Down-regulation of long non-coding RNA HOTAIR inhibits invasion and migration of oesophageal cancer cells via up-regulation of microRNA-204

Abstract

Oesophageal cancer is a progressive tumour with high mortality. However, therapies aimed at treating oesophageal cancer remain relatively limited. Accumulating studies have highlighted long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), microRNA-204 (miR-204) and homeobox C8 (HOXC8) in the progression of oesophageal cancer. Herein, we tried to demonstrate the function of HOTAIR, miR-204 and HOXC8 in oesophageal cancer and their relationship. Differentially expressed genes involved in oesophageal cancer were identified. The endogenous expression of HOTAIR and miR-204 in oesophageal cancer cell lines was altered to elucidate their effects and to identify the interaction among HOTAIR, miR-204 and HOXC8. We also explored the underlying regulatory mechanisms of HOTAIR and miR-204 with siRNA against HOTAIR, miR-204 mimic or miR-204 inhibitor. Cell proliferation, migration, invasion and apoptosis were subsequently detected. Xenograft in nude mice was induced to evaluate tumourigenicity. miR-204 was down-regulated, while HOTAIR and HOXC8 were up-regulated in the oesophageal cancer tissues. HOTAIR could competitively bind to miR-204 and miR-204 could further target HOXC8. The oesophageal cancer cells treated with si-HOTAIR or miR-204 mimic exhibited decreased expression levels of HOXC8, Vimentin and MMP-9, but increased E-cadherin level. Silenced HOTAIR or elevated miR-204 inhibited proliferation, migration and invasion, along with stimulated apoptosis of oesophageal cancer cells. In summary, our results show that lncRNA HOTAIR could specifically bind to miR-204 as a competing endogenous RNA and regulate miR-204 and HOXC8. Hence, down-regulation of HOTAIR could inhibit progression of oesophageal cancer, indicating a novel target for oesophageal cancer treatment.

Keywords: HOX transcript antisense RNA; MicroRNA-204; homeobox C8; invasion; long non-coding RNA; migration; oesophageal cancer; proliferation.

Figure 1

microRNA‐204 (miR‐204) has low expression level while HOTAIR and HOXC8 have high expression level in oesophageal cancer tissues. (A), HOTAIR is highly expressed in oesophageal cancer tissues based on the data from TCGA database; (B) HOXC8 is highly expressed in oesophageal cancer tissues based on the data from TCGA database; (C) miR‐204 is lowly expressed in oesophageal cancer tissues based on the data from TCGA database; (D) possible network of HOTAIR in oesophageal cancer as a ceRNA. (E,F), immunohistochemistry reveals that the positive expression rate of HOXC8 is significantly elevated in oesophageal cancer tissues (400x); (G) RT‐qPCR confirmed that HOTAIR is highly expressed while the miR‐204 is lowly expressed in oesophageal cancer tissues. The data are analysed by paired t test; n = 46. The experiment was independently repeated three times; *P < 0.05 vs the adjacent normal tissues

Figure 2

HOTAIR could bind to microRNA‐204 (miR‐204) and HOXC8 is the target gene of miR‐204. (A and C) miR‐204 binds to the HOTAIR predicted using the target prediction program and verified by the determination of luciferase activity; (B and D) miR‐204 binds to the 3'UTR of HOXC8 predicted using the target prediction program and verified by the determination of luciferase activity; (E) the RNA pull‐down assay demonstrates miR‐204 could directly bind to HOXC8; (F) the RIP assay indicates that HOXC8 could directly bind to Ago2 protein; (G) HOTAIR is mainly expressed in the cytoplasm (×400); the data are presented as mean ± SD, and analysed by one‐way ANOVA among multiple groups; pairwise comparison was conducted by paired t test. N = 3. The experiment was independently repeated three times. *P < 0.05 vs the NC group. ^# P < 0.05 vs the Ago2 group

Figure 3

EC9706 and ECA109 cell lines exhibit highest expression of HOTAIR (A) and miR‐204 (B), respectively. *P < 0.05 vs the HEEC cell line. ^# P < 0.05 vs the EC9706 cell line. ^& P < 0.05 vs the ECA109 cell line. n = 3. The measurement data were expressed by mean ± SD and analysed by one‐way ANOVA. The experiment was independently repeated three times

Figure 4

Down‐regulation of HOTAIR decreases the expression of HOXC8 and alters the protein expression levels related to cell proliferation, migration and invasion through up‐regulation of miR‐204. (A‐C), the expression of HOTAIR and miR‐204, and mRNA and protein expression of HOXC8, E‐cadherin, MMP‐9 and Vimentin in the EC9706 cell line; (D‐F), expression of HOTAIR and miR‐204 and mRNA and protein expression of HOXC8, E‐cadherin, MMP‐9 and Vimentin in ECA109 cell line; *P < 0.05 vs the control group. ^# P < 0.05 vs the blank group. ^& P < 0.05 vs the si‐HOTAIR group. ^$ P < 0.05 vs the miR‐204 mimic group. The data are presented as mean ± SD, and analysed by one‐way ANOVA. n = 3. The experiment was independently repeated three times

Figure 5

Down‐regulation of HOTAIR inhibits proliferation of oesophageal cancer cells through up‐regulation of miR‐204. (A) the EC9706 cell line treated by si‐HOTAIR shows the lowest OD values from MTT assay. (B) the ECA109 cell line treated with si‐HOTAIR and miR‐204 mimic shows the lowest OD values from MTT assay. *P < 0.05 vs the control group. ^# P < 0.05 vs the blank group. ^& P < 0.05 vs the si‐HOTAIR group. ^$ P < 0.05 vs the miR‐204 mimic group. The data are presented as mean ± SD. The values at different time‐points were compared using repeated measurement ANOVA. n = 3. The experiment was independently repeated three times

Figure 6

Down‐regulation of HOTAIR or up‐regulation of miR‐204 suppresses migration and invasion of oesophageal cancer cells through up‐regulation of miR‐204. (A,B), the EC9706 cell line treated by si‐HOTAIR shows the lowest migration ability detected by scratch test. (C,D), Transwell assay (×200) reveals that the EC9706 cells treated by si‐HOTAIR shows the lowest invasion ability. (E,F), the ECA109 cells treated by si‐HOTAIR and miR‐204 mimic shows the lowest migration ability detected by scratch test. (G,H), Transwell assay (×200) reveals that the ECA109 cells treated by si‐HOTAIR and miR‐204 mimic shows the lowest invasion ability. *P < 0.05 vs the control group. ^# P < 0.05 vs the blank group. ^& P < 0.05 vs the si‐HOTAIR group. ^$ P < 0.05 vs the miR‐204 mimic group. The data are presented as mean ± SD, and analysed by one‐way ANOVA. N = 3. The experiment was independently repeated three times

Figure 7

Down‐regulation of HOTAIR accelerates cell cycle progression and induces apoptosis of oesophageal cancer cells through up‐regulation of miR‐204. (A and E), cell cycle distribution of EC9706 and ECA109 cell lines detected by flow cytometry; (B and F) the percentage of PI‐stained cells at the G0/G1, S, and G2/M phases in the EC9706 and ECA109 cell lines; (C and G), oesophageal cancer cells of the EC9706 and ECA109 cell lines in the scatter plots in which the upper left quadrant identifies the necrotic cells (annexin V−/PI+), the upper right quadrant identifies the late apoptotic cells (annexin V+/PI+), the lower left quadrant identifies the live cells (annexin V−/PI−), and the lower right quadrant identifies the early apoptotic cells (annexin V+/PI−). (D and H), the percentage of early and late apoptotic EC9706 and ECA109 cells. *P < 0.05 vs the control group. ^# P < 0.05 vs the blank group. ^& P < 0.05 vs the si‐HOTAIR group. ^$ P < 0.05 vs the miR‐204 mimic group. The data are presented as mean ± SD, and analysed by one‐way ANOVA. n = 3. The experiment was independently repeated three times

Figure 8

Down‐regulation of HOTAIR suppresses tumour formation through up‐regulation of miR‐204. (A), tumour volume of nude mice after injection of transfected EC9706 cells. (B), tumour volume of nude mice after injection of transfected ECA109 cells. *P < 0.05 vs the control group. ^# P < 0.05 vs the blank group. ^& P < 0.05 vs the si‐HOTAIR group. ^$ P < 0.05 vs the miR‐204 mimics group. The data are presented as mean ± SD. The values at different time‐points were compared using repeated measurement ANOVA. N = 6. The experiment was independently repeated three times

Figure 9

The mechanism diagram depicting that lncRNA HOTAIR functions as a ceRNA of miR‐204 to increase the expression of HOXC8, and thus enhancing progression of oesophageal cancer