Aloe-emodin mediates the inhibitory effect of LncRNA D63785 on the PI3K/Akt/mTOR pathway in nasopharyngeal carcinoma

Abstract

Background: Long non-coding RNAs (lncRNAs) are dysregulated in nasopharyngeal carcinoma (NPC), yet their interplay with pharmacological agents like aloe-emodin (AE) remains unclear. This study explores AE's anti-NPC mechanisms via lncRNA D63785 and the PI3K/Akt/mTOR pathway.

Methods: NPC cells (CNE1, C666-1) were treated with AE, followed by qRT-PCR and Western blotting to assess lncRNA D63785 and PI3K/Akt/mTOR pathway proteins. siRNA-mediated lncRNA D63785 knockdown combined with functional assays (CCK-8, EdU, colony/wound-healing) evaluated AE's effects on proliferation, migration, and pathway activity. In vivo validation used nude mouse xenografts.

Results: LncRNA D63785 was overexpressed in NPC cells (p < 0.01). AE suppressed lncRNA D63785 expression, concurrently reducing PI3K/Akt/mTOR phosphorylation (p < 0.05). siRNA knockdown partially reversed AE's inhibition of NPC cell viability, proliferation, and migration. In vivo, AE attenuated tumor growth (p < 0.05), correlating with lncRNA D63785 downregulation and PI3K/Akt/mTOR dephosphorylation.

Conclusion: AE exerts anti-NPC effects by targeting the lncRNA D63785-PI3K/Akt/mTOR axis, offering a novel therapeutic strategy. These findings bridge AE's pharmacological activity with lncRNA regulatory networks in NPC pathogenesis.

Keywords: AE; LncRNA D63785; NPC; PI3K/Akt/mTOR signaling pathway; proliferation and migration.

FIGURE 1

AE downregulated lncRNA D63785 and inhibited the PI3K/Akt/mTOR pathway in CNE1 and C666-1 NPC cells in a concentration-dependent manner. AE significantly inhibits the expression of lncRNA D63785 in NPC cells, and this effect is enhanced with increasing concentrations. (A) Expression of LncRNA D63785 in normal nasal mucosal epithelial cells and multiple nasopharyngeal carcinoma cells. Comparison of LncRNA D63785 self-expression between normal human nasal mucosal epithelial cells HNEpC and nasopharyngeal carcinoma CNE1, 5-8F, C666-1, HONE1 cells. The data shown is mean ± standard deviation, n = 3. ^nsP>0.05,*P < 0.05, **P < 0.01, compared to the HNEpC group. (B,C) Changes in LncRNA D63785 under the action of aloe-emodin. The control group and different concentration gradients of aloe-emodin were treated on C666-1 cells for 48 h, and the expression of LncRNA D63785 was detected by real-time quantitative PCR (B). The expression of LncRNA D63785 was detected by real-time quantitative PCR in CNE1 cells treated with control group and different concentration gradients of aloe-emodin for 48 h (C). The data shown is mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, compared to the control (0) group. (D–F) The effect of aloe-emodin on the PI3K/Akt/mTOR signaling pathway in nasopharyngeal carcinoma cells. (D) The expression of pathway proteins in C666-1 and CNE1 cells was detected by Western blot after 48 h of treatment with control group and different concentration gradients of aloe-emodin. (E,F) Bar chart of phosphorylation protein expression of C666-1 and CNE1 cells under the action of control group and different concentration gradients of aloe-emodin. The data shown is mean ± standard deviation, n = 3. ^nsP>0.05,*P < 0.05, **P < 0.01, compared to the control (0) group.

FIGURE 2

AE inhibited NPC cell vibility may be mediated by LncRNA D63785. (A) Expression of LncRNA D63785 in nasopharyngeal carcinoma cells transfected with siRNA. The control group, transfected empty plasmid group, and transfected siRNA group were treated with nasopharyngeal carcinoma C666-1 and CNE1 cells for 48 h, and the expression of LncRNA D63785 was detected by real-time quantitative PCR. The data shown is mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, compared to the control group. (B,C) Knockdown of LncRNA D63785 in NPC cells inhibits cell activity. The control group, empty group (NC) siRNA group, AE group, and siRNA + AE group were treated for 48 h, and the viability of C666-1 (B) and CNE1 (C) was detected by CCK-8. The data shown is mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, compared to the control group; ^#P < 0.05,^##P < 0.01, compared to the siRNA + AE group. (D–G) The control group, empty group (NC), siRNA group, AE group, and siRNA + AE group were treated for 48 h, and the proliferation rate of nasopharyngeal carcinoma cells was detected by EdU incorporation experiment. (H–J) The control group, empty group (NC), siRNA group, AE group, and siRNA + AE group were treated for 48 h, and the proliferation rate of nasopharyngeal carcinoma cells was detected through colony formation assay. Bar = 100 μm. The data shown is mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, compared to the control group; ^#P < 0.05, ^##P < 0.01, compared to the siRNA + AE group.

FIGURE 3

AE exerted its inhibitory effect on NPC cell migration ability through LncRNA D63785. (A,B) The control group, empty group (NC) siRNA group, AE group, and siRNA + AE group were treated for 48 h, and the scratch healing experiment was used to detect the migration ability of nasopharyngeal carcinoma cells C666-1 (A) and CNE1 (B). Bar = 100 μm. (C,D) Histogram of migration ability of C666-1 (C) and CNE1 (D) cells. The data shown is mean ± standard deviation, n = 3. *P < 0.05, *P < 0.01, compared to the control group; ^#P < 0.05, ^##P < 0.01, compared to the siRNA + AE group.

FIGURE 4

The effects of aloe-emodin and knocking down LncRNA D63785 on the PI3K/Akt/mTOR signaling pathway in nasopharyngeal carcinoma cells. (A) The control group, empty group (NC) siRNA group, AE group, and siRNA + AE group were treated for 48 h, and the expression of pathway proteins was detected by Western blot. Histogram of C666-1 (B) and CNE1 (C) cells phosphorylated proteins p-PI3K, p-Akt, and p-mTOR expression. The data shown is mean ± standard deviation, n = 3. ^nsP>0.05, *P < 0.05, **P < 0.01, compared to the control group; ^#P < 0.05, ^##P < 0.01, compared to the siRNA + AE group.

FIGURE 5

AE inhibits the expression of LncRNA D63785 and phosphorylation of PI3K/Akt/mTOR pathway proteins in subcutaneous transplanted tumors of NPC in nude mice. (A) Expression of LncRNA D63785 in the control group and aloe-emodin group. The data shown is mean ± standard deviation, n = 3, **P < 0.01, compared to the control group. (B,C) Expression of PI3K/Akt/mTOR signaling pathway proteins in the control group and aloe-emodin group. The data shown is mean ± standard deviation, n = 3, **P < 0.01, compared to the control group.

FIGURE 6

Graphical abstract.