The expression of SNHG14 on retinoblastoma tissues and cells
The expression of SNHG14 in human retinoblastomas tissues and cells line was analyzed though qRT-PCR, and the data showed the level of SNHG14 was significantly increased in retinoblastoma tissues compared to in normal retina samples (Fig 1A). Similar, a notably up-regulation of SNHG14 expression in retinoblastoma cell lines (including WERI-RB1, SO-Rb50, HXO-Rb44 and Y79) compared to the normal retinoblastoma epithelial cell line APRE-19 was observed (Fig 1B). Thus, SNHG14 increase might be associated the development and progression.
Knockdown of SNHG14 inhibits proliferation and promotes apoptosis in retinoblastoma cells
To verify the effects of SNHG14 on retinoblastoma cells, HXO-Rb44 and Y79 cells were transfected with sh-SNHG14 or sh-NC, and a down-regulation of SNHG14 expression was observed in HXO-Rb44 and Y79 cells (Fig 2A). Subsequently, the effects of SNHG14 on cell proliferation and apoptosis were evaluated. CCK-8 assay showed knockdown of SNHG14 inhibited the proliferation of HXO-Rb44 and Y79 cells (Fig 2B, C). Meanwhile, the abrogation of SNHG14 induced apoptosis of HXO-Rb44 and Y79 cells (Fig 2D); besides that, SNHG14 silence reduced the level of Bcl-2, while elevated the level of cleaved-casp-3 and Bax in HXO-Rb44 and Y79 cells (Fig 2E), further suggesting SNHG14 silence promoted cell apoptosis. Taken together, Knockdown of SNHG14 inhibited the progression of retinoblastoma.
SNHG14 targeted miR-204-5p to promote retinoblastoma tumor progression
To investigate the underlying molecular mechanism of action of SNHG14 in retinoblastoma progression, the potential miRNA targets of SNHG14 were explored by bioinformatics analysis via TargetScan software, and SNHG14 was predicted to bind to miR-204-5p with a putative binding site (Fig 3A). MiR-204-5p was found to be significantly decreased in retinoblastoma tissues and cells compared to the normal control (Fig 3B, D), and was negatively correlated with SNHG14 expression in retinoblastoma tissues (r =-0.401; p=0.028) (Fig 3C). Subsequently, the dual luciferase reporter analysis showed that luciferase activity was conspicuously inhibited in HXO-Rb44 and Y79 cells transfected with WT-SNHG14 and miR-204-5p compared with the control, while there was no change in MUT-SNHG14 group (Fig 3E, F), indicating the direct interaction of SNHG14 and miR-204-5p. Additionally, we also found knockdown of SNHG14 significantly up-regulated the expression level of miR-204-5p (Fig 3G). Thus, we confirmed that SNHG14 targetedly suppressed miR-204-5p expression.
Base on the relationship between miR-204-5p and SNHG14, we detected whether the activity of SNHG14 on retinoblastoma cell was mediated by miR-204-5p. First, HXO-Rb44 and Y79 cells were transfected with anti-miR-204-5p or anti-NC, and anti-miR-204-5p transfection significantly reduced the level of miR-204-5p in HXO-Rb44 and Y79 cells compared with anti-NC transfection (Fig. 3H). Next, sh-NC, sh-SNHG14, sh-SNHG14 + anti-NC or sh-SNHG14 + anti-miR-204-5p was transfected into HXO-Rb44 and Y79 cells to conducted rescue assay. We discovered that miR-204-5p inhibition markedly reversed SNHG14 knockdown-mediated proliferation inhibition (Fig. 3I, J) and apoptosis promotion (Fig. 3K-M) in HXO-Rb44 and Y79 cells. These data indicated knockdown of SNHG14 regulated retinoblastoma progression by binding to miR-204-5p.
The expression and effects of HMGA1 on retinoblastoma
The expression level of HMGA1 were measured, and HMGA1 was significantly up-regulated in retinoblastoma tissues at mRNA and protein levels (Fig 4A, C); besides, HMGA1 mRNA expression was positively associated with SNHG14 (r=0.411, p=0.024) (Fig 4B). Also, relative to the normal APRE-19 cells, HMGA1 was elevated in HXO-Rb44 and Y79 cells (Fig 4D, E). Subsequently, HXO-Rb44 and Y79 cells were transfected with the sh-NC and sh-HMGA1 to explore the role of HMGA1 in retinoblastoma, and HMGA1 expression was significantly declined by sh-HMGA1 in HXO-Rb44 and Y79 cells (Fig 4F, G). After that, cell proliferative vitality was detected using CCK8 assays, and results illustrated HMGA1 knockdown inhibited Y79 and HXO-Rb44 cell proliferation (Fig 4H, I). Then flow cytometry and western bolt analysis indicated down-regulation of HMGA1 induced apoptosis of HXO-Rb44 and Y79 cells (Fig 4J, K). These results suggested HMGA1 silencing inhibited cell progression in retinoblastoma.
HMGA1 is a target of miR-204-5p
Considering the aberrantly expression of miR-204-5p and HMGA1 in retinoblastoma cells, we further studied the relationship between miR-204-5p and HMGA1. Though an online software TargetScan, we found HMGA1 might be a target of miR-204-5p (Fig 5A). Then a decline of luciferase activity in HXO-Rb44 and Y79 cells co-transfected with HMGA1 3’UTR-WT and miR-204-5p confirmed the direct interaction of miR-204-5p and HMGA1 (Fig 5B, C). Importantly, the expression of HMGA1, both mRNA and protein, was inhibited by miR-204-5p overexpression, but was elevated by miR-204-5p down-regulation in HXO-Rb44 and Y79 cells (Fig 5D-G). Thus, we verified that miR-204-5p targetedly suppressed HMGA1 expression.
SNHG14 inhibits proliferation and induces apoptosis by regulating the expression of HMGA1 via miR-204-5p in retinoblastoma cells
Given SNHG14 sponged miR-204-5p, and miR-204-5p targeted HMGA1, we investigated whether SNHG14 could regulate HMGA1 through miR-204-5p. The expression of HMGA1 was detected in HXO-Rb44 and Y79 cells transfected with sh-NC, sh-SNHG14, sh-SNHG14 + anti-NC or sh-SNHG14 + anti-miR-204-5p. Results exhibited that the level of HMGA1 was suppressed by SNHG14 knockdown, while was rescued by miR-204-5p inhibition (Fig 6A-B), suggesting SNHG14 regulated HMGA1 expression via miR-204-5p. Subsequently, HXO-Rb44 and Y79 cells were transfected with pcDNA or HMGA1, and HMGA1 expression was significantly increased in the HMGA1 transfected cells (Fig 6C), showing the successful transfection (Fig 6D). Next, sh-NC, sh-SNHG14, sh-SNHG14 + pcDNA, or sh-SNHG14 + HMGA1 was transfected into HXO-Rb44 and Y79 cells to performed rescues assay. We discovered that HMGA1 overexpression abated the inhibition on cell proliferation (Fig 6E, F) and the promotion on apoptosis (Fig 6G-I) mediated by SNHG14 knockdown in HXO-Rb44 and Y79 cells. Altogether, SNHG14 regulate retinoblastoma cell tumorigenesis by HMGA1.
SNHG14 knockdown inhibits retinoblastoma tumor growth in vivo
The role of SNHG14 in tumor growth in vivo was explored. As presented in Fig. 7A, B, SNHG14 knockdown impeded tumor growth, reflected by the decrease of tumor volume (Fig. 7A) and weight (Fig. 7B) in sh-SNHG14 group relative to the sh-NC group. Subsequently, molecular analysis in tumor masses showed SNHG14 silence reduced the levels of SNHG14 (Fig. 7C) and HMGCR (Fig. 7C, D), but elevated the level of miR-204-5p (Fig. 7C). Thus, we concluded that SNHG14 knockdown inhibits retinoblastoma tumor growth in vivo by regulating HMGCR and miR-204-5p.