LncRNA SNHG12 is up-regulated in DDP-resistant cervical cancer cell lines
The differential expression of SNHG12 between cervical cancer tissues and normal tissues using GEPIA2 (http://gepia2.cancer-pku.cn/#index) and UALCAN (http://ualcan.path.uab.edu/) based on TCGA database. The results showed that SNHG12 was dramatically higher in cervical cancer patients than in healthy people (Figs. 1A). The upregulation of SNHG12 wasn’t found to be associated with the stage (Figs. 1B) and subtype (Figs. 1C) of cervical cancer. Furthermore, it could be observed that the overall survival rate in the high expression group (n = 77) was lower than that in the low expression group (n = 227) from The Cancer Genome Atlas (TCGA) database (http://kmplot.com/analysis/index.php) (Fig. 1D). Our previous studies have shown that SNHG12 was up-regulated in cervical cancer tissues and cell lines , then we further explored whether SNHG12 was involved in the DDP resistance of cervical cancer cells. Interestingly. Then DDP-resistant HeLa/DDP and CaSki/DDP cell line were established with gradually increasing DDP concentration. SNHG12 expression was also significantly increased in both HeLa/DDP and CaSki/DDP cells compared with their parental cells by qRT-PCR, respectively (Fig. 1E). Therefore, these prompted us to suspect that SNHG12 might participate in DDP-resistance of cervical cancer.
Inhibition of SNHG12 suppressed the drug resistance of cervical cancer cells to DDP, whereas
To evaluate the role of SNHG12 in the regulation of cervical cancer chemoresistance to DDP, the expression of SNHG12 was knocked down by shRNA in both HeLa/DDP and CaSki/DDP cells. SNHG12 was dramatically decreased after SNHG12 silencing in both HeLa/DDP and CaSki/DDP cells (Fig. 2A). After transduction, the effects of SNHG12 on the proliferation and apoptosis of DDP-resistant cells were assessed. Upon 8 µg/ml DDP treatment, the IC50 value of SNHG12 knockdown was markedly lower than that of SCR group (Fig. 2B). Furthermore, apoptosis assay showed that SNHG12 silencing induced DDP-resistant cells apoptosis (Fig. 2C). Moreover, knockdown of SNHG12 led to a significant repressed anchorage-independent growth upon DDP treatment (Fig. 2D). In contrast, SNHG12 was overexpressed in HeLa and CaSki cells by transfecting SNHG12 and confirmed by qRT-PCR (Fig. 2E). As expected, overexpression of SNHG12 could enhanced the IC50 value (Fig. 2F) and colony formation (Fig. 2G), and inhibited apoptosis (Fig. 2H) of cervical cancer cells. In summary, these results revealed that SNHG12 played an important role in the chemoresistance of cervical cancer cell to DDP.
SNHG12 served as a molecule sponge for miR-503-5p
It is widely acknowledged that lncRNAs can act as competitive RNA (ceRNA) through sponging with miRNAs in various cancers . To determine the potential mechanism of SNHG12, online software program starbase v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php) was used to predict the possible miRNAs. To better understand the role of SNHG12 in the development of cervical cancer, LinkedOmics was used to analyze its related genes. The top 50 genes that were significantly positively or negatively correlated with SNHG12 are indicated with a heat map (Fig. 3A). Further the enrichment functions of GO annotations (Fig. 3B) and KEGG pathways (Fig. 3C) analyzed using GSEA showed that ribosome played a crucial role in biological process of SNHG12. By comparing all of the candidate genes predicted by starbase v2.0, miR-503-5p was selected due to the tumor suppressive roles of miR-503-5p in several cancers (Fig. 3D) [21–23]. To further investigate whether SNHG12 was a functional target of miR-503-5p, luciferase reporter was performed and our results showed that overexpressed miR-503-5p could downregulate the wild-type luciferase activity of SNHG12 but not the mutant of SNHG12 (Fig. 3E). Furthermore, miR-503-5p overexpression resulted in a significant decrease of SNHG12 expression (Fig. 3F), whereas SNHG12 knockdown increased miR-503-5p expression (Fig. 3G). Moreover, miR-503-5p was significantly decreased in cervical cancer tissues compared to the paired adjacent normal tissues (Fig. 3H). A negative correlation between SNHG12 and miR-503-5p expressions was observed in cervical cancer tissues (Fig. 3I). All these findings suggested that SNHG12 was a sponge of miR-503-5p and negatively regulated the expression of miR-503-5p.
MiR-503-5p was required for SNHG12-mediated drug sensitivity of DDP-tolerated cervical cancer cells
To determine the functional significance of miR-503-5p in the SNHG12-induced phenotype, a series of restoration assays were performed in DDP resistance of cervical cancer cells. Anti-miR-503-5p was used to inhibit miR-503-5p expression in SNHG12-depleted DDP-tolerant cervical cancer cells and miR-503-5p was significantly decreased after miR-503-5p inhibitor transfection (Fig. 4A). As illustrated in Fig. 4B and 4C, decreased IC50 value of SNHG12-depleted DDP-tolerant cervical cancer cells was increased by miR-503-5p inhibitor transfection. Furthermore, inhibition of miR-503-5p could significantly abrogate the effect of SNHG12 on DDP-tolerant cervical cancer cells. Colony formation assay could not be performed because of the few numbers of colonies after SNHG12 silencing. Taken together, these results implied that miR-503-5p was required for the biological functions of SNHG12 in cervical cancer cells.
WEE1 was directly targeted by miR-503-5p and partly controlled by SNHG12
Using online software program, TargetScan, starbase v2.0and miRDB, a total of 145 genes were identified to potentially regulate by miR-503-5p (Fig. 5A). Investigating genetic model of drug response (iGMDR) (https://igmdr.modellab.cn) was used to assess the drug sensitivity of WEE1 in cervical cancer . The results showed the related genes (Fig. 5B) and pathways (Fig. 5C) involved in drug response of WEE1. By comparing all candidate genes predicted by the programs, WEE1 was selected for further experiments due to the ontogenetic role of WEE1 in cervical cancer (Fig. 5D). Luciferase reporter assays were performed and the results revealed that miR-503-5p could dramatically suppress the luciferase activity of the reporter gene in recombinant plasmids containing the wild-type 3′UTRs of WEE1 in HEK-293T cells but did not affect the activities of mutant (Fig. 5E). In concordance with these results, the mRNA and protein expression of WEE1 was significantly decreased in miR-503-5p-overexpressed cervical cancer cells (Fig. 5F). Similar results were observed in SNHG12-depleted DDP-tolerant cervical cancer cells (Fig. 5G). Moreover, introduction of anti-miR-503-5p partly abrogated the downregulation of WEE1 induced by SNHG12 silencing in both HeLa/DDP and CaSki/DDP cells (Fig. 5H). In addition, the mRNA level of WEE1 was upregulated in cervical cancer tissues (Fig. 5I). A dramatically negative correlation was noted between miR-503-5p and WEE1 expression (Fig. 5J) and a positive correlation was also noted between SNHG12 and WEE1 expression in cervical cancer tissues (Fig. 5K). Taken together, these data indicated that the miR-503-5p/WEE1 axis mediated the effect of SNHG12 on cervical cancer cells DDP resistance.
SNHG12 silencing decreases cervical cancer cell resistance to DDP therapy in vivo
To investigate the in vivo effect of SNHG12 on cervical cancer DDP resistance, we generated xenografts using SNHG12 stale knockdown HeLa/DDP cells. Once the tumors reached a diameter of 1.0 cm, mice was divided into two groups and treated with either DDP or saline solution, respectively (Fig. 6A). DDP treatment led to a significant reduction of tumor volume compared to that of the group without DDP treatment (Fig. 6B). Furthermore, the growth curse and weight of the tumors with DDP treatment were decreased compared with those in the group without DDP treatment (Fig. 6C and D). In addition, the expression of c-Myc was remarkably decreased in SNHG12 silencing xenografts tissues compared to the PBS-treated xenografts sections (Fig. 6E), which further confirmed that SNHG12 has a positive effect on both the growth and drug resistance in vitro and in vivo.