Non-coding RNAs participate in the regulatory network of CLDN4 via ceRNA mediated miRNA evasion

Abstract

Thousands of genes have been well demonstrated to play important roles in cancer progression. As genes do not function in isolation, they can be grouped into "networks" based on their interactions. In this study, we discover a network regulating Claudin-4 in gastric cancer. We observe that Claudin-4 is up-regulated in gastric cancer and is associated with poor prognosis. Claudin-4 reinforce proliferation, invasion, and EMT in AGS, HGC-27, and SGC-7901 cells, which could be reversed by miR-596 and miR-3620-3p. In addition, lncRNA-KRTAP5-AS1 and lncRNA-TUBB2A could act as competing endogenous RNAs to affect the function of Claudin-4. Our results suggest that non-coding RNAs play important roles in the regulatory network of Claudin-4. As such, non-coding RNAs should be considered as potential biomarkers and therapeutic targets against gastric cancer.Non-coding RNAs can modify the expression of proteins in cancer networks. Here the authors reveal a regulatory network in gastric cancer whereby claudin-4 expression is reduced by specific miRNAs, which are in turn bound by specific lncRNAs acting as competing endogenous RNAs (ceRNAs), resulting in increased claudin-4 expression.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1

CLDN4 is identified as a target of miR-596 and miR-3620-3p. a Hierarchical clustering analysis of mRNAs, lncRNAs, and miRNAs that were differentially expressed between GC tissues and non-tumorous adjacent tissues (>2.0-fold; P < 0.05; filtered to show the top 30 up-regulated or down-regulated results for mRNAs and lncRNAs). Expression values are represented in shades of red and green, indicating expression above and below the median expression value across all tissues, respectively. b The mRNA-lncRNA -miRNA networks in the GC. The networks include cell adhesion pathway, pathway in cancer, tight junction pathway, and cell cycle pathway. Genes colored in green are protein-coding RNAs associated with GC. Genes colored in blue are lncRNAs and genes colored in red are miRNAs associated with GC. c Predicted binding sites for miR-596, miR-3620-3p, and miR-4292 on the CLDN4 transcript. The white nucleotides are the seed sequences of miRNAs. d Real-time PCR analysis of CLDN4 expression in GC cells treated with mimics of miR-596, miR-3620-3p, miR-4292, and negative control. e GC cell line SGC-7901 was transfected with the mimics of miR-596, miR-3620-3p, miR-4292, and negative control. Reduced CLDN4 expression was shown by western blotting analysis and normalized to β-tubulin. f Luciferase activities were measured in GC cells co-transfected with luciferase reporter containing CLDN4 and the mimics of miR-596, miR-3620-3p, miR-4292, or mutant. Data are presented as the relative ratio of renilla luciferase activity and firefly luciferase activity. g The relative expressions of CLDN4 were determined by real-time PCR and western blotting. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 2

CLDN4 reinforces the proliferative and invasive capacity of GC cells in vitro. a Cell proliferation was assessed daily for 4 days using the Cell Counting Kit-8 (CCK-8) assay in CLDN4 overexpressing SGC-7901 cells. b Transwell assays were used to evaluate the involvement of CLDN4 for invasion in CLDN4 overexpressing SGC-7901 cells. c Scrape motility assays were monitored at 0, 6, 12, and 24 h in CLDN4 overexpressing SGC-7901 cells. d Cell proliferation assessed in CLDN4 overexpressing AGS cells. e Transwell assays assessed in CLDN4 overexpressing AGS cells. f Scrape motility assays in CLDN4 overexpressing AGS cells. g Cell proliferation assessed in CLDN4 knockdown SGC-7901 cells. h Transwell assays assessed in CLDN4 knockdown SGC-7901 cells. i Scrape motility assays in CLDN4 knockdown SGC-7901 cells. j Cell proliferation assessed in CLDN4 knockdown AGS cells. k Transwell assays assessed in CLDN4 knockdown AGS cells. l Scrape motility assays in CLDN4 knockdown AGS cells. In b, e, h, and k, cells were incubated for 24 h, and counted under the microscope. Original magnification ×200. Scale bars = 100 μm. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 3

CLDN4 induces EMT in vitro. a Phase-contrast micrographs of CLDN4 overexpressing cells, pEX2-NC cells and SGC-7901 cells. Scale bars = 50 μm. b, c The transcriptional and translational levels of EMT related markers. The real-time PCR and western blotting were performed at 48 h after the CLDN4 overexpressing cells treated with mimics of miR-596 or miR-3620-3p. d Immunofluorescence microscopy of the localization and expression of EMT and invasion markers in CLDN4 overexpressing cells. Scale bars = 50 μm. e The relative expression levels of CLDN4 in human GC tissues compared with their matched non-tumorous adjacent tissues. f Kaplan–Meier analysis of the correlation between CLDN4 expression levels and overall survival. g, h The relative expression levels of miR-596 and miR-3620-3p in human GC tissues compared with their matched non-tumorous adjacent tissues. i, j The correlation between CLDN4 transcriptional levels and miR-596 or miR-3620-3p transcriptional levels were measured in the same set of patients by Spearman correlation analysis. k Representative images of CLDN4, miR-596 and miR-3620-3p expression from GC tissues and non-tumorous adjacent tissues by ISH assays. Original magnification × 200. Scale bars = 100μm. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test and Wilcoxon’s Sign Rank Test is used to evaluate the differential expression between GC tissues and their matched non-tumorous adjacent tissues. *P < 0.05, **P < 0.01

Fig. 4

LncRNAs can crosstalk with miRNAs through direct binding. a Relative expression of lncRNAs in GC cells treated with mimics of miR-596 or miR-3620-3p were measured by real-time PCR. b Luciferase activity in GC cells co-transfected with luciferase reporter containing lncRNA-KRTAP5-AS1 and the mimics of miR-596, miR-3620-3p or mutant. Data are presented as the relative ratio of renilla luciferase activity and firefly luciferase activity. c Luciferase activity in GC cells co-transfected with luciferase reporter containing lncRNA-TUBB2A and the mimics of miR-3620-3p or mutant. Data are presented as the relative ratio of renilla luciferase activity and firefly luciferase activity. d The schematic diagram and real-time PCR results of the MS2-RIP method used to identify the binding between lncRNAs and miRNAs in both SGC-7901 and AGS cells. e The schematic diagram of the RNA pull down method used to identify the binding between lncRNAs and miRNAs in both SGC-7901 and AGS cells. GC cell lysates were incubated with biotin-labeled lncRNA-KRTAP5-AS1, lncRNA-TUBB2A, lncRNA-KRTAP5-AS1-mut, and lncRNA-TUBB2A-mut. MiRNA real-time PCR was performed after pull down process. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 5

LncRNA-KRTAP5-AS1 and lncRNA-TUBB2A enhance the proliferative and invasive capacity of GC cells in vitro. a Cell proliferation was assessed daily for 4 days using the CCK-8 assay in lncRNA-KRTAP5-AS1 overexpressing SGC-7901 cells. b Transwell assays were used to evaluate the involvement of lncRNA-KRTAP5-AS1 for invasion in lncRNA-KRTAP5-AS1 overexpressing SGC-7901 cells. c Cell proliferation assessed in lncRNA-KRTAP5-AS1 overexpressing AGS cells. d Transwell assays assessed in lncRNA-KRTAP5-AS1 overexpressing AGS cells. e Cell proliferation assessed in lncRNA-KRTAP5-AS1 knockdown SGC-7901 cells. f Transwell assays assessed in lncRNA-KRTAP5-AS1 knockdown SGC-7901 cells. g Cell proliferation assessed in lncRNA-KRTAP5-AS1 knockdown AGS cells. h Transwell assays assessed in lncRNA-KRTAP5-AS1 knockdown AGS cells. i Cell proliferation assessed in lncRNA-TUBB2A overexpressing SGC-7901 cells. j Transwell assays assessed in lncRNA-TUBB2A overexpressing SGC-7901 cells. k Cell proliferation assessed in lncRNA-TUBB2A overexpressing AGS cells. l Transwell assays assessed in lncRNA-TUBB2A overexpressing AGS cells. m Cell proliferation assessed in lncRNA-TUBB2A knockdown SGC-7901 cells. n Transwell assays assessed in lncRNA-TUBB2A knockdown SGC-7901 cells. o Cell proliferation assessed in lncRNA-TUBB2A knockdown AGS cells. p Transwell assays assessed in lncRNA-TUBB2A knockdown AGS cells. In b, d, f, h, j, l, n, p, cells were incubated for 24 h, and counted under the microscope. Original magnification × 200. Scale bars = 100 μm. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 6

LncRNA-KRTAP5-AS1 and LncRNA-TUBB2A act as ceRNAs. a Luciferase activity in GC cells co-transfected with psiCHECK2-CLDN4 and ceRNAs. Data were presented as the relative ratio of renilla luciferase activity and firefly luciferase activity. b The schematic diagram and real-time PCR results of the RIP based on Ago2 showed that lncRNAs can compete with the CLDN4 transcript for the binding of miRNAs. c The relative expression levels of CLDN4 were determined by real-time PCR and western blotting after co-transfecting ceRNAs. d Cell proliferation was assessed daily for 4 days using the CCK-8 assay in CLDN4 overexpressed cells transfected the plasmids of pcDNA3.1-KRTAP5-AS1 and pcDNA3.1 -TUBB2A. e Transwell assays were used to assay the involvement of CLDN4 for invasion in CLDN4 overexpressing cells. Cells were incubated for 24 h, and counted under the microscope. Original magnification ×200. Scale bars = 100 μm. f, g The transcriptional and translational levels of EMT related markers. The real-time PCR and western blotting were performed at 48 h after the CLDN4 overexpressing cells treated with pcDNA3.1-KRTAP5-AS1 and pcDNA3.1-TUBB2A. Data are shown as mean ± s.d., n = 3. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 7

CLDN4 promotes proliferation in vivo. a In vivo tumor lumps of xenograft mouse models composed of CLDN4 overexpressing cells, which were treated with miR-596 or miR-3620-3p as well as pcDNA3.1-KRTAP5-AS1 or pcDNA3.1-TUBB2A. Mice were sacrificed at the 19th day after injection and each tumor lump was removed from the body. bImages of the tumor lumps of each group at the endpoint of the experiment described in a. c The tumor growth curves of in vivo tumor volumes. Data are mean ± s.d. of the tumor volumes, n = 8, *P < 0.05. d The mean tumor weight of each group. Data are shown as mean ± s.d. of the tumor weights, n = 8. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 8

The effects of CLDN4 and ncRNAs on tumor growth. SGC-7901 cells were subcutaneously injected at both right and left armpit regions of nude mice. After tumor formation, plasmids of CLDN4, lncRNAs and agomirs of miRNAs were injected into tumors of right side and their negative controls into left side. a Treatment of CLDN4 promoted xenograft tumor growth. b, c Treatment of miR-596 and miR-3620-3p suppressed xenograft tumor growth. d, e Treatment of pcDNA3.1-KRTAP5-AS1 and pcDNA3.1-TUBB2A promoted xenograft tumor growth. f, g miR-596 and miR-3620-3p treated tumors showed less CLDN4 expression. h miR-596 and miR-3620-3p treated tumors showed less IncRNA-KRTAP5-AS1 expression. i miR-3620-3p treated tumors showed less IncRNA-TUBB2A expression. Data are shown as mean ± s.d., n = 5 for each group. The data statistical significance is assessed by Student’s t-test. *P < 0.05, **P < 0.01

Fig. 9

The metastasis promoting effect of CLDN4 could be regulated in vivo. a, b Transverse section of 18F-FDG PET images of mice at the 56 day after tail vein injection with 1 × 106 SGC-7901 cell clones and the max SUVs were analyzed in each group. Data are mean ± s.d. of the tumor volumes, n = 8, *P < 0.05. c The gross lesion of lung tissues isolated from the mice. d The microscopic images of lung tissue sections stained by hematoxylin and eosin. Scale bars = 500μm. e The number of metastatic nodules in the lungs from 56 days after tail vein injection in a (five sections evaluated per lung). Data are mean ± s.d. of the tumor volumes, n = 8, *P < 0.05. f, g Transverse section of 18F-FDG PET images of mice at the 56 day after tail vein injection with 1 × 106 HGC-27 cell clones and the max SUVs were analyzed in each group. Data are shown as mean ± s.d. of the tumor volumes, n = 8. The data statistical significance is assessed by Student’s t-test. *P < 0.05

Fig. 10

The mechanism graph of the regulatory network and function of CLDN4. CLDN4 could promote proliferation, metastasis or EMT processes of GC, which could be inhibited by miR-596, miR-3620-3p and enhanced by lncRNA-KRTAP5-AS1, lncRNA-TUBB2A as ceRNAs