Long noncoding RNA 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) acting as crucial regulators in tumorigenesis. However, its biological functions of lncRNAs in colorectal cancer (CRC) have not been systematically clarified. Methods An unbiased screening was performed to identify disregulated lncRNAs revealed to be implicated in CRC carcinogenesis according to an online-available data dataset. In situ hybridization (ISH), RT-qPCR and RNA fluorescence in situ hybridization (RNA-FISH) were applied to detect RP11-757G1.5 expression in CRC tissues and cell lines. The associations of RP11-757G1.5 with clinicopathological characteristics were analyzed. Their effects on prognosis were analyzed by the Kaplan-Meier analysis, Log-rank test, Univariate and Multivariate Cox regression analysis. The potential biological function of RP11-757G1.5 in CRC was investigated by Colony formation, Edu cell proliferation, Flow cytometry, Wound healing and Transwell assays. Bioinformatics binding site analysis, Luciferase reporter assay, Ago2 immunoprecipitation assays, RNA pull-down assay, RT-qPCR and Western blotting were utilized to demonstrate the mechanism of RP11-757G1.5 acts as a molecular sponge of miR-139-5p to regulate the expression of YAP1. Finally, we further explore the potential role of RP11-757G1.5 in CRC orthotopic xenografts in vivo. Results We discovered a novel oncogenic lncRNA RP11-757G1.5, that was overexpressed in CRC tissues, especially in aggressive cases. Moreover, up-regulation of RP11-757G1.5 strongly correlated with poor clinical outcomes of patients with CRC. Functional analyses revealed that RP11-757G1.5 promoted cell proliferation in vitro and in vivo. Furthermore, RP11-757G1.5 stimulated cell migration and invasion in vitro and in vivo. Mechanistic studies illustrated that RP11-757G1.5 regulated the expression of YAP1 through sponging miR-139-5p and inhibiting its activity thereby promoting CRC progression and development. Conclusions Altogether, these results reveal a novel RP11-757G1.5/miR-139-5p/YAP1 regulatory axis that participates in CRC carcinogenesis and progression.


Background
Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with approximately 1.3 million new cancer cases and over 0.6 million deaths reported each year [1]. The occurrence and development of CRC involve a series of complex changes at the genetic and epigenetic levels [2]. In terms of clinical treatment, high rates of metastasis, cancer recurrence, and chemoresistance, make CRC challenging to treat at any stage. As previous studies have described that resistin exposure can block cells in the G1 phase, thereby prolonging their resistance and delaying the progression of p53 non-functional colon cancer cells [3]. In the field of molecular targeted therapy, anti-epidermal growth factor receptor monoclonal antibodies, such as cetuximab or panitumumab, combined with chemotherapy have made some progress for the treatment of patients with RAS and BRAF wild-type metastatic CRC [4]. Immunotherapy has also made great progress in the therapy of CRC, two kinds of programmed cell death 1 (PD-1) blocking antibodies, pembrolizumab and nivolumab, show efficacy in patients with metastatic CRC who have defects in repair of mismatch repair and high microsatellite instability [5,6]. But it remains inadequate to ameliorate the clinical outcomes and prognosis of colorectal cancer patients, thus, this highlights the need for the development and further necessitating new therapeutic strategies.
Recently, lncRNAs (long non-coding RNAs) have been obseverved in a number of studies to be a key player in the incidence and growth of colorectal cancer. LncRNAs, which are also known as non-protein coding RNA molecules, consist of more than 200 nucleotides. They are said to control a wide range of functions which include post-transcriptional and chromatin modification. These type of RNAs are linked as well to the advancement of numerous cancers not excluding liver [7], colorectal [8], gastric [9], and small cell lung cancers [10]. MicroRNAs (miRNAs), a collection of non-coding RNAs, regulate a few stages of tumor genesis and cancer development and gene expression [11,12]. Aside from this, a number of human diseases are also closely linked to the atypical expression of miRNAs; especially in cancer cases. For example, Anti-miR-203, by acting on SOCS3, will hinder breast cancer evolution and stemness [13]. Previous studies have exhibited that the suppression of miR-203 can inhibit tumor growth and cell proliferation in ERpositive breast carcinegenic cells by inhibiting Cyclin D1 and pStat3. In ovarian cancer cases, the upregulation of MiR-205 was observed and the overexpression of miR-205 was associated with tumor metastasis [14]. Suggestions made by recent studies show that the modulation of gene expression by some IncRNAs is carried out through the suppression of miRNA levels. Precisely, lncRNAs act as competitive endogenous RNAs (ceRNA) by binding to miRNAs, rendering them inaccessible for binding with mRNA. This mooching of miRNAs boosts the suppression of gene expression by miRNAs.
Here, we uncovered RP11-757G1.5, a novel lncRNA that is highly expressed in CRC tissues. Results of the Kaplan-Meier analysis, Log-rank test, Univariate as well as Multivariate analyses, all showed that up-regulated RP11-757G1.5 was linked with higher expression, infiltration and metastasis of CRC cells in vitro and in vivo. Results also showed that this was linked with poor prognosis of patients with CRC. Our outcomes demonstrated that RP11-757G1.5 acts by directly interacting with miR-139-5p, behaving as an miRNA smokescreen to epigenetically activate downstream gene YAP1 expression, and as a result this mechanism promotes proliferation and migration in patients diagnosed with CRC. As a whole, our findings illuminate the therapeutic prospect of a novel RP11-757G1.5/miR-139-5p/YAP1 axis in CRC progression in the future.

Clinical samples
All CRC tissues and adjacent non-cancer control tissues performed in our study were collected from surgical specimens of CRC patients residing in the Department of general surgery of the second affiliated hospital of Nanchang University. All patient samples were obtained per patient's written informed consent. Table 1

Cell culture
The following CRC cell lines were acquired from the American type culture collection (ATCC): HT-29, HCT-116, SW480, SW620, LoVo and Caco-2, and finally, the normal human colonic epithelial cell line NCM460. All cells were cultivated in DMEM (Invitrogen) and enhanced with 10% Gibco FBS (Cat. No. 10100147), 1% Lglutamine (Thermo Fisher Scientific, Cat. No. 21051024), 25 units/ml penicillin (Gibco, Cat. No. 15140148) and 25 g/ml streptomycin (Gibco, Cat. No. 15140148). All cell lines were detected and authenticated as bacteria, mycoplasma free and short tandem repeat profiling as per ATCC's guidelines within the past 3 months. All the primers used in vector construction are shown in Table 2.

RNA extraction and RT-qPCR assay
RNA extraction was carried out using Trizol (Invitrogen, Cat. No. 15596-026). Reverse transcription was carried out using the Superscript III transcriptase kit (Invitrogen, Cat. No. 18080-044). RT-qPCR was conducted on the Biorad CFX96 system using SYBR green. RT-qPCR was executed through the following process: 55°C for 3 min, 95°C for 7.5 min, and subsequently 50 cycles at 95°C for 10 s, and 65°C for 2 min. The next step was done at 95°C for 2 min, 50°C for 1 min, and 50°C for 10 s. GAPDH was used as the reference gene. The Pure-Link® miRNA kit was used for mining of miRNAs. The RT-qPCR formula was as follows: 95°C for 3 min, followed by 50 cycles at 95°C for 10 s, and 55°C for 50 s. The reference genes were U6 and/or β-actin. All sequences for RT-qPCR are outlined in Table 3.

Isolation of cytoplasmic and nuclear RNA
The cytoplasmic & nuclear RNA purification kit (Norgen, Cat. No. 21000) was utilized to carry out nuclear RNA extraction and purification as per the manufacturer's instructions.
Cell proliferation assay CRC proliferation was assessed by colony formation and Edu incorporation assays. To carry out colony formation analysis, 350 transfected cells/well were seeded into 6well plates and cultured for 2 weeks. Next, colonies were fixed with 4% paraformaldehyde before staining with 0.5% crystal violet and the total number of colonies was counted. Edu assays were done using a commercial kit (Ribobio, Cat. No. C10310) according to the manufacturer's instructions as described previously [21]. All assays were executed in threefold.

In vitro cell migration and invasion assay
Cell horizontal migration was examined using woundhealing assays. When cells were approximately 95% confluent, media was extracted and a 10 μl tip used to scrape the monolayer perpendicularly. Cell fragmetns were mined from the cells by washing 3 times with PBS, after which the cells were put back in culture. The cells were imaged at 0 and 48 h after wounding using an inverted microscope (Olympus Corp, Tokyo, Japan). Wound healing capacity was determined by the size of the gaps measured under a microscope.
For cell vertical migration and invasion assays, the transfected cells were suspended once again in serumfree media and seeded into the upper chambers and then cultured for 72 h. A key point that needed extra attention was that, for cell invasion assays, 2 × 10 4 cells were seeded in the upper chambers of transwell plates which were coated with Corning Matrigel (BD Biocoat, Cat. No. 354234) for 2 h prior to seeding, while the migration experiment was not coated with Corning Matrigel. Further Steps were the identical, 700 μl of cell culture media supplemented with 10% FCS was then added into the lower chambers and the cells incubated at 37°C in normal cell culture conditions for 10-15 h. Cells that invaded the lower chamber were fixed with by methanol for 15 min at room temperature and stained with 0.1% (w/v) crystal violet in the dark. Assays were executed in threefold.

RNA pull-down assay
HCT-116 cells were lysed in 1 ml of cell lysis buffer for 72 h. 1.5 μL of RNAse inhibitor, 10 μL of streptavidin agarose beads and 500 pM of antisense oligos were added and the cells rotated overnight at 4°C. The beads were washed 5 times using cell lysis buffer. Afterwards, purified RNAs were extracted and analyzed by RT-qPCR to illustrate the presence of binding targets.

Ago2 immunoprecipitation assay
Transfected cells were lysed with RIPA lysis buffer and centrifuged for 20 min at 12000 rpm. 2 μl of Ago2 antibody and 10 μl of beads were added and the supernatant rotated overnight at 4°C. The resulting mixture was rinsed 3 times with lysis buffers and RNA using Trizol reagent (Invitrogen, Cat. No. 15596-026).

Western blotting analysis
Proteins were parted by electrophoresis on 8% or 10% SDS-PAGE gels and then transferred onto 0.45 μm PVDF membranes. The membranes were then obstructed with non-fat milk for 1-2 h at room temperature. All antibodies were diluted in Primary Antibody Dilution Buffer (Solarbio, Cat. No. A1810) at 1:1000. Finally, protein bands were perceived on X-ray film through ECL luminescence reagent (Solarbio, Cat. No. SW2010).

In vivo studies
Thirty-two 6-8-week-old nude mice were acquired from the Shanghai laboratory animal company. HCT-116 and SW480 cells expressing a luciferase reporter (pcDNA3.1luciferase) and stably expressing pcDNA3.1-757G1.5 and sh-757G1.5#1, were generated. Afterwards, 1 × 10 6 HCT-116 and SW480 cells per mouse (mixed with Matrigel at a 1:1 ratio), were injected subcutaneously or intravenously for the diagnosis of tumor growth and metastasis. Tumor development and metastasis were observed weekly by means of an IVIS Fluorescent Imaging System (IVIS Spectrum). The mice were sacrificed after 6 weeks and tumors collected for analysis.

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

Results
Overexpression of RP11-757G1.5 in CRC associates with poor prognosis To further ascertain lncRNAs that are differentially expressed in CRC, we primarily analyzed GSE63675 from GEO, which comprises of lncRNA data for 43 CRC tissues and 6 neighboring non-tumor control tissues. Of note, CRC tissues expressed 8 notably differentially expressed lncRNAs as compared with neighboring non-tumor tissues (Fig. 1a). Amid them, lncRNA RP11-757G1.5 was chosen for further analysis. Principally, in situ hybridization (ISH) was applied to gauge level of the expression of RP11-757G1.5 in tissues. As presented in Fig. 1b, RP11-757G1.5 was gradually strongly stained with staging and lymph node metastasis in CRC tissues comparing to adjacent tissues. Also, RP11-757G1.5 was intensively stained in CRC cells' cytoplasm. Next, we assessed the expression of RP11-757G1.5 in CRC tissues by RT-qPCR and it was ascertained that this lncRNA was markedly upregulated in CRC tissues relative to the non-tumor control tissue (p < 0.001, Fig. 1c). Successively, we appraised the link between high RP11-757G1.5 expression and clinicopathological features of the disease. Results were similar to ISH and pointed out that elevated RP11-757G1.5 expression was directly linked with significant lymph node metastasis and advanced TNM staging (Fig. 1d, e, S1A and S1B). To assess the importance of this relationship, we distributed 112 CRC patients into 2 sets in relation to the extent of RP11-757G1.5 expression: RP11-757G1.5-high and RP11-757G1.5-low. Pearson chi-square or Fisher's Exact tests revealed that elevated RP11-757G1.5 levels were linked with greater tumor size (p = 0.003), lymph node metastasis (p = 0.008) and advanced TNM staging (p < 0.001). A relationship between RP11-757G1.5 levels and other clinical features was not observed (  Table 4). Wholly, these findings recognized that high RP11-757G1.5 levels correlate with poor CRC clinical outcomes.

RP11-757G1.5 promotes CRC cell proliferation and cell cycle progression in vitro
Next, we assessed the expression pattern of RP11-757G1.5 in standard NCM460 and CRC cell lines (HT-29, HCT-116, SW480, SW620, LoVo, Caco-2) by RT- Fig. 1 LncRNA RP11-757G1.5 expression is upregulated in CRC tissues and is associated with poor prognosis. a Heat-maps of lncRNAs that were differentially expressed between CRC tissues and matched adjacent normal samples. RP11-757G1.5 was the most appropriate lncRNA to select in eight lncRNAs. The color scale shown below illustrates the relative RNA expression levels; red represents high expression, and blue represents low expression. b The expression of RP11-757G1.5 was determined by in situ hybridization at different lymph node metastasis, TNM stages of CRC patients compare with corresponding adjacent normal tissues. Scale bar, 50 μm. c Comparison of RP11-757G1.5 in CRC tissues (n = 64) and normal tissues (n = 56) by RT-qPCR. d-e RP11-757G1.5 expression at different lymph node metastasis (Normal: normal adjacent tissues; N0: no lymph node metastasis; N1: 1~3 regional lymph node metastasis; N2: ≥4 regional lymph node metastases, n = 18) and TNM stages (n = 15) of CRC patients. f-g Kaplan-Meier survival analysis of the overall survival and disease-free survival in two groups defined by low and high expression of RP11-757G1.5 in patients with CRC. The median expression of RP11-757G1.5 was used as cut-off. p = 0.0047 and p = 0.0129 by Log-rank test. *p < 0.05, **p < 0.01 by Student's t-test. Data are representative of at least three independent experiments qPCR. The outcomes revealed markedly higher levels of RP11-757G1.5 in CRC cell lines compared to NCM460 (p < 0.05, Fig. 2a). RP11-757G1.5 was expressed was most 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 accordance, the results of fluorescence in situ hybridization (FISH) also pointed out that lncRNA RP11-757G1.5 is mainly found in the cytoplasm (Fig. 2b). The possible biological function of RP11-757G1.5 in CRC was evaluated by observing the overexpression of RP11-757G1.5 in HCT-116 cells (Fig.  2c), while it was knocked down in SW480 cells (Fig. 2d).
RT-qPCR was conducted to confirm the success of the evaluation, and Fig. 2e and f (p < 0.05) show the outcomes.
The outcomes illustrated that RP11-757G1.5-overexpression leads to a reduction in the expression of epithelial cell marker E-cadherin and an augmention in the expression of another epithelial cell marker N-cadherin (p < 0.05, Fig. 4e, f). Therefore, we initially verified RP11-757G1.5 promotes metastasis through affecting the EMT process of CRC cells. Moreover, to further verify the cancer-promoting effect of RP11-757G1.5 in CRC. We selected SW620 (High metastasis) and HT-29 (Low metastasis) two cell lines of CRC, then successfully performed RP11-757G1.5 knockdown and overexpression, respectively. The results confirmed that RP11-757G1.5 acts as an oncogene which could promote metastasis of CRC (p < 0.05, Figure S4A, S4B). Collectively, these results strongly propose that the RP11-757G1. 5

Deregulation of lncRNA RP11-757G1.5 suppresses cell proliferation and invasion in CRC orthotopic xenografts
In this section, we explored the role of RP11-757G1.5 in CRC in vivo by building orthotopic xenograft mouse models. In short, HCT-116 cells overexpressing RP11-757G1.5 or with SW480 knockdown were xenografted subcutaneously into mice. The stimulating effect of RP11-757G1.5 knockdown on tumor evolution and migration was inspected by use of the IVIS imaging system. Results of this investigation showed that tumor luciferase activity in the pcDNA3.1-757G1.5 expressing cells was elevated in those transfected with an empty vector (Fig. 7a). Meanwhile, RP11-757G1.5-silencing exhibited approximately opposite effects (Fig. 7f). Moreover, we discovered that RP11-757G1.5 overexpression enhanced tumor growth (Fig. 7a-e). RP11-757G1.5 knockdown hindered tumor growth (Fig. 7f-j). In addition, overexpression of RP11-757G1.5 stimulated metastasis to the liver and spleen. However, when interfering with RP11-757G1.5, the metastasis of tumor cells to organs in the liver and spleen was significantly reduced (Fig. 7k-o). Evaluating the results, we could see that the elevation of RP11-757G1.5 behaves as an oncogene and contributes to tumorigenesis and liver, spleen metastasis of CRC in vivo.

Discussion
Accumulating evidences have greatly underscored lncRNAs are tumor-associated biological molecules and have been found to serve multiple functions in the h YAP1 was up-regulated in CRC tissue and cell lines as determined by a RT-qPCR, normalized to para-tumor tissue group and NCM460 group, respectively. i-k Correlation between RP11-757G1.5, miR-139-5p, and YAP1 expression in CRC and normal colon specimens as detected by RT-qPCR (n = 41). m Western blot assays were performed to 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 by Student's t-test. Data are representative of at least three independent experiments tumorigenesis and progression of CRC. Apart from well characterized lncRNAs, HOTAIR [28], MALAT1 [29] and H19 [30], the investigation of other prospective essential lncRNAs that play a role in CRC pathogenesis is also worthwhile. To ascertain these potential functional lncRNAs, we initially considered openly available CRC-associated microarray data. A novel lncRNA RP11-757G1.5 was discovered, which was seen to be greatly expressed both in CRC tissues and cell lines relative to the findings in adjacent non-cancer tissues. Followed by, the associations of RP11-757G1.5 with clinic and pathological characteristics were analyzed. Moreover, their effects on prognosis were analyzed by the Kaplan-Meier analysis, Log-rank test, Univariate and Multivariate Cox regression analysis. Meaningfully, our data implies that larger tumor size is positively correlated with the higher expression of RP11-757G1.5, along with lymph node metastases, high TNM staging, and poor clinical outcomes of CRC. In tumor evolution, lncRNAs regulate a wide array of biological processes, including tumor growth and progression of CRC. As such, this RNA group has the likelihood to be beneficial for application in the diagnosis, treatment, and prediction of CRC clinical outcomes. Subsequently, to further distinguish the effect of RP11-757 g1.5 on the biological behavior of CRC cells, we carried out cell proliferation and cell metastasis experiments, including Colony formation, Edu cell proliferation, Flow cytometry, Wound healing and Transwell assays. Loss-of-function assays displayed that subdual of RP11-757G1.5 markedly inhibited CRC proliferation, invasion, and migration in vitro. On the contrary, RP11-757G1.5-overexpressing promoted these processes. The mechanism is related to the regulation of Cyclin D1 and PCNA to promote proliferation, Ecadherin and N-cadherin to enhance cell metastasis. Nevertheless, more specific molecular mechanism needs further study. In vivo experiments establish that deregulation of RP11-757G1.5 represses cell accretion and infiltration in CRC orthotopic xenografts. Furthermore, overexpression of RP11-757G1.5 stimulated metastasis to the liver and spleen. Overall, these results suggested RP11-757G1.5 as an oncogene in CRC. LncRNAs have been advocated to exercise their biological tasks by behaving as ceRNAs, which by sponging miRNAs render them inaccessible for interaction with target miRNA. For instance, the lncRNA DANCR, has been described to behave as a ceRNA for miR-335-5p and miR-1972, thereby enhancing ROCK1-mediated osteosarcoma pathogenesis [31]. In hepatocellular carcinoma, the lncRNA MCM3AP-AS1 could enhance cancer development by affecting the miR-194-5p/FOXA1 axis [32]. In other studies, LINC00152 was described to promote CRC proliferation, metastasis, and 5-Fu resistance by impeding miR-139-5p [15]. A different study found that the substantial downregulation of miR-139-5p in CRC tissues inhibited CRC growth, proliferation and metastasis while in contrast encouraged cell death and cell Fig. 7 Deregulation of lncRNA RP11-757G1.5 suppresses CRC cell proliferation and invasion in CRC orthotopic xenografts. a-e Representative IVIS images of tumor size (a), macroscopic appearance (b), tumor growth curves (c), tumor weight (d), and metastasis (e) in pcDNA3.1-757G1.5 group vs control group. Scale bar, 100 μm. f-j Representative IVIS images of tumor size (f), macroscopic appearance (g), tumor growth curves (h), tumor weight (i), and metastasis (j) in sh-757G1.5#1 group vs control group. k-l Representative macroscopic appearance and IVIS image of metastatic foci (white arrows) in the liver (k) and spleen (l). Each sample was run in triplicate and in multiple experiments. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 by Student's t-test. Data were shown as mean ± SD for three independent experiments cycle arrest by targeting Notch1 signaling [20]. There are an array of microRNAs which have been stated to encourage CRC carcinogenesis by regulating YAP1 signaling [24,33,34]. Consequently, in the present study we hypothesized that lncRNA RP11-757G1.5 may regulate CRC advancement by targeting miR-139-5p-YAP1 action. To assess the likelihood of this, we used bioinformatics analysis and luciferase reporter assays to assess the interaction between possible MREs and RP11-757G1.5. Predictably, we saw that miR-139-5p did repress the binding of RP11-757G1.5-WT to its targets, while not RP11-757G1.5-Mut. Investigation of the subcellular locality of RP11-757G1.5 in CRC cells showed that it was in a position in the cytosol similar to that of miR-139-5p. In addition, RP11-757G1.5 silencing distinctly enhanced miR-139-5p levels in CRC cells. Additional study revealed that RP11-757G1.5 levels inversely associated with miR-139-5p levels. Subsequent luciferase reporter assays, Ago2 immunoprecipitation assays and RNA pull-down showed that RP11-757G1.5-WT physically interacted with miR-139-5p, thereby sponging miR-139-5p in molecular level. Moreover, we have presented convincing data to support that RP11-757G1.5 can encourage the proliferation and metastasis of CRC cells by sponging miR-139-5p in vitro.
YAP1 plays a part in several cellular processes and cancers [35][36][37]. However, it is still unclear by which mechanism lncRNA regulates the function of YAP1. Here, we identified YAP1 as a direct target of miR-139-5p in CRC by luciferase reporter assays. Particularly, we found that RP11-757G1.5 and miR-139-5p display contrasting functions in CRC, with RP11-757G1.5 stimulating cancer progression and miR-139-5p subduing it. In CRC, a positive correlation was also observed between RP11-757G1.5 and YAP1, while an inverse link 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. Finally, in an in vivo animal study, we proved once more that RP11-757G1.5 positively triggered tumor growth and liver, spleen metastasis in mice. Unfortunately, it is uncertain whether the RP11-757G1.5/miR-139-5p/YAP1 pathway is involved in the CRC tumorigenesis in vivo.
The pathogenesis of CRC is a multi-step and slow biological process which involves a vast number of molecular events and complicated mechanisms. The ceRNA network which is comprised by RP11-757G1.5/miR-139-5p/YAP1 is just the tip of an iceberg in colorectal cancer. We have cause to consider that multitudinous downstream targets and more precise mechanisms of RP11-lncRNAs are still worthy of further exploring.

Conclusion
In the present study, we uncovered a novel carcinogenic lncRNA that is radically upregulated in CRC. Data in our study point out that RP11-757G1.5 is related with poor CRC prognosis and its silencing suppresses CRC cell proliferation, migration, and invasion in vitro and in vivo. Mechanically, we demonstrate that RP11-757G1.5 exerts its oncogenic function by sponging miR-139-5p, thus upregulating YAP1 expression. Jointly, our findings offer insights into the potential of RP11-757G1.5 as a prognostic biomarker and therapeutic target in CRC.