LncRNA SNHG7 Functions as an Oncogene in Cervical Cancer by Sponging miR-485-5p to Modulate JUND Expression

Abstract

Background: Long non-coding RNA (LncRNA) SNHG7 is involved in the development of multiple cancers. However, its role in cervical cancer (CC) has not been elucidated. This study aimed to explore the function of SNHG7 in CC progression and the underlying mechanisms.

Materials and methods: The expression levels of SNHG7 and miR-485-5p in CC tissues and cell lines were measured by qPCR. Functional experiments including CCK-8 assay, wound healing assay, transwell assay, flow cytometry, Western blot, luciferases reporter assay and immunoprecipitation (RIP) were performed to explore the SNHG7/miR-485-5p/JUND pathway. Additionally, in vivo study was carried out by establishing tumor xenograft models.

Results: We found that SNHG7 was markedly enhanced in CC tissues and cell lines, and associated with poor clinical characteristics. In vitro, knockdown of SNHG7 inhibited CC cell proliferation, migration and invasion, as well as aggravated cell apoptosis. As to mechanism investigation, rescue experiments revealed that miR-485-5p inhibitor could partially reverse the effects on CC cells induced by SNHG7 knockdown. SNHG7 upregulated JUND expression via miR-485-5p. Moreover, tumor xenograft models were established to confirm the findings in vivo.

Conclusion: SNHG7 promoted CC progression through miR-485-5p/JUND axis. The SNHG7/miR-485-5p/JUND pathway might provide a novel therapeutic target for CC treatment.

Keywords: CC; JUND; cell proliferation; cervical cancer; lncRNA SNHG7; miR-485-5p.

Figure 1

The upregulation of SNHG7 in CC tissues and cell lines. (A) Relative expression of SNHG7 in CC tissues and adjacent normal tissues was detected by qPCR. (B) Relative expression of SNHG7 in CC cell lines (Hela, SIHA, C-33A and HT-3) and normal cervical cells (Ect1/E6E7) was detected by qPCR. (C) The Kaplan–Meier curves of overall survivals with high and low SNHG7 expressions. **P<0.01.

Figure 2

Knockdown of SNHG7 reduced CC cell proliferation, migration and invasion. (A) Relative expression of SNHG7 in EC cells was detected after SNHG7 siRNAs (si-SNHG7#1 and si-SNHG7#2) were transfected. (B) CCK-8 was conducted to estimate the cell viability. (C) Flow cytometry was performed to determine the cell apoptotic rate. (D) Wound healing assay was carried out to estimate the cell migration. (E) Transwell assay was conducted to measure the cell invasion ability. **P<0.01.

Figure 3

SNHG7 sponged miR-485-5p. (A) Predicted binding sites between SNHG7 and miR-485-5p from Starbase. (B) miR-NC mimics or miR-385a-5p mimics was co-transfected with SNHG7 mut or SNHG7 wt, and luciferase reporter assay was performed 48 h later. (C) si-NC or si-SNHG7 was transfected into EC cells, and the relative expression of miR-485-5p was detected by qPCR 48 h later. (D) miR-NC mimics or miR-485-5p mimics was transfected into EC cells, and the relative expression of miR-485-5p was detected by qPCR 48 h later. (E) The direct interaction between SNHG7 and miR-485-5p was detected by RIP with Ago-2 antibody. (F) The relative expression of miR-485-5p in CC tissues and adjacent normal tissues was measured by qPCR. (G) The relationship between SNHG7 and miR-485-5p in CC patients was analyzed by Pearson’s correlation analysis. **P<0.01.

Figure 4

SNHG7-induced EC cell activity changes was mediated by miR-485-5p. (A) miR-485-5p expression was measured by qPCR with miR-485-5p inhibitor transfection. Then, EC cells were transfected with si-SNHG7 in the presence of miR-NC or miR-485-5p mimics, (B) cell viability was measured by CCK-8 assay; (C) cell apoptosis was determined by flow cytometry; (D) migration was detected by wound healing assay; (E) cell invasion was detected by transwell assay; (F) statistical assays were presented in bar graphs. **P<0.01 versus si-NC+miR-NC; ^##P<0.01 versus si-SNHG7+miR-NC.

Figure 5

miR-485-5p directly targeted JUND. (A) Predicted binding sites between miR-485-5p and JUND from TargetScan. (B) miR-NC mimics or miR-485-5p mimics was co-transfected with JUND mut or JUND wt, and luciferase reporter assay was performed 48 h later. (C) miR-NC or miR-485-5p mimics was transfected into EC cells, and the relative expression of JUND was detected by Western blot 48 h later. (D) The relative expression of JUND in CC tissues and adjacent normal tissues was measured by qPCR. (E) The relationship between miR-485-5p and JUND in CC patients was analyzed by Pearson’s correlation analysis. **P<0.01.

Figure 6

JUND was regulated by SNHG7/miR-485-5p axis. si-SNHG7 was co-transfected with si-NC or miR-485-5p inhibitor, then the mRNA and protein expression levels of JUND were estimated by qPCR (A) and Western blot (B, C). (D) The relationship between SNHG7 and JUND in CC patients was analyzed by Pearson’s correlation analysis. **P<0.01 versus si-NC+miR-NC; ^##P<0.01 versus si-SNHG7+miR-NC.

Figure 7

SNHG7 knockdown suppressed cervical tumor growth via miR-485-5p/JUND axis in vivo. Xenograft models were established by injecting sh-NC or sh-SNHG7 transfected HeLa cells into in the flanks nude mice, then (A) tumor volume was measured weekly; (B) tumor weight was measured 5 weeks later; (C) miR-485-5p mRNA expression was detected by qPCR; (D) JUND protein expression was detected by Western blot; (E) Ki67 was detected by IHC. **P<0.01 versus sh-NC. **P<0.01.

Figure 8

Diagram showing the regulation mechanism of SNHG7 in CC progression.