Long Noncoding RNA HOTAIR Promotes Epithelial-Mesenchymal Transition and Is a Suitable Target to Inhibit

Abstract
Objectives: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer.
Methods: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination.
Results: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination – and significantly prolonged the survival – in the orthotopic tumor mouse model.
Conclusion: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.

Materials and Methods

Results
HOTAIR Was Expressed at Significantly High Levels in a Metastatic Cell Line, 60As6

Results
E-Cadherin Was Significantly Decreased, and N-Cadherin and Vimentin Significantly Increased, in 60As6 Cells

Results
Preparation of a Stable HOTAIR Knockdown Line in 60As6 Cells via shRNA
The Stable HOTAIR Knockdown Line in 60As6 Cells Reversed EMT Progression

Results
Preparation of a Stable HOTAIR-Overexpressing Cell Line in HSC-60 Cells
The Stable HOTAIR-Overexpressing Cell Line in HSC-60 Cells Promoted EMT

Results
HOTAIR Targets miR-217 via Direct Binding

Results
miR-217 Directly Targets ZEB1

Results
In vivo Phenotypes

Results
Proposal of Antiperitoneal Dissemination Therapy Targeting HOTAIR

Results
HOTAIR-miR-217-ZEB1 Expression Levels in Cancer Tissues from Patients

Discussion
Thus, in 60As6 cells, our highly metastatic line derived from a patient, the expression of HOTAIR was significantly increased (Fig. 1) compared with that in the parental line with low metastatic properties (HSC-60).
We also demonstrated using the patient clinical samples that the HOTAIR expression was significantly higher in primary cancer tissues from patients with peritoneal metastasis than in the cancer tissues from patients without peritoneal metastasis (Fig. 9). The HOTAIR expression was further increased in the peritoneal cancer tissues (Fig. 9). These findings are important in 2 ways.
(i) They suggest that HOTAIR expression could feasibly be used to clinically diagnose peritoneal metastasis of scirrhous gastric cancers.
(ii) They suggest that HOTAIR can be targeted to inhibit peritoneal metastasis of scirrhous gastric cancers (opening the possibility of a novel antiperitoneal dissemination therapy).
Elevated HOTAIR in 60As6 contributes to the promotion of EMT (Fig. 2-4), and the signals of elevated HOTAIR are transduced to downregulate the miR-217 level and further to dysregulate ZEB1 expression (as a result, upregulation of ZEB1; Fig. 5-7).

Discussion
That upregulation of ZEB1 promotes EMT and cancer metastasis is well known. In our in vivo study, we obtained 2 excellent results as follows.
(i) When the 60As6-HOTAIR shRNA line (stably expressed line) was orthotopically inoculated into the stomach wall in nude mice, we observed an inhibition of peritoneal dissemination and prolonged survival rate (Tables 1, 2). (ii) In tumor-bearing mice with orthotopic inoculation of 60As6 without any transfection of the shRNA, when we intravenously injected the complex of siRNA targeting HOTAIR and atelocollagen, a marked antiperitoneal dissemination effect was shown (Fig. 8).
In the treated mice, the HOTAIR expression was reduced to increase miR-217, and to decrease ZEB1 (Fig. 8), as observed in the in vitro study. Further, HOTAIR knockdown in the mice led to a reversal of the EMT status (Fig. 8). We also showed that the overall survival rate of the treated mice was significantly longer (73 ± 13 days) than that of the control mice (36 ± 9 days), as shown in Table 3.

Conclusions
We demonstrated that high expression of HOTAIR in 60As6 promotes EMT to assist the peritoneal dissemination of scirrhous gastric cancers. The EMT-associated pathway (HOTAIR-miR-217-ZEB1 axis) might be targeted to inhibit peritoneal dissemination as a novel strategy for the treatment of scirrhous gastric cancers (Fig. 10).