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Ginsenoside Rh2 upregulates long noncoding RNA STXBP5-AS1 to sponge microRNA-4425 in suppressing breast cancer cell proliferation

ABSTRACT:

Background

Ginsenoside Rh2, a major saponin derivative in ginseng extract, is recognized for its anticancer activities. Compared to coding genes, studies on long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) that are regulated by Rh2 in cancer cells, especially on competitive endogenous RNA (ceRNA) are sparse.

Methods

LncRNAs whose promoter DNA methylation level was significantly altered by Rh2 were screened from methylation array data. The effect of STXBP5-AS1, miR-4425, and RNF217 on the proliferation and apoptosis of MCF-7 breast cancer cells was monitored in the presence of Rh2 after deregulating the corresponding gene. The ceRNA relationship between STXBP5-AS1 and miR-4425 was examined by measuring the luciferase activity of a recombinant luciferase/STXBP5-AS1 plasmid construct in the presence of mimic miR-4425.

Results

Inhibition of STXBP5-AS1 decreased apoptosis but stimulated growth of the MCF-7 cells, suggesting tumor-suppressive activity of the lncRNA. MiR-4425 was identified to have a binding site on STXBP5-AS1 and proven to be downregulated by STXBP5-AS1 as well as by Rh2. In contrast to STXBP5-AS1, miR-4425 showed pro-proliferation activity by inducing a decrease in apoptosis but increased growth of the MCF-7 cells. MiR-4425 decreased luciferase activity from the luciferase/STXBP5-AS1 construct by 26%. Screening the target genes of miR-4425 and Rh2 revealed that Rh2, STXBP5-AS1, and miR-4425 consistently regulated tumor suppressor RNF217 at both the RNA and protein level.

Conclusion

LncRNA STXBP5-AS1 is upregulated by Rh2 via promoter hypomethylation and acts as a ceRNA, sponging the oncogenic miR-4425. Therefore, Rh2 controls the STXBP5-AS1/miR-4425/RNF217 axis to suppress breast cancer cell growth. Graphical abstract Image 1

SUBMITTER: Park J

PROVIDER: S-EPMC8570952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The efficacy of ginsenoside Rh2 (Rh2) in cancer therapy has been reported; however, its function in lung cancer remains unknown. To analyze the role of Rh2 in the inhibition of lung cancer cell proliferation in the present study, protein expression levels of E-cadherin, vimentin, ?-catenin, Smo, Gli1, and ?-catenin were assessed by western blotting, whilst mRNA expression levels of TCF7 FZD8, Smo, Gli1, Gli2, and Gli3 were determined by reverse transcription-quantitative PCR in the A549 cell line. Phosphorylation sites were detected by proteomic methods and cell proliferation was analyzed by MTT assay. The present study revealed that Rh2 treatment significantly inhibited cell proliferation. Western blotting indicated that the expression levels of E-cadherin were increased and vimentin was downregulated in Rh2-treated cells compared with control cells. Treatment of A549 cells with Rh2 suppressed phosphorylation of five distinct proteins and increased phosphorylation of nine proteins. Among them, the phosphorylation of ?-catenin at S641 was significantly induced. Rh2 treatment suppressed the expression levels of key genes involved in Wnt (Wnt3, transcription factor 7 and frizzled class receptor 8) and hedgehog [smoothened, frizzled class receptor (Smo), GLI family zinc finger (Gli)1, Gli2, and Gli3] signaling. Immunoblotting results indicated that ?-catenin, Smo and Gli1 protein expression levels were also suppressed by treatment with Rh2 compared with control treatment. Expression of ?-catenin S641D, a phosphomimetic form of ?-catenin, inhibited the accumulation of ?-catenin and Gli1 and inhibited cell proliferation and invasion. Furthermore, knockdown of ?-catenin (CTNNB1) or Gli1 with specific small interfering RNAs inhibited cell proliferation, whereas overexpression of these genes had an opposite effect. Additionally, overexpression of ?-catenin or Gli1 activated cell proliferation, even in the presence of Rh2, suggesting that Rh2 affects A549 cell proliferation through inhibition of Wnt and hedgehog signaling by phosphorylation of ?-catenin at S641. Together, these data suggested that Rh2 treatment may inhibit the proliferation of A549 lung cancer cells. Further exploration of the underlying mechanism by which Rh2 inhibits cell proliferation is warranted.