Overexpression of RP11-757G1.5 in CRC associates with poor prognosis
To identify lncRNAs that are differentially expressed in CRC, we analyzed microarray dataset GSE63675 from GEO, which consists of lncRNAs data for 43 CRC tissues and 6 adjacent non-tumor control tissue. Notably, 8 lncRNAs were differentially expressed in CRC tissues relative to adjacent non-tumor tissues (Fig. 1A). Among them, lncRNA RP11-757G1.5 was selected for further analysis. Next, we evaluated the expression of RP11-757G1.5 in CRC tissues by RT-qPCR and observed that this lncRNA was significantly upregulated in CRC tissues relative to the non-tumor control tissue (p < 0.001, Fig. 1B). Subsequently, we evaluated the relationship between high RP11-757G1.5 expression and clinicopathological features of the disease. Results indicated that elevated RP11-757G1.5 correlated with greater lymph node metastasis and advanced TNM staging (Fig. 1C-D). To assess the significance of this association, we divided 112 CRC patients into 2 groups depending on the level of RP11-757G1.5 expression: RP11-757G1.5-high and RP11-757G1.5-low. Pearson chi-square analysis or Fisher's Exact tests revealed that elevated RP11-757G1.5 levels correlated with larger tumor size (p = 0.003), lymph node metastasis (p = 0.008) and advanced TNM staging (p < 0.001). No apparent association was observed between RP11-757G1.5 levels and other clinical features (Table 1). Next, Kaplan-Meier analysis and log-rank test were done to establish the relationship between RP11-757G1.5 and CRC survival time. This analysis showed that patients in the RP11-757G1.5-high group exhibited a significantly shorter survival rate relative to those in the RP11-757G1.5-low group (45.741 ± 3.539 vs 69.818 ± 3.662 months; log rank = 4.178, p = 0.0047, Fig. 1E). Moreover, high RP11-757G1.5 levels correlated with poor disease-free survival (log rank = 9.561, p = 0.0129, Fig. 1F). Univariate and multivariate analyses revealed RP11-757G1.5 expression as an independent prognostic indicator of CRC (hazard ratio (HR) = 3.441, 95% confidence interval (CI) = 1.471–8.005, p = 0.008; HR = 2.015, 95% CI = 1.018–5.856, p = 0.019, Table 2). Taken together, these findings show that high RP11-757G1.5 levels correlate with poor CRC clinical outcomes.
Table 2
Multivariate analysis of clinicopathological factors for disease-specific survival
Variable | Subset | Univariateanalysis | | Multivariate analysis | |
| | p-value | HR (95% CI) | p-value | HR (95% CI) |
Gender | Male/female | 0.771 | 0.734 (0.415–1.859) | -- | -- |
Age at diagnosis(years) | < 60/≥60 | 0.474 | 0.626 (0.537–2.552) | -- | -- |
Differentiation | Well + moderately/poorly | 0.331 | 1.422 (0.614–2.683) | -- | -- |
Tumor size (cm) | < 5/≥5 | 0.002** | 2.742 (0.326–4.663) | 0.017* | 3.471 (0.527–6.028) |
Depth of invasion | T1 + T2/T3 + T4 | 0.095 | 1.116 (0.549–4.241) | -- | -- |
Location | Colon/rectum | 0.852 | 0.632 (0.412–1.743) | -- | -- |
Lymph node status | N0/N1 + N2 | 0.153 | 2.571 (1.306–4.663) | 0.216 | 2.154(1.146–6.915) |
TNM stage | I + II/III + IV | 0.003** | 13.145 (5.044–34.378) | 0.004** | 7.603(2.521–33.109) |
RP11-757G.15 | High/low | 0.008** | 3.441(1.471–8.005) | 0.019* | 2.015 (1.018–5.856) |
RP11-757G1.5 promotes CRC cell proliferation in vitro
Next, we evaluated the expression of RP11-757G1.5 in normal NCM460 and CRC cell lines (HT-29, HCT-116, SW480, SW620, LoVo, Caco-2) by RT-qPCR. The results revealed significantly higher levels of RP11-757G1.5 in CRC cell lines relative to NCM460 (p < 0.05, Fig. 2A). Among the CRC cell lines, RP11-757G1.5 expression was highest in SW480 (p < 0.001) and least in HCT-116 (p < 0.05). These 2 CRC cell lines were therefore selected for downstream experiments. In addition, fluorescence in situ hybridization (FISH) results suggested that lncRNA RP11-757G1.5 mainly located in the cytoplasm (Fig. 2B). To investigate the biological function of RP11-757G1.5 in CRC, RP11-757G1.5 was overexpressed in HCT-116 cells (Fig. 2C) and knocked down in SW480 cells (Fig. 2D). To minimize the chances of off-targeting, 3 shRNAs (sh-RP11-757G1.5#1 (or sh-757G1.5#1) and sh-RP11-757G1.5#2 (or sh-757G1.5#2)) were used (Fig. 2D). RT-qPCR was conducted to confirm the success of RP11-757G1.5 overexpression and knockdown, and results are shown in Fig. 2E-F, (p < 0.05). Further analyses showed that RP11-757G1.5 overexpression significantly enhanced HCT-116 proliferation and colony formation while these processes were suppressed by RP11-757G1.5 silencing in SW480 cells (Fig. 3A-D). Western blot analysis showed that overexpression of RP11-757G1.5 in HCT-116 enhanced Cyclin D1 and PCNA expression, factors known to promote cell proliferation (Fig. 3E) while RP11-757G1.5 knockdown diminished their expression (Fig. 3F). Taken together, these datasets suggest that RP11-757G1.5 may promote CRC progression.
RP11-757G1.5 promotes CRC cell migration and invasion in vitro
Next, we performed cell invasion and migration assays to determine the role of RP11-757G1.5 in CRC metastasis. We found that RP11-757G1.5 overexpression promoted HCT-116 cell migration and invasion (Fig. 4A-C). Conversely, RP11-757G1.5 knockdown suppressed these processes in SW480 cells (Fig. 4B-D). Taken together, these results suggest that the RP11-757G1.5 promotes CRC cell migration and invasion in vitro.
LncRNA RP11-757G1.5 acts as a molecular sponge of miR-139-5p to regulate YAP1 expression in CRC cells
To understand the mechanisms by which RP11-757G1.5 promotes CRC, we evaluated its subcellular localization HCT-116 and observed that it predominantly resided in the cytosol (Fig. 5A). This suggests it may function at the post-transcriptional level. Multiple studies have reported that lncRNAs may act as a sponge by binding competitively to miRNA, thereby modulating gene expression by making the unavailable for interaction with their target miRNA responsive elements (MREs). To investigate whether RP11-757G1.5 performs this role, we examined possible binding sites between miR-139-5p and RP11-757G1.5 by cloning the full-length RP11-757G1.5 containing the presumptive miR-139-5p binding sites (Fig. 5B). This analysis was done on the background that miR-139-5p and RP11-757G1.5, exhibit opposing functions in CRC. We therefore constructed a luciferase reporter plasmid (pLuc) containing RP11-757G1.5 (pLuc-RP11-757G1.5-WT) or a mutant bearing mutation in the miR-139-5p seed sequence (pLuc-RP11-757G1.5-Mut). Next, we examined the association between miR-139-5p and RP11-757G1.5 by luciferase reporter assays and observed that overexpressing miR-139-5p (or its miR-139-5p mimic) markedly suppressed pLuc-RP11-757G1.5-WT activity relative to the mutant reporter (Fig. 5C), confirming that miR-139-5p binds and inhibits RP11-757G1.5. To test whether this interaction is physical, we carried out RNA immunoprecipitation (RIP) with an anti-Ago2 antibody. We found enrichment for both RP11-757G1.5 and miR-139-5p in the Ago2 complex (Fig. 5E).
RP11-757G1.5 modulates YAP1 expression by competitively binding miR-139-5p
Several lines of evidence have shown that microRNAs modulate CRC pathogenesis via YAP1 regulation[16-18]. Using bioinformatics analysis, we found that miRNAs sequences are compatible with recognition sequences on RP11-757G1.5 and the 3'-UTR of YAP1, suggesting that miR-139-5p may interact with the RP11-757G1.5 sequence and its 3'-UTR (Figure 5B). Next, we tested whether the effects of RP11-757G1.5 on CRC pathogenesis are mediated by the miR-139-5p/YAP1 pathway. Thus, we evaluated the interaction between RP11-757G1.5, miR-139-5p, and YAP1 by luciferase assays and observed that, relative to the empty vector control, RP11-757G1.5 overexpression reversed the miR-139-5p-mediated inhibition of pLuc-NOTCH1-3'UTR luciferase output (Figure 5D), indicating that RP11-757G1.5 suppresses miR-139-5p-mediated inhibition of YAP1 by competitively binding miR-139-5p. Additionally, we found that YAP1 was elevated in CRC tissues and cell lines (Figure 5G-H). RP11-757G1.5 knockdown remarkably suppressed endogenous YAP1 levels in CRC cells (Figure 5F-L). Conversely, YAP1 expression was elevated by RP11-757G1.5 overexpression in CRC cells (Figure 5F-L). Next, we examined the relationship between RP11-757G1.5, miR-139-5p, and YAP1 expression in the GSE63675 dataset. The results showed that RP11-757G1.5 expression negatively correlated with miR-139-5p levels (r=−0.402, p=0.003), but positively correlated with YAP1 levels (r=0.380, p=0.006). Additionally, a negative correlation was observed between miR-139-5p and YAP1 expression in the GSE63675 dataset (r=−0.297, p=0.004) (Figure 5K). To investigate whether the effects of RP11-757G1.5 on YAP1 levels depend on miR-139-5p, HCT-116 cells were co-transfected with the miR-139-5p mimic and pcDNA3.1-RP11-757G1.5 (or pcDNA3.1-757G1.5) and the effects of this approach on YAP1 were evaluated. Results revealed a significantly higher protein levels of YAP1 in HCT-116 cells relative to cells transfected with miR-139-5p mimic. RP11-757G1.5 knockdown markedly reversed the inhibitory effects of miR-139-5p on YAP1 expression in SW480 cells, as revealed by western blot analysis (Figure 5M).
RP11-757G1.5 promotes tumor progression in CRC via miR-139-5p/YAP1 axis
MiR-139-5p and YAP1 have been reported to modulate CRC progression [9, 19-21]. We therefore wondered whether RP11-757G1.5 regulates the pathogenesis of CRC via miR-139-5p/YAP1 axis. To investigate this, we evaluated how miR-139-5p and YAP1 affect RP11-757G1.5-driven cell proliferation. This analysis revealed that miR-139-5p overexpression or YAP1 knockdown blocked RP11-757G1.5-driven CRC cell proliferation (Figure 6A-D). Next, western blotting was performed to investigate whether miR-139-5p and YAP1 affect CyclinD1 and PCNA levels in the context of RP11-757G1.5-driven cell proliferation. Results showed that miR-139-5p expression or YAP1 knockdown significantly reversed the effects of RP11-757G1.5 overexpression on CyclinD1 and PCNA expression (Figure 6E-F). Next, we assessed how the miR-139-5p/YAP1 axis affects RP11-757G1.5 driven migration and invasion of CRC cells. The results revealed that miR-139-5p expression or YAP1 silencing suppressed cell migration and invasion caused by RP11-757G1.5 overexpression in CRC cells (Figure 6G-J). Collectively, these data demonstrate that RP11-757G1.5 acts as an oncogene in CRC, at least in part by sponging miR-139-5p, thereby modulating YAP1 levels.
Deregulation of lncRNA RP11-757G1.5 suppresses cell proliferation and invasion in CRC orthotopic xenografts
Next, we investigated the role of shRP11-757G1.5 in CRC in vivo. Briefly, HCT-116 cells overexpressing RP11-757G1.5 or with SW480 knockdown were xenografted into mice subcutaneously. The effect of RP11-757G1.5 knockdown on tumor growth, tumor growth and metastasis was monitored using IVIS imaging system. This analysis revealed that tumor luciferase activity in the pcDNA3.1-757G1.5 expressing cells was higher than in those transfected with an empty vector (Figure 7A). Additionally, we found that RP11-757G1.5 overexpression enhanced tumor growth and metastasis (Figure 7A-E). RP11-757G1.5 knockdown suppressed tumor growth and metastasis (Figure 7F-J). Additionally, overexpression of RP11-757G1.5 promoted metastasis to the liver and spleen (Figure 7K-L). Taken together, these observations indicate that RP11-757G1.5 is an oncogene in CRC.