LINC00115 promotes gastric cancer partly by the miR-212-5p/ATPAF1 axis

LncRNAs are known to be key regulators in the initiation and development of diverse cancers. Whether LINC00115 is involved in the regulation of gastric cancer (GC) progression remains unclear. Here, we aimed to show the function of LINC00115 in GC. RT-qPCR was used to measure gene expression in GC tissues and cells. Colony formation, EdU, TUNEL, and wound healing assays were used to analyze cellular processes in GC. The in vivo GC xenograft model was established. We observed that LINC00115 was highly expressed in GC. Functionally, silencing LINC00115 inhibited GC cell proliferation, and migration but facilitated GC apoptosis. Mechanistically, LINC00115 sponged miR-212-5p, while miR-212-5p targeted ATPAF1 in GC cells. Rescue assays showed ATPAF1 overexpression countervailed the inhibitory role of LINC00115 depletion in GC progression in vitro and in vivo. Overall, LINC00115 promoted GC progression by upregulating ATPAF1 via miR-212-5p.

Key words
LINC00115; gastric cancer; miR-212-5p; ATPAF1

Authorship

QINGXI ZHU

conceived and designed the experiments

carried out the experiments

analyzed the data

drafted the manuscript

Tongren Hospital of Wuhan University (Wuhan Third Hospital), Department of Gastroenterology, No.241, Pengliuyang Road, Wuhan 430060, Hubei, ChinaTongren Hospital of Wuhan University (Wuhan Third Hospital)ChinaWuhan, Hubei, ChinaTongren Hospital of Wuhan University (Wuhan Third Hospital), Department of Gastroenterology, No.241, Pengliuyang Road, Wuhan 430060, Hubei, China

https://orcid.org/0000-0001-7154-9939

JIE TAN

conceived and designed the experiments

carried out the experiments

analyzed the data

drafted the manuscript

https://orcid.org/0000-0002-8861-1134

TING ZHAN

carried out the experiments

https://orcid.org/0000-0003-0622-7890

MENG LIU

carried out the experiments

https://orcid.org/0000-0002-3332-1316

YANLI ZOU

carried out the experiments

analyzed the data

drafted the manuscript

https://orcid.org/0000-0001-8818-9001

WEIJIE LIU

carried out the experiments

analyzed the data

drafted the manuscript

https://orcid.org/0000-0002-5763-7286

Correspondence to: Yanli Zou, Weijie Liu E-mail: haisizou@163.com, 172077205@qq.com *These authors contributed equally to the work.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (5)

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Figure 1
LINC00115 knockdown suppressed GC cell growth. (a) The LINC00115 level in GC tissues and nontumor issues was detected by RT-qPCR. (b) RT-qPCR was used to evaluate the LINC00115 level in GC cells. (c) The knockdown efficiency of LINC00115 was measured by RT-qPCR in HGC-27 and AGS cells. (d) Colony formation and (e) EdU assays were performed to assess cell proliferation after transfection of sh-NC or sh-LINC00115. (f) Cell apoptosis of HGC-27 and AGS in the two groups was detected by TUNEL assay. (g) Wound healing assay was performed to assess the migratory capability of HGC-27 and AGS cells after transfection. (h) The protein levels of matrix metalloproteinases (MMP2 and MMP9) in sh-LINC00115 and sh-NC groups were detected by western blot. **p<0.01, ***p<0.001.

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Figure 2
LINC00115 sponged miR-212-5p in GC cells. (a) Subcellular fractionation assay was applied to determine the location of LINC00115 in HGC-27 and AGS cells. (b) Three miRNAs sharing binding sites with LINC00115 were found; pulldown assay was conducted to select the most suitable miRNA for LINC00115. (c) and (d) The miR-212-5p level in GC tissues and cells was measured by RT-qPCR. (e) Binding site of miR-212-5p on LINC00115. (f) Overexpression efficiency of miR-212-p in GC cells. (g) Luciferase reporter assay was conducted to test the binding of LINC00115 to miR-212-5p. (h) The expression correlation between LINC00115 and miR-212-5p in GC tissues by Pearson correlation analysis. **p<0.01, ***p<0.001.

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Figure 3
ATPAF1 was targeted by miR-212-5p. (a) The expression levels of potential target mRNAs in GC cells transfected with miR-212-5p mimics were detected by RT-qPCR. (b) The protein level of ATPAF1 after miR-212-5p upregulation was detected by western blot. (c) Binding site of miR-212-5p on ATPAF1. (d) The binding between ATPAF1 and miR-212-5p was determined by luciferase reporter assay. (e) Downregulation efficiency of miR-212-5p was tested by RT-qPCR. (f) The effect of LINC00115 silencing together miR-212-5p downregulation on ATPAF1 mRNA expression and protein level. (g) and (h) The ATPAF1 level in GC tissues and cells. (i) The relationship between ATPAF1 expression and the overall survival of GC patients was predicted with online tool (https://kmplot.com). **p<0.01, ***p<0.001.

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Figure 4
LINC00115 knockdown restrained GC cellular processes through the regulation of ATPAF1. (a) The transfection efficiency of ATPAF1 overexpression in HGC-27 cells. HGC-27 cell proliferation after transfection with sh-NC, sh-LINC00115, or sh-LINC00115+ATPAF1 was detected by (b) colony formation and (c) EdU assays. (d) The apoptosis of HGC-27 cells in the groups of sh-NC, sh-LINC00115, or sh-LINC00115+ATPAF1 was assessed with TUNEL assay. (e) Wound healing assay was adopted to evaluate the migration of HGC-27 cells in the three groups. (f) Western blot analysis detected the protein levels of matrix metalloproteinases (MMP2 and MMP9) in HGC-27 cells transfected with the indicated plasmids. ***p<0.001.

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Figure 5
LINC00115 silencing suppressed tumorigenesis in vivo by downregulating ATPAF1. (a) Representative images of the xenograft tumors in subcutaneous xenograft mouse model injected with HGC-27 cells transfected with sh-NC, sh-LINC00115 and sh-LINC00115+ATPAF1. (b) and (c) Tumor volume and weight of the xenograft in each group. ***p<0.001.

Figure 1 LINC00115 knockdown suppressed GC cell growth. (a) The LINC00115 level in GC tissues and nontumor issues was detected by RT-qPCR. (b) RT-qPCR was used to evaluate the LINC00115 level in GC cells. (c) The knockdown efficiency of LINC00115 was measured by RT-qPCR in HGC-27 and AGS cells. (d) Colony formation and (e) EdU assays were performed to assess cell proliferation after transfection of sh-NC or sh-LINC00115. (f) Cell apoptosis of HGC-27 and AGS in the two groups was detected by TUNEL assay. (g) Wound healing assay was performed to assess the migratory capability of HGC-27 and AGS cells after transfection. (h) The protein levels of matrix metalloproteinases (MMP2 and MMP9) in sh-LINC00115 and sh-NC groups were detected by western blot. p<0.01, *p<0.001.

Figure 2 LINC00115 sponged miR-212-5p in GC cells. (a) Subcellular fractionation assay was applied to determine the location of LINC00115 in HGC-27 and AGS cells. (b) Three miRNAs sharing binding sites with LINC00115 were found; pulldown assay was conducted to select the most suitable miRNA for LINC00115. (c) and (d) The miR-212-5p level in GC tissues and cells was measured by RT-qPCR. (e) Binding site of miR-212-5p on LINC00115. (f) Overexpression efficiency of miR-212-p in GC cells. (g) Luciferase reporter assay was conducted to test the binding of LINC00115 to miR-212-5p. (h) The expression correlation between LINC00115 and miR-212-5p in GC tissues by Pearson correlation analysis. p<0.01, *p<0.001.

Figure 3 ATPAF1 was targeted by miR-212-5p. (a) The expression levels of potential target mRNAs in GC cells transfected with miR-212-5p mimics were detected by RT-qPCR. (b) The protein level of ATPAF1 after miR-212-5p upregulation was detected by western blot. (c) Binding site of miR-212-5p on ATPAF1. (d) The binding between ATPAF1 and miR-212-5p was determined by luciferase reporter assay. (e) Downregulation efficiency of miR-212-5p was tested by RT-qPCR. (f) The effect of LINC00115 silencing together miR-212-5p downregulation on ATPAF1 mRNA expression and protein level. (g) and (h) The ATPAF1 level in GC tissues and cells. (i) The relationship between ATPAF1 expression and the overall survival of GC patients was predicted with online tool (https://kmplot.com). p<0.01, *p<0.001.

Figure 4 LINC00115 knockdown restrained GC cellular processes through the regulation of ATPAF1. (a) The transfection efficiency of ATPAF1 overexpression in HGC-27 cells. HGC-27 cell proliferation after transfection with sh-NC, sh-LINC00115, or sh-LINC00115+ATPAF1 was detected by (b) colony formation and (c) EdU assays. (d) The apoptosis of HGC-27 cells in the groups of sh-NC, sh-LINC00115, or sh-LINC00115+ATPAF1 was assessed with TUNEL assay. (e) Wound healing assay was adopted to evaluate the migration of HGC-27 cells in the three groups. (f) Western blot analysis detected the protein levels of matrix metalloproteinases (MMP2 and MMP9) in HGC-27 cells transfected with the indicated plasmids. ***p<0.001.

Figure 5 LINC00115 silencing suppressed tumorigenesis in vivo by downregulating ATPAF1. (a) Representative images of the xenograft tumors in subcutaneous xenograft mouse model injected with HGC-27 cells transfected with sh-NC, sh-LINC00115 and sh-LINC00115+ATPAF1. (b) and (c) Tumor volume and weight of the xenograft in each group. ***p<0.001.

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LINC00115 promotes gastric cancer partly by the miR-212-5p/ATPAF1 axis

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[1] Correspondence to: Yanli Zou, Weijie Liu E-mail: haisizou@163.com, 172077205@qq.com *These authors contributed equally to the work.