LncRNA RP11-757G1.5 sponges miR-139-5p and upregulates YAP1 thereby promoting the proliferation and liver, spleen metastasis of colorectal cancer

Background Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play a crucial role in tumorigenesis. However, the biological functions of lncRNAs in colorectal cancer (CRC) remain unclear. Microarray dataset analysis, FISH, RT-qPCR was used to detect RP11-757G1.5 expression in CRC tissues. The biological function of RP11-757G1.5 in CRC was determined by colony formation, Edu cell proliferation, wound healing and transwell assays. Bioinformatics binding site analysis, Luciferase reporter assay, Ago2 immunoprecipitation assays and western blot were performed to demonstrate the mechanism of RP11-757G1.5 acts as a molecular sponge of miR-139-5p. Moreover, we further explore the potential role of RP11-757G1.5 in CRC orthotopic xenografts. We discovered a novel lncRNA RP11-757G1.5, that was overexpressed in CRC tissues,especially in aggressive cases. Moreover, upregulation of RP11-757G1.5 strongly correlated with poor prognosis of patients with CRC. Functional analyses revealed that RP11-757G1.5 promotes cell proliferation in vitro and in vivo . Furthermore, RP11-757G1.5 enhanced cell migration and invasion in vitro and in vivo . Mechanistic studies demonstrated that RP11-757G1.5 regulated the expression of YAP1 through sponging miR-139-5p and inhibiting its activity thereby promoting CRC progression and development. RP11-757G1.5/miR-139-5p/YAP1

LncRNAs are a class of non-protein coding RNA molecules that consist of > 200 nucleotides. lncRNAs have been reported to regulate a broad range of functions including post-transcriptional and chromatin modification. They are also associated with the development of various cancers including liver [3], colorectal [4], gastric [5]and small cell lung cancer [6]. MicroRNAs (miRNAs) are a class of noncoding RNAs known to modulate various aspects of tumorigenesis and cancer progression [7,8], including gene expression. Evidence from recent studies show that some lncRNAs modulate gene expression by suppressing miRNA levels. Specifically, lncRNAs bind to miRNA, making them unavailable for interaction with mRNA, thereby acting as competitive endogenous RNA (ceRNA). This sponging of miRNAs lifts the suppression of gene expression by miRNAs.
Here, we uncovered RP11-757G1.5, a novel lncRNA that is highly expressed in CRC tissues. Kaplan-Meier analysis, log-rank test, univariate and multivariate analyses indicated that upregulated RP11-757G1.5 correlated with higher proliferation, invasion of CRC cells in vitro and in vivo, as well as poor prognosis of CRC. Our findings show that RP11-757G1.5 directly interacts with miR-139-5p, acting as a miRNA decoy to regulate YAP1 expression. Taken together, our findings highlight the therapeutic potential of RP11-757G1.5 in CRC.

Clinical samples
CRC tissues and adjacent non-cancer control tissue were obtained from patients diagnosed with CRC at the Department of general surgery, at the second affiliated hospital of Nanchang University. All patient specimens were collected with patients' written informed consent. The clinical information of all participants is summarized in Table 1. Ethical approval for this study was provided by the clinical research ethics committee of the second affiliated hospital of Nanchang University. Table 1 The correlation of the expression of RP11-757G1. 5

RNA extraction and RT-qPCR assay
RNA extraction was done using Trizol (Invitrogen, Cat. No. 15596-026). Reverse transcription was done using Superscript III transcriptase kit (Invitrogen, Cat. No. 18080-044). RT-qPCR was conducted on the Biorad CFX96 system using SYBR green. Primer sequences used are shown in Table 4. RT-qPCR was done using the following protocol: 55°C for 3 minutes, 95°C for 7.5 minutes, followed by 50 cycles at 95°C for 10 seconds, and 65°C for 2 minutes. The extension step was done at 95°C for 2 minutes, 50°C for 1 minute, and 50°C for 10 seconds. GAPDH was used as the reference gene. The PureLink® miRNA kit was used for extraction of miRNAs. The RT-qPCR protocol was as follows: 95°C for 3 minutes, followed by 50 cycles at 95°C for 10 seconds, and 55°C for 50 seconds. The reference genes were U6 and/or β-actin. Table 4 The sequences for RT-qPCR

Isolation of cytoplasmic and nuclear RNA
Cytoplasmic and nuclear RNA isolation and purification were done using cytoplasmic & nuclear RNA purification kit (Norgen, Cat. No. 21000) following manufacturer's instructions.

Cell invasion assay
Cell migration was examined using wound-healing assays. When cells were about 100% confluent, media was removed and a 10μl tip used to scrape the monolayer vertically. Cells were then washed 3 times with PBS 1X to remove cell fragments and then put back in culture. The cells were imaged at 0 and 48 hours after wounding using an inverted microscope (Olympus). Wound healing capacity was estimated based on the size of the gaps measured under the microscope.

RNA pull-down assay
HCT-116 cells were lysed in 1 ml of cell lysis buffer for 72 hours. 1.5μL of RNAse inhibitor, 10μL of streptavidin agarose beads and 500pM of antisense oligos were added and the cells rotated overnight at 4°C. The beads were washed 5 times using cell lysis buffer. The RNA was then analyzed by RT-qPCR analysis.

Ago2 immunoprecipitation assay
Transfected cells were lysed with RIPA lysis buffer and centrifuged for 20 minutes at 12000 rpm. 2μl of AGO2 antibody and 10μl of beads were added and the supernatant rotated overnight at 4°C. The mixture was washed 3 times with lysis buffer and RNA using Trizol reagent (Invitrogen, Cat. No.

Western blotting
Proteins were separated by electrophoresis on 8% or 10%) SDS-PAGE gels and then transferred onto 0.45μm PVDF membranes. The membranes were then blocked with non-fat milk for xx at xx. Both antibodies were diluted in xx at 1:1000.

Immunohistochemistry analysis
IHC analysis of YAP1 was done using the Dako Envision TM FLEX + system (Dako, Glostrup, Denmark) as described previously [15].

In vivo studies
Twenty-four 6-8-week-old nude mice were purchased from the Shanghai laboratory animal company.

Statistical analyses
Data are presented as the mean ± S.D. Student's t-test, the Mann-Whitney U-test and the χ2 test were used to analyze differences between groups. Survival rates were evaluated using Kaplan-Meier analysis and compared by the log-rank test. HRs and 95% CIs were calculated using Cox proportional hazards model. P-value < 0.05 was considered statistically significant.

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).

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.

Discussion
Accumulating evidence has underscored the significance of lnRNAs in the tumorigenesis and progression of CRC.
Besides the widely studied lncRNAs, HOTAIR [22], MALAT1 [23] and H19 [24], other lncRNAs participate in CRC pathogenesis. To identify such lnRNAs, we analyzed publicly available CRC microarray data. We uncovered a novel lncRNA RP11-757G1.5, which was highly expressed in CRC tissues and cell lines relative to the adjacent non-cancer tissues. Our data indicate that high RP11-757G1.5 expression positively correlates with larger tumor size, lymph node metastases, and high TNM staging as well as CRC poor clinical outcomes. LncRNAs modulate a wide range of biological processes, including tumor development and progression. This RNA class, therefore, has the potential for application in the diagnosis, treatment, and prediction of CRC clinical outcomes.
Loss-of-function assays showed that silencing of RP11-757G1.5 significantly suppressed CRC proliferation, invasion, and migration in vitro and in vivo, suggesting an oncogenic role for RP11-757G1.5 in CRC.
lncRNAs have been suggested to exert their biological functions by acting as ceRNAs, which by sponging miRNAs render them unavailable for interaction with target mRNA. The lncRNA DANCR, has been reported to act as a ceRNA for miR-335-5p and miR-1972, thereby enhancing ROCK1-mediated osteosarcoma pathogenesis [25]. The lncRNA MCM3AP-AS1 enhanced hepatocellular carcinoma by targeting the miR-194-5p/FOXA1 axis [26].
Another study found that miR-139-5p was significantly downregulated in CRC tissues which inhibited CRC development, proliferation and metastasis but promoted apoptosis and cell cycle arrest by targeting Notch1 signaling [14]. A variety of microRNAs have been reported to promote CRC carcinogenesis by modulating YAP1 signaling [27][28][29]. We therefore hypothesized that lncRNA RP11-757G1.5 might modulate CRC progression by targeting miR-139-5p-YAP1 activity. To test this possibility, we used luciferase reporter assays to assess the interaction between possible MREs and lncRNA RP11-757G1.5. As expected, we observed that miR-139-5p repressed the binding of lncRNA RP11-757G1.5 to its targets.
YAP1 has been implicated in various cellular processes and cancers [30,31,32]. However, the mechanism through which lncRNA regulates the function of YAP1 remains unclear. Here, we identified YAP1 as a direct target of miR-139-5p in CRC. Notably, we find that RP11-757G1.5 and miR-139-5p exhibit opposing functions in CRC, with RP11-757G1.5 promoting cancer progression and miR-139-5p suppressing it. Additionally, in CRC, a positive correlation was observed between RP11-757G1.5 and YAP1, while an inverse relationship was found between miR-139-5p and YAP1. Restoration of YAP1 suppressed the proliferation, migration and invasion of cells induced by RP11-757G1.5 knockdown.

Conclusion
Here, we uncovered a novel lncRNA that is significantly upregulated in CRC. Our data indicate that RP11-757G1.5 correlates with poor CRC prognosis and its silencing suppresses CRC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, we show that RP11-757G1.5 exerts its oncogenic function by sponging miR-139-5p, thereby upregulating YAP1 expression. Collectively, our findings highlight the potential of RP11-757G1.5 as a biomarker and therapeutic target in CRC.

Declarations
Natural Science Youth Foundation of Jiangxi Province (20192BAB215036), the Foundation for Fostering Young Scholar of Nanchang Universiy (PY201822), and Project of Jiangxi Provincial Innovation fund for graduate students (Nos. YC2019-B014).

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.    test YAP1 expression after HCT-116 cells were transfected with miR-139-5p mimic or co-transfected with miR-139-5p mimic and pcDNA3.1-757G1.5. Meanwhile, SW480 cells were transfected with miR-139-5p inhibitor or co-transfected with miR-139-5p inhibitor and sh-757G1.5#1. Data from Western Blot assay has been represented as a quantification graph normalized to the levels of GAPDH together with the statistical tests. *p<0.05, **p<0.01, ***p<0.001.  Each sample was run in triplicate and in multiple experiments for mean ± SEM. *p<0.05, **p<0.01.