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. 2021 Dec;12(1):627-639.
doi: 10.1080/21655979.2021.1882164.

Long non-coding RNA LINC00997 silencing inhibits the progression and metastasis of colorectal cancer by sponging miR-512-3p

Zhiliang Shi  1 , Chenglong Shen  1 , Cheng Yu  1 , Xiaoling Yang  1 , Jiazhe Shao  1 , Jian Guo  1 , Xinguo Zhu  2 , Guoqiang Zhou  1
Affiliations

Long non-coding RNA LINC00997 silencing inhibits the progression and metastasis of colorectal cancer by sponging miR-512-3p

Zhiliang Shi et al. Bioengineered. 2021 Dec.

Abstract

We aimed to study the role of LINC00997 in the metastasis of colorectal cancer (CRC). LINC00997 and miR-512-3p expression in the primary colorectal cancer (NCRC) tissues and metastatic colorectal cancer (MCRC) tissues were detected using RT-qPCR. The Cancer Genome Atlas database was used to evaluate LINC00997 levels in the NCRC and MCRC tissues, and the correlations of LINC00997 expression with distant metastasis (M), regional lymph node metastasis (N), age and tumor stage were analyzed. Subsequently, RT-qPCR was performed to determine the expression of metastasis-related genes in MCRC tissues and analyze the correlation of LINC00997 or miR-512-3p level with the protein expression of metastasis-related genes. In vitro, LINC00997 expression in several CRC cell lines was examined. After LINC00997 silencing, cell invasion and migration were evaluated with Transwell and wound healing assays, respectively. The expression of metastasis- and EMT-related proteins was measured. Additionally, the potential interaction between LINC00997 and miR-512-3p was verified using a luciferase reporter assay. Rescue assays were conducted to clarify the regulatory effects of LINC00997 and miR-512-3p on CRC development. Results revealed that LINC00997 was frequently overexpressed in MCRC tissues, which was positively related to the tumor metastasis and stage. Additionally, LINC00997 was significantly elevated in CRC cells and LINC00997 silencing inhibited the invasion, migration and EMT of CRC cells, which was restored by miR-512-3p inhibitor. Luciferase reporter assay confirmed that LINC00997 could target miR-512-3p. In conclusion, LINC00997 regulated the metastasis of CRC by targeting miR-512-3p, providing some insights into the regulatory mechanism of CRC.

Keywords: Colorectal cancer; linc00997; metastasis; miR-512-3p; migration.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
LINC00997 is highly expressed in MCRC tissues. (a) The expression of LINC00997 in NCRC tissues and the corresponding adjacent tissues was determined using RT-qPCR assay. ***P < 0.001. (b) LINC00997 level in MCRC tissues and para-carcinoma tissues was detected with RT-qPCR. ***P < 0.001. (c) The relative expression ratio of LINC00997 in the Tumor-MCRC/Normal-MCRC group and Tumor-CRC/Normal-NCRC group. *P < 0.05 vs. Tumor-NCRC/Normal-NCRC
Figure 2.
Figure 2.
LINC00997 expression is associated with the clinicopathological features of CRC. (a) The Cancer Genome Atlas database (TCGA) was used to evaluate the expression of LINC00997 in CRC tumor tissues and adjacent non-tumor tissues. (b) LINC00997 expression in the MCRC and NCRC tissues was tested using the TCGA database. (c) The correlation of LINC00997 expression with distant metastasis (m), regional lymph node metastasis (n), age and tumor stage was analyzed using TCGA database. (d-f) RT-qPCR was employed to assess the expression of VIM, MMP2 and MMP7 in the MCRC tissues and the corresponding beside tissues. ***P < 0.001. (g-i) The correlation between LINC00997 expression and VIM, MMP2 and MMP7 level
Figure 3.
Figure 3.
LINC00997 silencing suppressed the migration and invasion of CRC cells. (a) LINC00997 level in several CRC cell lines was detected using RT-qPCR. **P < 0.01, ***P < 0.001 vs. HIEC. (b) The level of LINC00997 was tested with RT-qPCR after transfection with shRNA-LINC00997 into HCT116 cells. **P < 0.01, ***P < 0.001. (c and d) Cell migration was tested using wound healing assay. (e and f) The invasive ability of HCT116 cells was determined with Transwell assay. (g) Western blot analysis was used to examine the expression of MMP2 and MMP7. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 4.
Figure 4.
LINC00997 silencing inhibited the epithelial-mesenchymal transition (EMT) of HCT116 cells. The expression of (a) E-cadherin and (b) N-cadherin in HCT116 cells was evaluated using immunofluorescence assay. (c) The expression of EMT-related proteins was measured by means of western blot analysis. **P < 0.01, ***P < 0.001
Figure 5.
Figure 5.
MiR-512-3p could be directly targeted by LINC00997. (a) Binding region between LINC00997 and miR-512-3p. (b) RT-qPCR was employed to evaluate the expression of miR-512-3p after LINC00997 silencing. (c) The expression of miR-512-3p was assessed after transfection with miR-512-3p mimic. (d) A luciferase reporter assay was performed to determine the relative luciferase activity. **P < 0.01 vs. mimic-NC. (e) The expression of miR-512-3p in NCRC tissues and the corresponding adjacent tissues was determined using RT-qPCR assay. ***P < 0.001. (f) MiR-512-3p level in MCRC tissues and para-carcinoma tissues was detected with RT-qPCR. ***P < 0.001. (g-i) The correlations between LINC00997 expression and VIM, MMP2 and MMP7 levels
Figure 6.
Figure 6.
MiR-512-3p knockdown restored the inhibitory effects of LINC00997 silencing on the migration and invasion of HCT116 cells. (a and b) Wound healing assay was utilized for the evaluation of cell migration. (c and d) Transwell assay was applied for the measurement of cell invasion. (e) The expression of MMP2 and MMP7 was examined using western blotting. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 7.
Figure 7.
MiR-512-3p knockdown attenuated the impacts of LINC00997-downregulation on the EMT of HCT116 cells. The level of (a) E-cadherin and (b) N-cadherin in HCT116 cells was detected with immunofluorescence assay. (c) The expression of EMT-related proteins was measured using western blot analysis. **P < 0.01, ***P < 0.001
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