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LncRNA NEAT1 promotes endometrial cancer cell proliferation, migration and invasion by regulating the miR-144-3p/EZH2 axis

Wei Wang
Wei Wang
, 
Liang Ge
Liang Ge
, 
Xiao-Juan Xu
Xiao-Juan Xu
, 
Ting Yang
Ting Yang
, 
Yue Yuan
Yue Yuan
, 
Xiao-Ling Ma
Xiao-Ling Ma
 and 
Xue-Hong Zhang
Xue-Hong Zhang
   | Nov 20, 2019
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Article Category: Research Article
Published Online: Nov 20, 2019
Page range: 434 - 442
Received: May 22, 2019
Accepted: Sep 21, 2019
DOI: https://doi.org/10.2478/raon-2019-0051
© 2019 Wei Wang, Liang Ge, Xiao-Juan Xu, Ting Yang, Yue Yuan, Xiao-Ling Ma, Xue-Hong Zhang, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

NEAT1 was highly expressed in EC tissues and cells. (A) NEAT1 was highly expressed in EC tissues. The transcription level of the NEAT1 gene in EC tissues was measured by qRT-PCR. Cancer tissues and adjacent tissues were from human EC patients. (B) The transcription of NEAT1 was upregulated in various EC cell lines. qRT-PCR was used to examine the expression of NEAT1 in various EC cell lines (HEC-1A, HEC-1B, Ishikawa, RL-95-2, and JEC), and hESCs, which were used as a control group.* indicates P < 0.05; ** indicates P < 0.01; ***indicates P < 0.001. The Data are presented as the mean ± SD.
NEAT1 was highly expressed in EC tissues and cells. (A) NEAT1 was highly expressed in EC tissues. The transcription level of the NEAT1 gene in EC tissues was measured by qRT-PCR. Cancer tissues and adjacent tissues were from human EC patients. (B) The transcription of NEAT1 was upregulated in various EC cell lines. qRT-PCR was used to examine the expression of NEAT1 in various EC cell lines (HEC-1A, HEC-1B, Ishikawa, RL-95-2, and JEC), and hESCs, which were used as a control group.* indicates P < 0.05; ** indicates P < 0.01; ***indicates P < 0.001. The Data are presented as the mean ± SD.

Figure 2

NEAT1 promoted EC cell proliferation, migration, and invasion. (A) Effects of NEAT1-shRNA on NEAT1 expression. The transcription of NEAT1 was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h respectively. (B, C) Effects of NEAT1 knockdown on the proliferation of EC cells (HEC-1A and Ishikawa). The MTT assay results showed the effects on cell growth as measured by cell vitality on three consecutive days. (D, E) Effects of NEAT1 knockdown on the cell proliferation of EC cells. A colony formation assay was used to detect cell proliferation. Figure 2D is one representative result of the colony formation assay and Figure 2E shows one quantitative result repeated at least three times. (F, G) Effects of NEAT1 knockdown on the migration of EC cells. A transwell assay was used to measure the cell migration ability. Figure 2F is a representative result from the transwell assays, and Figure 2G shows one quantitative result with at least three replicates. (H, I) Effects of NEAT1 knockdown on the invasion of EC cells. A transwell assay was used to measure the cell invasion ability. Figure 2H is a representative result from the transwell assays and Figure 2I shows one quantitative result with at least three replicates.* indicates P < 0.05, ** indicates P < 0.01. The data are presented as the mean ± SD.
NEAT1 promoted EC cell proliferation, migration, and invasion. (A) Effects of NEAT1-shRNA on NEAT1 expression. The transcription of NEAT1 was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h respectively. (B, C) Effects of NEAT1 knockdown on the proliferation of EC cells (HEC-1A and Ishikawa). The MTT assay results showed the effects on cell growth as measured by cell vitality on three consecutive days. (D, E) Effects of NEAT1 knockdown on the cell proliferation of EC cells. A colony formation assay was used to detect cell proliferation. Figure 2D is one representative result of the colony formation assay and Figure 2E shows one quantitative result repeated at least three times. (F, G) Effects of NEAT1 knockdown on the migration of EC cells. A transwell assay was used to measure the cell migration ability. Figure 2F is a representative result from the transwell assays, and Figure 2G shows one quantitative result with at least three replicates. (H, I) Effects of NEAT1 knockdown on the invasion of EC cells. A transwell assay was used to measure the cell invasion ability. Figure 2H is a representative result from the transwell assays and Figure 2I shows one quantitative result with at least three replicates.* indicates P < 0.05, ** indicates P < 0.01. The data are presented as the mean ± SD.

Figure 3

NEAT1 is a molecular sponge of miR-144-3p. (A) Sequences of miR-144-3p and wild-type NEAT1 (NEAT1 WT) and its mutant (NEAT1 MUT). NEAT1 WT can target miR-144-3p, whereas NEAT1 MUT lost this ability. (B) miR-144-3p was downregulated in EC tissues. The transcription level of miR-144-3p in EC tissues was measured by qRT-PCR. Cancer tissues and adjacent tissues were from human EC patients. (C) The transcription of miR-144-3p was upregulated in various EC cell lines. qRT-PCR was used to examine the expression of miR-144-3p in various EC cell lines (HEC-1A, HEC-1B, Ishikawa, RL-95-2 and JEC), and hESCs, which were used as a control group. (D) Effects of NEAT1 knockdown on miR-144-3p expression. The transcription of miR-144-3p was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h. (E) NEAT1 targeted miR-144-3p. A dual luciferase activity assay was used to evaluate the binding ability of NEAT1 WT and NEAT1 MUT to miR-144-3p.* indicates P < 0.05, ** indicates P < 0.01. The data are presented as the mean ± SD.
NEAT1 is a molecular sponge of miR-144-3p. (A) Sequences of miR-144-3p and wild-type NEAT1 (NEAT1 WT) and its mutant (NEAT1 MUT). NEAT1 WT can target miR-144-3p, whereas NEAT1 MUT lost this ability. (B) miR-144-3p was downregulated in EC tissues. The transcription level of miR-144-3p in EC tissues was measured by qRT-PCR. Cancer tissues and adjacent tissues were from human EC patients. (C) The transcription of miR-144-3p was upregulated in various EC cell lines. qRT-PCR was used to examine the expression of miR-144-3p in various EC cell lines (HEC-1A, HEC-1B, Ishikawa, RL-95-2 and JEC), and hESCs, which were used as a control group. (D) Effects of NEAT1 knockdown on miR-144-3p expression. The transcription of miR-144-3p was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h. (E) NEAT1 targeted miR-144-3p. A dual luciferase activity assay was used to evaluate the binding ability of NEAT1 WT and NEAT1 MUT to miR-144-3p.* indicates P < 0.05, ** indicates P < 0.01. The data are presented as the mean ± SD.

Figure 4

miR-144-3p inhibited EC cell proliferation, migration, and invasion. (A) Effects of the miR-144-3p mimic on miR-144-3p expression. The transcription of miR-144-3p was measured by qRT-PCR after miR-144-3p mimics or mimic NC transfection into HEC-1A and Ishikawa cells for 24 h. (B, C) Effects of the miR-144-3p mimic on the proliferation of EC cells. MTT assay results showed effects on cell growth as measured by cell vitality on three consecutive days. (D, E) Effects of the miR-144-3p mimic on the proliferation of EC cells. A colony formation assay was used to detect cell proliferation. Figure 4D is a representative result from the colony formation assay and Figure 4E shows one quantitative result repeated at least three times. (F, G) Effects of the miR-144-3p mimic on the migration of EC cells. A transwell assay was used to measure the cell migration ability. Figure 4F is a representative result from the transwell assays and Figure 4G shows one quantitative result with at least three replicates. (H, I) Effects of the miR-144-3p mimic on the invasion of EC cells. A transwell assay was used to measure the cell invasion ability. Figure 4H is a representative result from the transwell assays, and Figure 4I shows one quantitative result with at least three replicates.** indicates P < 0.01. The data are presented as the mean ± SD.
miR-144-3p inhibited EC cell proliferation, migration, and invasion. (A) Effects of the miR-144-3p mimic on miR-144-3p expression. The transcription of miR-144-3p was measured by qRT-PCR after miR-144-3p mimics or mimic NC transfection into HEC-1A and Ishikawa cells for 24 h. (B, C) Effects of the miR-144-3p mimic on the proliferation of EC cells. MTT assay results showed effects on cell growth as measured by cell vitality on three consecutive days. (D, E) Effects of the miR-144-3p mimic on the proliferation of EC cells. A colony formation assay was used to detect cell proliferation. Figure 4D is a representative result from the colony formation assay and Figure 4E shows one quantitative result repeated at least three times. (F, G) Effects of the miR-144-3p mimic on the migration of EC cells. A transwell assay was used to measure the cell migration ability. Figure 4F is a representative result from the transwell assays and Figure 4G shows one quantitative result with at least three replicates. (H, I) Effects of the miR-144-3p mimic on the invasion of EC cells. A transwell assay was used to measure the cell invasion ability. Figure 4H is a representative result from the transwell assays, and Figure 4I shows one quantitative result with at least three replicates.** indicates P < 0.01. The data are presented as the mean ± SD.

Figure 5

NEAT1 promoted the expression of EZH2 by miR-144-3p. (A) Effects of NEAT1 knockdown on the transcription of EZH2 in EC cells. The transcription of the EZH2 gene was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h. (B, C) Effects of NEAT1 knockdown on the EZH2 protein level of in EC cells. The protein level of EZH2 was measured by Western blot after sh-NEAT1 and sh-NC transfection into HEC-1A and Ishikawa cells for 48 h. The indicated antibodies were used for Western blot analysis. Quantitative Western blot results are shown in Figure 5B with GAPDH as a control. Figure 4C shows one representative result and the right part shows one quantitative result repeated at least three times. (D) Effects of miR-144-3p mimic on the transcription of EZH2 in EC cells. The transcription of the EZH2 gene was measured by qPCR after miR-144-3p mimic and mimic NC transfection into HEC-1A and Ishikawa cells for 48 h. (E) Effects of the miR-144-3p mimic on the EZH2 protein level in EC cells. The EZH2 protein level was measured by Western blot after miR-144-3p mimics or mimic NC transfection into HEC-1A and Ishikawa cells for 48 h. The indicated antibodies were used for Western blot analysis. (F) Sequences of miR-144-3p and wild-type EZH2 (EZH2 3’-UTR WT) and its mutant (EZH2 3’-UTR MUT). EZH2 3’-UTR WT can be targeted by miR-144-3p, whereas miR-144-3p cannot bind to EZH2 3’-UTR MUT. (G) miR-144-3p directly targeted the 3’-UTR region of EZH2. A dual luciferase activity assay was used to evaluate the binding ability of miR-144-3p to EZH2 3’-UTR WT and EZH2 3’-UTR MUT.* indicates P < 0.05; ** indicates P < 0.01. The data are presented as the mean ± SD.
NEAT1 promoted the expression of EZH2 by miR-144-3p. (A) Effects of NEAT1 knockdown on the transcription of EZH2 in EC cells. The transcription of the EZH2 gene was measured by qRT-PCR after sh-NEAT1 and sh-NC were transfected into HEC-1A and Ishikawa cells for 48 h. (B, C) Effects of NEAT1 knockdown on the EZH2 protein level of in EC cells. The protein level of EZH2 was measured by Western blot after sh-NEAT1 and sh-NC transfection into HEC-1A and Ishikawa cells for 48 h. The indicated antibodies were used for Western blot analysis. Quantitative Western blot results are shown in Figure 5B with GAPDH as a control. Figure 4C shows one representative result and the right part shows one quantitative result repeated at least three times. (D) Effects of miR-144-3p mimic on the transcription of EZH2 in EC cells. The transcription of the EZH2 gene was measured by qPCR after miR-144-3p mimic and mimic NC transfection into HEC-1A and Ishikawa cells for 48 h. (E) Effects of the miR-144-3p mimic on the EZH2 protein level in EC cells. The EZH2 protein level was measured by Western blot after miR-144-3p mimics or mimic NC transfection into HEC-1A and Ishikawa cells for 48 h. The indicated antibodies were used for Western blot analysis. (F) Sequences of miR-144-3p and wild-type EZH2 (EZH2 3’-UTR WT) and its mutant (EZH2 3’-UTR MUT). EZH2 3’-UTR WT can be targeted by miR-144-3p, whereas miR-144-3p cannot bind to EZH2 3’-UTR MUT. (G) miR-144-3p directly targeted the 3’-UTR region of EZH2. A dual luciferase activity assay was used to evaluate the binding ability of miR-144-3p to EZH2 3’-UTR WT and EZH2 3’-UTR MUT.* indicates P < 0.05; ** indicates P < 0.01. The data are presented as the mean ± SD.
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