Long non-coding RNA SNHG6 promotes the growth and invasion of non-small cell lung cancer by downregulating miR-101-3p

Abstract

Background: The aim of this study was to determine the function of long non-coding RNA small nucleolar RNA host gene 6 (SNHG6) in non-small cell lung cancer (NSCLC) and its underlying mechanisms.

Methods: The association of SNHG6 or miR-101-3p with clinicopathological characteristics and prognosis in patents with NSCLC was assessed by TCGA dataset. Cell proliferation and invasion were evaluated by MTT and Transwell assays and SNHG6-specific binding with miR-101-3p was verified by bioinformatic analysis, luciferase gene report and RNA immunoprecipitation assays. qRT-PCR and Western blot was used to assess the effects of SNHG6 on the expression of miR-101-3p and chromodomain Y like (CDYL) in NSCLC cells. A xenograft tumor model in vivo was established to observe the effects of SNHG6 knockdown on tumor growth.

Results: We found that increased expression of SNHG6 was associated with pathological stage and lymph node infiltration, and acted as an independent prognostic factor of tumor recurrence in patients with NSCLC. Silencing SNHG6 expression repressed cell growth and invasion in vitro and in vivo, but overexpression of SNHG6 reversed these effects. Furthermore, SNHG6 was identified to act as a sponge of miR-101-3p, which could reduce cell proliferation and attenuate SNHG6-induced CDYL expression. Low expression of miR-101-3p or high expression of CDYL was related to poor survival in patients with NSCLC.

Conclusions: Our findings demonstrated that lncRNA SNHG6 contributed to the proliferation and invasion of NSCLC by downregulating miR-101-3p.

Keywords: Growth; SNHG6; invasion; miR-101-3p; non-small cell lung cancer.

Figure 1

Increased expression of lncRNA SNHG6 was associated with poor survival and tumor recurrence in LAC patients. (a) TCGA cohort indicated an increased expression level of SNHG6 in 58 paired and 515 unpaired LAC tissues. (b) qRT‐PCR also showed an elevated expression level of SNHG6 in 10 paired LAC samples. (c) The cutoff value of SNHG6 was acquired by ROC curve in LAC according to the SNHG6 expression, and the patients' survival time and survival status by Cutoff Finder. (d) Kaplan‐Meier analysis demonstrated that the patients with high SNHG6 expression harbored a poorer survival and a higher tumor recurrence as compared with those with low SNHG6 expression (low SNHG6 expression, high SNHG6 expression), (low SNHG6 expression, high SNHG6 expression).

Figure 2

SNHG6 promoted proliferation and invasion of NSCLC cells. (a) qRT‐PCR analysis showed that SNHG6 had a lower expression in A549 cell lines but a higher expression in NCI‐460 cell line (BEAS‐2B, A549, NCI‐H23, NCI‐H1993, NCI‐H522, NCI‐H460). (b) qRT‐PCR analysis of the overexpression efficiencies of SNHG6 plasmids in A549 cell line or knockdown efficiency of sh‐SNHG6 in NCI‐H460 cell line (pcDNA3.1, SNHG6, sh‐NC, sh‐SNHG6). (c), (d), MTT and Transwell analysis of the effects of SNHG6 overexpression or knockdown on cell proliferation and invasion in A549 and NCI‐H460 cells (pcDNA3.1, SNHG6, sh‐NC, sh‐SNHG6). (e) Western blot analysis of the effects of SNHG6 overexpression or knockdown on PCNA and MMP2 expression in A549 or NCI‐H460 cell line (pcDNA3.1, SNHG6, sh‐NC, sh‐SNHG6), ( pcDNA3.1, SNHG6, sh‐NC, sh‐SNHG6). *P < 0.05; **P < 0.01.

Figure 3

miR‐101‐3p was associated with SNHG6 expression and prognosis in LAC patients. (a) TCGA analysis of the expression levels of miR‐101‐3p/−26a‐5p/26b‐5p in 39 paired and 415 unpaired LAC tissues. (b) Pearson correlation analysis of a negative correlation of SNHG6 with miR‐101‐3p expression in LAC tissues. (c) qRT‐PCR indicated a decreased expression level of miR‐101‐3p in 10 paired LAC samples. () Adjacent normal and () LAC. (d) The cutoff value of miR‐101‐3p was acquired by ROC curve in LAC according to the miR‐101‐3p expression, and the patients' survival time and survival status by Cutoff Finder. (e) Kaplan‐Meier analysis demonstrated that the patients with low miR‐101‐3p expression had a poorer survival as compared with those with high miR‐101‐3p expression. () miR‐101‐3p high expression and () miR‐101‐3p low expression.

Figure 4

SNHG6 negatively regulated miR‐101‐3p expression in NSCLC cells. (a) The binding sites between miR‐101‐3p and WT or Mut SNHG6 3′UTR. (b) The luciferase activity of WT SNHG6 3′UTR was decreased by miR‐101‐3p mimic and increased by miR‐101‐3p inhibitor, but the luciferase activity of Mut SNHG6 3′UTR was unaffected by miR‐101‐3p inhibitor or inhibitor in A549 and NCI‐H460 cells. () miR‐NC, () miR‐101‐3p‐mimic, () NC and () miR‐101‐3p‐inhibitor. (c) qRT‐PCR analysis of the overexpression efficiency of miR‐101‐3p mimic in A549 cell line or knockdown efficiency of miR‐101‐3p inhibitor in NCI‐H460 cell line. () miR‐NC, () miR‐101‐3p‐mimic, () NC and () miR‐101‐3p‐inhibitor. (d) qRT‐PCR analysis of the effects of SNHG6 overexpression or knockdown on miR‐101‐3p expression in A549 or NCI‐H460 cell line. () pcDNA3.1, () SNHG5, () sh‐NC and () sh‐SNHG6. (e) RIP assay and qRT‐PCR analysis of the increased enrichment levels of SNHG6 and miR‐101‐3p pulled down from Ago2 or IgG protein in A549 and NCI‐H460 cells. (f) MTT analysis of the cell viability after cotransfection with SNHG6 plasmids and miR‐101‐3p mimic in A549 cell line or sh‐SNHG6 and miR‐101‐3p inhibitor in NCI‐H460 cell line.() pcDNA3.1+miR‐NC, () SNHG6+miR‐NC, () pcDNA3.1+miR‐101‐3p mimic and () SNHG6+miR‐101‐3P mimic. () sh‐NC+NC, () sh‐SNHG6+NC, () sh‐NC+miR‐101‐3p inhibitor and () sh‐SNHG6+miR‐101‐3p inhibitor. *P < 0.05, **P < 0.01.

Figure 5

Identification of the targets of miR‐101‐3p in NSCLC tissues. (a) Bioinformatic identification of eight targets of miR‐101‐3p. (b),(c) TCGA analysis of the expression levels of eight targets of miR‐101‐3p in 58 paired and 515 unpaired NSCLC tissues. (d) Pearson analysis of the negative correlation of CDYL with miR‐101‐3p 412 expression in NSCLC tissues. (e) The cutoff value of CDYL was acquired by ROC curve in NSCLC according to the CDYL expression, and the patients' survival time and survival status by Cutoff Finder. (f) Kaplan‐Meier analysis demonstrated that the patients with high CDYL expression had a poorer survival as compared with those with low CDYL expression. () CDYL high expression and () CDYL low expression.

Figure 6

miR‐101‐3p reversed SNHG6‐induced CDYL expression in NSCLC cells. (a) The binding sites between miR‐101‐3p and WT or Mut CDYL 3′ UTR. (b) The luciferase activity of WT CDYL 3′UTR was decreased by miR‐101‐3p mimic and increased by miR‐101‐3p inhibitor, but the luciferase activity of Mut CDYL 3′UTR was unaffected by miR‐101‐3p inhibitor or inhibitor in A549 and NCI‐H460 cells. () miR‐NC, () miR‐101‐3p‐mimic, () NC and () miR‐101‐3p‐inhibitor. (c),(d) qRT‐PCR and western blot analysis of the CDYL expression levels after cotransfection with SNHG6 plasmids and miR‐101‐3p mimic in A549 cell line or sh‐SNHG6 and miR‐101‐3p inhibitor in NCI‐H460 cell line. () pcDNA3.1+miR‐NC, () SNHG6+miR‐NC, () pcDNA3.1+miR‐101‐3p mimic and () SNHG6+miR‐101‐3P mimic. () sh‐NC+NC, () sh‐SNHG6+NC, () sh‐NC+miR‐101‐3p inhibitor and () sh‐SNHG6+miR‐101‐3p inhibitor. *P < 0.05; **P < 0.01.

Figure 7

SNHG6 knockdown inhibited tumor growth in vivo. (a) Schematic representation of the comparison of subcutaneous xenograft tumors between sh‐NC and sh‐SNHG6 groups. (b) Tumor size was examined every other day and the tumor growth curve was drawn in sh‐SNHG6 and sh‐NC groups (sh‐NC and sh‐SNHG6). (c),(d), Tumor volume and weight were lower in the sh‐SNHG6 group as compared with the sh‐NC group. (e), IHC analysis demonstrated that Ki‐67 proliferation index was decreased in the sh‐SNHG6 group as compared with the sh‐NC group. *P < 0.05.