Downregulation of long noncoding RNA LINC01419 inhibits cell migration, invasion, and tumor growth and promotes autophagy via inactivation of the PI3K/Akt1/mTOR pathway in gastric cancer

Abstract

Background: Accumulating evidence has highlighted the crucial role of long noncoding RNAs (lncRNAs) in the tumorigenesis of gastric cancer (GC), which is the most common gastrointestinal malignancy. The present study aimed to identify the capacity of lncRNA LINC01419 (LINC01419) in GC progression, with the potential mechanism explored.

Methods: Highly expressed lncRNAs were identified by in silico analysis, with the LINC01419 expression in GC tissues measured using reverse transcription-quantitative PCR (RT-qPCR). The GC cells were subsequently transfected with siRNA against LINC01419 or Rapamycin (the inhibitor of the mTOR pathway), or both, in order to measure cell migration and invasion in vitro as well as tumor growth and metastasis in vivo. Moreover, the expression of PI3K/Akt1/mTOR pathway-associated factors was determined.

Results: LINC01419, highly expressed in GC samples of the Gene Expression Omnibus database, was observed to be markedly upregulated in GC tissues. Moreover, LINC01419 silencing, or PI3K/Akt1/mTOR pathway inhibition, exhibited an inhibitory role in GC cell migration and invasion in vitro, coupled with promoted cell autophagy in vitro, and inhibited tumor growth and metastasis in vivo. It was also revealed that LINC01419 silencing blocked the PI3K/Akt1/mTOR pathway, as proved by decreased extents of Akt1 and mTOR phosphorylation.

Conclusions: In conclusion, LINC01419 inhibition may suppress GC cell invasion and migration, and promote autophagy via inhibition of the PI3K/Akt1/mTOR pathway. This provides significant theoretical basis and possibilities for further elucidation of the molecular mechanism of GC and finding new molecular-targeted therapeutic regimens.

Keywords: LINC01419; RNA; autophagy; gastric cancer; invasion and migration; long noncoding PI3K/Akt1/mTOR pathway.

Figure 1.

Highly expressed LINC01419 is involved in GC identified by bioinformatics prediction and RT-qPCR. (a) Heatmap of differentially expressed lncRNAs in GSE45001 dataset. (b) Expression of LINC01419 in tumor and paracancerous tissues of patients with GC; *p < 0.05 versus the paracancerous tissues. (c) Expression of LINC01419 in metastatic and nonmetastatic tissues of patients with GC; *p < 0.05 versus nonmetastatic tissues. (d) Expression of LINC01419 in gastric epithelial and GC cell lines; *p < 0.05 versus the CES-1 cell line; statistical data were measurement data, and described as mean ± standard deviation; the paired t test was used for comparison between the two groups, and the one-way ANOVA was used for comparison among multiple groups followed by Tukey’s post hoc test. ANOVA, one-way analysis of variance; GC, gastric cancer; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Figure 2.

The PI3K/Akt1/mTOR pathway is blocked through LINC01419 gene silencing. (a) LINC01419 expression in siRNA-NC, siRNA1, siRNA2, and siRNA3; *p < 0.05 versus siRNA-NC. (b) Grey value of Akt1, p-Akt1, mTOR, p-mTOR, Beclin1, LC3BI, and LC3BII protein bands in the MGC-803 cells transfected with LINC01419 siRNA, Rapamycin and LINC01419 siRNA + Rapamycin. (c) Protein expression of Beclin1, LC3BII/I, as well as the extents of Akt1 and mTOR phosphorylation in the MGC-803 cells transfected with LINC01419 siRNA, Rapamycin and LINC01419 siRNA + Rapamycin evaluated by Western blot analysis. (d) LINC01419 expression and mRNA expressions of Beclin1, LC3B, Akt1, and mTOR in the MGC-803 cells transfected with LINC01419 siRNA, Rapamycin and LINC01419 siRNA + Rapamycin determined by RT-qPCR; *p < 0.05 versus the control and NC groups; ^#p < 0.05 versus the Rapamycin group; PI3K/Akt1/mTOR, phosphatidylinositol 3-kinase/Akt1/mammalian target of rapamycin; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; the experiment was repeated three times to obtain the mean value. ANOVA, one-way analysis of variance; NC, negative control.

Figure 3.

MGC-803 cell migration ability is inhibited by LINC01419 silencing and mTOR inhibition. (a) Wound healing images of MGC-803 cell through the scratch test. (b) Statistical chart of cell migration rate; *p < 0.05 versus the control and NC groups; ^#p < 0.05 versus the Rapamycin group; mTOR, mammalian target of rapamycin; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; the experiment was repeated three times to obtain the mean value. ANOVA, one-way analysis of variance; NC, negative control.

Figure 4.

MGC-803 cell invasion ability is prevented by LINC01419 silencing and mTOR inhibition. (a) Cells passing through the bottom chamber were stained with crystal violet. (b) Statistical results of the number of the cells passing through the bottom chamber; *p < 0.05 versus the control and NC groups; ^#p < 0.05 versus the Rapamycin group; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; the experiment was repeated three times to obtain the mean value. ANOVA, one-way analysis of variance; mTOR, mammalian target of rapamycin; NC, negative control.

Figure 5.

LINC01419 siRNA and Rapamycin treatment promotes MGC-803 cell autophagy. (a) Immunofluorescence staining, where LC3B indicates the localization of LC3B in the cells, DAPI indicates nuclear localization, and Merge indicates localization of both. (b) Fluorescence intensity of LC3B in cells transfected with LINC01419 siRNA, Rapamycin, and LINC01419 siRNA + Rapamycin. (c) Observation of autophagic structure of GC cells in each group by transmission electron microscope, where arrows indicate autophagy lysosomes. (d) Gray value of CatB and CatD protein bands in response to LINC01419 siRNA, Rapamycin, and LINC01419 siRNA + Rapamycin treatment assessed by Western blot analysis. (e) Protein expression of CatB and CatD in response to LINC01419 siRNA, Rapamycin, and LINC01419 siRNA + Rapamycin treatment assessed by Western blot analysis; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; *p < 0.05 versus the NC group; ^#p < 0.05 versus the Rapamycin group; the experiment was repeated three times. ANOVA, one-way analysis of variance; Cat, cathepsins; DAPI, 4’,6-diamidino-2-phenylindole; LC3B, light chain 3B; mTOR, mammalian target of rapamycin; NC, negative control.

Figure 6.

GC growth is suppressed by LINC01419 silencing and mTOR inhibition. (a) GC volumes in mice treated with LINC01419 siRNA, Rapamycin and LINC01419 siRNA + Rapamycin. (b) Statistical chart of tumor volume. (c) Tumor weight in mice treated with LINC01419 siRNA, Rapamycin, and LINC01419 siRNA + Rapamycin; *p < 0.05 versus the NC group; ^#p < 0.05 versus the Rapamycin group; mTOR, mammalian target of rapamycin; siRNAs, small interfering RNAs; GC, gastric cancer; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; the experiment was repeated three times to obtain the mean value. ANOVA, one-way analysis of variance; GC, gastric cancer; mTOR, mammalian target of rapamycin.

Figure 7.

LINC01419 combined with Rapamycin downregulates the expressions of MMP2 and VEGF. (a) Expression of MMP2 in the presence of LINC01419 siRNA, Rapamycin, or both. (b) Expression of VEGF in the presence of LINC01419 siRNA, Rapamycin, or both; *p < 0.05 versus the NC group; ^#p < 0.05 versus the Rapamycin group; measurement data were presented as mean ± standard deviation and compared by one-way ANOVA followed by Tukey’s post hoc test; the experiment was repeated three times. ANOVA, one-way analysis of variance; GC, gastric cancer; mTOR, mammalian target of rapamycin; NC, negative control; siRNAs, small interfering RNAs.