HOTAIR contributes to the carcinogenesis of gastric cancer via modulating cellular and exosomal miRNAs level

Abstract

Gastric cancer (GC) is one of the most leading malignancies. Long noncoding RNA is related to GC. In this study, 11 miRNAs in the exosomes and six lncRNAs in the tissues was examined by qRT-PCR. Correlation analysis was used to analyze the relationship between miRNAs in exosome and lncRNAs in the tissues. Four miRNAs level in GC tissues were examined by qRT-PCR. MTT was used to determine cell viability. Flow cytometry was used to quantify the apoptotic cells. Transwell assay was used to examine the migration and invasion capacity. Dual-luciferase assay was used to examine the interaction between HOTAIR and miR-30a or -b. Capillary formation was used to determine the capillary formation capacity. Weak negative correlations were found between HOTAIR and miR-30a or -b in GC tissue samples. Interestingly, strong negative correlations were identified between the HOTAIR level in GC tissue samples and the miR-30a or -b levels in plasma exosomes. HOTAIR knockdown GC cells exhibited decreased migration, invasion, proliferation, and upregulated apoptosis, which released more miR-30a and -b into the exosomes. KRAS was upregulated when co-cultured with exosomes from HOTAIR overexpressed cells, and promoted GC cells proliferation, migration, and invasion. Meanwhile, HUVEC cells expressed increased VEGF-A and formatted more capillaries. Subsequently, we identified a 10mer target site of miR-30a or -b in HOTAIR sequence, and the overexpression of HOTAIR induced the degradation of miR-30a or -b, indicating a ceRNA role of HOTAIR. We report the negative correlation between the plasma miRNAs level and GC tissue HOTAIR expression for the first time and unveiled the ceRNA role of HOTAIR in GC. HOTAIR functions as an onco-lncRNA regulating the level of miR-30a and -b in both GC cells and exosomes. These findings may give insight into understanding the mechanism of GC pathogenesis and provide new biomarkers for clinical diagnosis.

Fig. 1. Plasma exosomal miRNAs level in GC patients.

Exosomes were isolated from plasma samples of 87 GC patients by stepwise centrifugation. The isolation of exosomes was confirmed by detecting CD63 and CD81 by immunoblotting (a). b Electronic microscopic image of exosomes. c Total RNAs were extracted by Trizol reagents and miRNAs level were determined by qRT-PCR. Results were analyzed by student’s t-test and P < 0.05 was considered statistically significant.

Fig. 2. The level of miRNAs in GC tissue samples and the correlation analysis.

a The expression of four miRNAs and four lncRNAs were detected in GC tissue samples and adjacent normal controls by qRT-PCR. The results were representing as heat map. Results were analyzed by student’s t-test and P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01. The correlations between tissue HOTAIR level and tissue (b) or plasma exosomal (c) miRNAs’ expressions were analyzed by χ2-analysis. d The overall survival curves are presented according to the expression level of HOTAIR in GC patients.

Fig. 3. HOTAIR modulates the degradation of miR-30a, -b, and miR-16 through direct interaction.

a a, schematic diagram of the predicted interaction between wild-type HOTAIR and miRNAs, and the dual-luciferase assay was used to confirm the interaction; b, schematic diagram of the predicted interaction between mutated HOTAIR and miRNAs. Results were analyzed by One-way ANOVA and P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01. b RNA pulldown. BGC-823 or SGC-7901 cell lysates were incubated with biotin labeled HOTAIR probe or control DNA oligo for 2 h, and then incubated with streptavidin beads for 1 h. Total RNA were extracted after four times washes and the level of HOTAIR and miRNAs was examined by RT-PCR. Results were analyzed by student’s t-test and P < 0.05 was considered statistically significant. **P < 0.01. c BGC-823 and SGC-7901 cells were treated with actinomycin D for 8 h. Total RNA was extracted and the level of c-myc and GAPDH were examine by RT-PCR. d BGC-823 and SGC-7901 cells were transfected with wild-type or mutant HOTAIR expression vector for 48 h followed by 8 h treatment with actinomycin D. The level of miR-30a, -b, and miR-16 was examined by qRT-PCR. Results were analyzed by student’s t-test and P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.

Fig. 4. The biological function of HOTAIR in GC cells.

a Two HOTAIR siRNAs were transfected into GC cells separately. 48 h after transfection, total RNA was extracted and the HOTAIR level was determined by qRT-PCR. In vitro cell migration and invasion assay were processed using traditional transwell plate. b MTT assay was used to determine the relative cell viability. c The numbers of apoptotic cells in HOTAIR knocked down GC cells were counted by flow cytometry after annexin V-FITC and PI staining. Results were analyzed by One-way ANOVA and p < 0.05 was considered as significant. *P < 0.05, **P < 0.01.

Fig. 5. HOTAIR modulated proliferation and apoptosis through regulating exosomal miRNAs.

a, b BGC-823 or SGC-7901 cells were transfected with reporter vector and then cultured with exosomes from HOTAIR knockdown or overexpressed BGC-823 cells. 48 h after co-culture, cells were lysed and luciferase activities were detected. c, d BGC-823 or SGC-7901 cells were cultured with exosomes from HOTAIR knockdown or overexpressed BGC-823 cells. 48 h after co-culture, cell proliferation and apoptosis were detected. Results between three groups were analyzed by One-way ANOVA, and results between two groups were analyzed by student’s t-test. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.

Fig. 6. HOTAIR modulated the angiogenesis through altering exosomal miRNA.

HUVEC cells were transfected with reporter vector and then cultured with exosomes from HOTAIR knockdown (a) or overexpressed (b) BGC-823 cells. Forty-eight hour after co-culture, cells were lysed and luciferase activities were detected. c–e HUVEC cells were cultured with exosomes from HOTAIR knockdown or overexpressed BGC-823 or SGC-7901 cells. Forty-eight after co-culture, cell viability was detected. Meanwhile, capillary formation assay was used to determine the angiogenetic capacity of HUVEC cells. Results between two groups were analyzed by student’s t-test, and results between more than two groups were analyzed by One-way ANOVA. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.

Fig. 7. Exosomes from HOTAIR knockdown GC cells repressed GC tumorigenesis.

A total of 5 × 10⁶ BGC-823 cells in were injected subcutaneously into the shoulder to establish a human GC cell xenograft mouse model. Exosomes were injected directly into the tumor at days 14, 21, 24, and 28 after tumor cell injection. The Ki-67 level was determined by immunohistochemistry, and the level of cleaved PARP1 and caspase-3 was detected by immunoblotting. Results were analyzed by student’s t-test. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01.