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

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…

Fig. 2. Overexpression and stable knockdown of RP11-757G1.5 in HCT-116 and SW480 cells. a Relative expression of RP11-757G1.5 in six CRC cell lines and a colonic epithelial cell. b Fluorescence in situ hybridization (FISH) assay was conducted to determine the subcellular localization of RP11-757G1.5 in HCT-116 and SW480 cells. Nuclei are stained blue (DAPI), and RP11-757G1.5 are stained green. Scale bar, 25 μm. c-d Representative images of HCT-116 and SW480 cells transfected with pcDNA3.1-757G1.5 and sh-757G1.5#1; sh-757G1.5#2, respectively. Scale bar, 25 μm. e-f The validation of overexpression and knockdown efficacy of RP11-757G1.5 in CRC cell lines by RT-qPCR. *p < 0.05, **p < 0.01 by Student’s t-test, Data are representative of at least three independent experiments

Fig. 3. RP11-757G1.5 promotes CRC cell proliferation in vitro. a-b Effects of RP11-757G1.5 overexpression and knockdown on colony formation in CRC cells. c-d Effects of RP11-757G1.5 overexpression and downregulation on CRC cell proliferation were measured by a Edu assay. Scale bar, 25 μm. e-f Cell cycle analysis was conducted in HCT-116 and SW480 cells transfected with pcDNA3.1-757G1.5 and sh-757G1.5#1; sh-757G1.5#2, respectively. g-h Western blotting was performed to detect proliferation-associated antigen Cyclin D1 and PCNA expression in HCT-116, SW480 cells transfected with pcDNA3.1-757G1.5 or sh-757G1.5, respectively. *p < 0.05, **p < 0.01 by Student’s t-test. Data are representative of at least three independent experiments

Fig. 4. RP11-757G1.5 accelerates CRC cell migration and invasion in vitro. a-b Wound-healing assay was used to investigate the horizontal migration ability with RP11-757G1.5 overexpression or knockdown in CRC cells, and relative gap distance was calculated and plotted on a histogram. c-d The transwell assays were applied in both HCT-116 and SW480 cells to evaluate the CRC cell vertical migration and invasion abilities prior transfected with pcDNA3.1-757G1.5 or sh-757G1.5, respectively, and the number of cells was calculated and plotted on a histogram. Scale bar, 25 μm. e-f Immunoblotting analysis was performed to determine epithelial-mesenchymal transition marker E-cadherin and N-cadherin expression in HCT-116, SW480 cells transfected with pcDNA3.1-757G1.5 or sh-757G1.5, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test. Data are representative of at least three independent experiments

Fig. 5. RP11-757G1.5 sponges miR-139-5p and modulate YAP1 expression. a Subcellular localization of RP11-757G1.5 was determined by RT-qPCR in HCT-116 and SW480 cell lines. b miR-139-5p-binding sequence in RP11-757G1.5 and YAP1 3’UTR. A mutation was generated in RP11-757G1.5 in the complementary site for miR-139-5p binding. c Luciferase activity of a luciferase reporter plasmid (pLuc) containing wild-type or mutant RP11-757G1.5 co-transfected with miR-139-5p was determined using the dual luciferase assay. d MiR-139-5p and pLuc plasmid containing YAP1 3’UTRs were co-transfected with pcDNA3.1-757G1.5 or empty vector into HCT-116 cells to verify whether RP11-757G1.5 can function as a ceRNA of miR-139-5p. e Cellular lysates from HCT-116 cells were used for RIP with an anti-Ago2 antibody or IgG antibody. The levels of RP11-757G1.5 and miR-139-5p were detected by RT-qPCR. f and l The expression levels of YAP1 in HCT-116 cells transfected with pcDNA3.1-757G1.5 and SW480 cells transfected with sh-757G1.5 were analyzed by RT-qPCR and western blotting. g The elevated expression of YAP1 in tissue level was detected by immunohistochemistry (IHC) test, normalized to para-tumor tissue group. Scale bar,…

Fig. 6. RP11-757G1.5 exerts tumor-promoting function in CRC by regulating the miR-139-5p/YAP1 axis. a-d The increased cell proliferation ability in pcDNA3.1-757G1.5 transfected CRC cells was abolished by ectopic miR-139-5p expression or YAP1 knockdown. The cell proliferation ability was measured by a colony formation and Edu proliferation assay. Scale bar, 25 μm. e Western blotting analysis of proliferation-associated antigens in HCT116 cells transfected with miR-139-5p mimic or co-transfected with miR-139-5p mimic and pcDNA3.1-757G1.5. f Western blotting was used to determine the changes of the proliferation-associated antigens in HCT-116 cells transfected with si-YAP1 (siRNA) or co-transfected with si-YAP1 and pcDNA3.1-757G1.5. Tubulin served as a loading control. g-j The increased cell migration and invasion ability in pcDNA3.1-757G1.5 transfected CRC cells were abolished by ectopic miR-139-5p expression or YAP1 knockdown. Cell migration and invasion were measured by wound healing and transwell assays. Scale bar, 25 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test. The data represented from at least three independent experiments

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