sh-NC. (C) Western blot analysis was performed to detect the protein expression levels of the EMT markers following SNHG17 knockdown. *P<0.05, **P<0.01 vs. A549 sh-NC; #P<0.05, ##P<0.01 vs. H1299 sh-NC. (D) Migration and invasion analyses of A549 and H1299 cells following overexpression of SNHG17. *P<0.05, **P<0.01 vs. pcDNA3.1; #P<0.05, ##P<0.01 vs. pcDNA3.1. (E) The MTT assay was performed to assess the proliferation of A549 and H1299 cells following transfection with pcDNA3.1 and pcDNA-SNHG17. **P<0.01 vs. pcDNA3.1; ##P<0.01 vs. pcDNA3.1. (F) Western blot analysis was performed to detect the protein expression levels of the EMT markers following overexpression of SNHG17. *P<0.05, **P<0.01 vs. A549 pcDNA3.1; #P<0.05 vs. H1299 pcDNA3.1. EMT, epithelial-to-mesenchymal transition; sh, short hairpin; NC, negative control.
miR-193a-5p is a target of SNHG17. (A) Bioinformatics analysis indicated the potential binding sites between SNHG17 and miR-193a-5p. (B) Correlation analysis indicated a negative correlation between miR-193a-5p and SNHG17 expression in LUAD tissues, according to the StarBase database. (C) miR-193a-5p expression was downregulated in LUAD, according to the StarBase database. ***P<0.001. (D) miR-193a-5p expression analysis in cancers was obtained from the Tumor-miRNA-Pathway database. (E) Reverse transcription-quantitative PCR analysis was performed to detect miR-193a-5p expression in LUAD tissues and adjacent normal tissues. ***P<0.001. (F) miR-193a-5p expression was detected in normal and LUAD cell lines. *P<0.05 vs. BEAS-ZB. (G) The dual-luciferase reporter assay was performed to verify the association between SNHG17 and miR-193a-5p expression. **P<0.01 vs. NC mimics. (H) The interaction between SNHG17 and miR-193a-5p was assessed using the RIP assay. **P<0.01 vs. IgG; #P<0.05, IgG. (I) The enrichment of miR-193a-5p was assessed in A549 and H1299 cells via the RNA pull down assay. *P<0.05 vs. Bio-NC; ##P<0.05 vs. Bio-NC. (J) miR-193a-5p expression level in cells transfected mimics or inhibitor. *P<0.05 vs. NC mimics, &&P<0.01 vs. NC inhibitor; #P<0.05 vs. NC mimics, $P<0.05 vs. NC inhibitor. (K) SNHG17 expression level in cells transfected mimics or inhibitor. **P<0.01 vs. NC mimics, &&P<0.01 vs. NC inhibitor; ###P<0.05 vs. NC mimics, $$P<0.05 vs. NC inhibitor. (L) Correlation analysis was performed to detect the correlation between miR-193a-5p and SNHG17 expression. miR, microRNA; LUAD, lung adenocarcinoma; NC, negative control; WT, wild-type; MUT, mutant.
miR-193a-5p regulates migration, invasion, proliferation and EMT of A549 and H1299 cells. Migration and invasion analyses of (A) A549 and (B) H1299 cells transfected with miR-193a-5p mimics or inhibitor. (C) The MTT assay was performed to assess the proliferation of A549 or H1299 cells transfected with miR-193a-5p mimics or inhibitor. (D) The expression levels of the EMT markers were detected in A549 or H1299 cells transfected with miR-193a-5p mimics or inhibitor. *P<0.05, **P<0.01 vs. NC mimics; #P<0.05, ##P<0.01, ###P<0.001 vs. NC inhibitor. miR, microRNA; EMT, epithelial-to-mesenchymal transition; NC, negative control; OD, optical density.
NETO2 acts as an oncogene in LUAD. (A) The 33 potential downstream target proteins of miR-193a-5p were predicted using the StarBase, TargetScan and miRDB databases. NETO2, one of the 33 target genes, was further studied in LUAD. The expression and potential function of NETO2 were analyzed, according to UALCAN. (B) Expression of NETO2 across TCGA cancers. (C) Expression of NETO2 in LUAD cancer tissues and normal tissues. ***P<0.001. (D) Expression of NETO2 in different cancer stages of LUAD. ***P<0.001 vs. normal. (E) Association between NETO2 and nodal metastasis status of patients with LUAD. ***P<0.001 vs. normal. (F) Survival analysis of patients with LUAD based on NETO2 expression. (G) NETO2 expression increased in the presence of TP53 mutation. ***P<0.001 vs. normal; ###P<0.001 vs. TP53-Mutant. (H) The reduced promoter methylation level of NETO2 in LUAD. ***P<0.001. NETO2, neuropilin and tolloid-like 2; LUAD, lung adenocarcinoma; miR, microRNA; TCGA, The Cancer Genome Atlas.
miR-193a-5p regulates NETO2 expression and the functions of NETO2 are affected by miR-193a-5p and SNHG17. (A) The potential binding and mutation sites between NETO2 and miR-193a-5p. (B) The dual-luciferase reporter assay revealed that NETO2 is directly targeted by miR-193a-5p. *P<0.05 vs. NC mimics. (C) NETO2 expression was upregulated in LUAD cancer samples, according to the StarBase database. ***P<0.001. (D) NETO2 expression was significantly upregulated in LUAD cells. **P<0.01, ***P<0.001 vs. BEAS-2B. (E and F) Transfection with miR-193a-5p mimics or inhibitor regulated NETO2 mRNA and protein expression levels, respectively. *P<0.05, **P<0.01 vs. NC mimics; ###P<0.001 vs. NC inhibitor. (G) Knockdown result of NETO2. **P<0.01 vs. si-NC. (H) NETO2 knockdown inhibited the migratory and invasive abilities of A549 and H1299 cells, the effects of which were reversed following transfection with miR-193a-5p inhibitor or pcDNA-SNHG17. *P<0.05, **P<0.01 vs. si-NC; &P<0.05, &&P<0.01 vs. si-NETO2. miR, microRNA; LUAD, lung adenocarcinoma; NC, negative control; si, small interfering; WT, wild-type; MUT, mutant.
NETO2 function is affected by miR-193a-5p and SNHG17. (A) The proliferative ability of A549 and H1299 cells was assessed following transfection with the indicated groups. *P<0.05, **P<0.01 vs. si-NC; &P<0.05, &&P<0.01 vs. si-NETO2. (B) Western blot analysis was performed to detect the protein expression levels of the epithelial-to-mesenchymal markers following transfection with the indicated groups. *P<0.05, **P<0.01, ***P<0.001 vs. si-NC; &P<0.05, &&P<0.01 vs. si-NETO2. (C) Correlation analysis between NETO2 and miR-193a-5p expression. (D) Correlation analysis between NETO2 and SNHG17 expression. (E and F) The effects of miR-193a-5p and SNHG17 on NETO2 expression. **P<0.01, ***P<0.001 vs. si-NC; &&P<0.01, &&&P<0.001 vs. si-NETO2. miR, microRNA; si, small interfering; NC, negative control; OD, optical density.
Association between SNHG17 expression and the clinicopathological characteristics of patients with lung adenocarcinoma (n=50).
SNHG17 expression | ||||
---|---|---|---|---|
Characteristic | Number of patients | Low (n=21) | High (n=29) | P-value |
Sex | 0.793 | |||
Female | 18 | 8 | 10 | |
Male | 32 | 13 | 19 | |
Age, years | 0.536 | |||
<60 | 26 | 12 | 14 | |
≥60 | 24 | 9 | 15 | |
TNM stage | 0.027 |
|||
I/II | 23 | 14 | 9 | |
III/IV | 27 | 7 | 20 | |
EGFR mutation | 0.089 | |||
No | 28 | 12 | 16 | |
Yes | 22 | 9 | 13 | |
PD-L1 expression | 0.006 |
|||
Low | 22 | 14 | 8 | |
High | 28 | 7 | 21 |
Data were analyzed using the χ2 test.
P<0.05
P<0.01. TNM, tumor-node-metastasis; EGFR, epidermal growth factor receptor; PD-L1, programmed death ligand 1.