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. 2021 Dec;22(6):818.
doi: 10.3892/ol.2021.13079. Epub 2021 Oct 5.

Long non-coding RNA SNHG17 promotes lung adenocarcinoma progression by targeting the microRNA-193a-5p/NETO2 axis

Zhiwei Zhang  1   2 , Yulan Yan  2   3 , Bin Zhang  1 , Yuchen Ma  1 , Chen Chen  1 , Changli Wang  1
Affiliations

Long non-coding RNA SNHG17 promotes lung adenocarcinoma progression by targeting the microRNA-193a-5p/NETO2 axis

Zhiwei Zhang et al. Oncol Lett. 2021 Dec.

Abstract

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.

Keywords: SNHG17; lung adenocarcinoma; microRNA-193a-5p; neuropilin and tolloid-like 2.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
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.
Figure 7.
Figure 7.
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.
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