Ham, Juyeon
(Department of Life Science, Dongguk University-Seoul)
,
Jeong, Dawoon
(Department of Life Science, Dongguk University-Seoul)
,
Park, Sungbin
(Department of Life Science, Dongguk University-Seoul)
,
Kim, Hyeon Woo
(Department of Life Science, Dongguk University-Seoul)
,
Kim, Heejoo
(Department of Life Science, Dongguk University-Seoul)
,
Kim, Sun Jung
(Department of Life Science, Dongguk University-Seoul)
Background: Ginsenoside Rg3, a derivative of steroidal saponins abundant in ginseng, has a range of effects on cancer cells, including anti-cell proliferation and anti-inflammation activity. Here, we investigate two long noncoding RNAs (lncRNAs), STXBP5-AS1 and RFX3-AS1, which are hypomethylated and...
Background: Ginsenoside Rg3, a derivative of steroidal saponins abundant in ginseng, has a range of effects on cancer cells, including anti-cell proliferation and anti-inflammation activity. Here, we investigate two long noncoding RNAs (lncRNAs), STXBP5-AS1 and RFX3-AS1, which are hypomethylated and hypermethylated in the promoter region by Rg3 in MCF-7 cancer cells. Methods: The lncRNAs epigenetically regulated by Rg3 were mined using methylation array analysis. The effect of the lncRNAs on the apoptosis and proliferation of MCF-7 cells was monitored in the presence of Rg3 or Korean Red Ginseng (KRG) extract after deregulating the lncRNAs. The expression of the lncRNAs and their target genes was examined using qPCR and Western blot analysis. The association between the expression of the target genes and the survival rate of breast cancer patients was analyzed using the Kaplan-Meier Plotter platform. Results: STXBP5-AS1 and RFX3-AS1 exhibited anti- and pro-proliferation effects, respectively, in the cancer cells, and the effects of Rg3 and KRG extract on apoptosis and cell proliferation were weakened after deregulating the lncRNAs. Of the genes located close to STXBP5-AS1 and RFX3-AS1 on the chromosome, STXBP5, GRM1, RFX3, and SLC1A1 were regulated by the lncRNAs on the RNA and protein level. Breast cancer patients that exhibited a higher expression of the target genes of the lncRNAs had a higher metastasis-free survival rate. Conclusion: The current study is the first to identify lncRNAs that are regulated by the presence of Rg3 and KRG extract and that subsequently contribute to inhibiting the proliferation of cancer cells.
Background: Ginsenoside Rg3, a derivative of steroidal saponins abundant in ginseng, has a range of effects on cancer cells, including anti-cell proliferation and anti-inflammation activity. Here, we investigate two long noncoding RNAs (lncRNAs), STXBP5-AS1 and RFX3-AS1, which are hypomethylated and hypermethylated in the promoter region by Rg3 in MCF-7 cancer cells. Methods: The lncRNAs epigenetically regulated by Rg3 were mined using methylation array analysis. The effect of the lncRNAs on the apoptosis and proliferation of MCF-7 cells was monitored in the presence of Rg3 or Korean Red Ginseng (KRG) extract after deregulating the lncRNAs. The expression of the lncRNAs and their target genes was examined using qPCR and Western blot analysis. The association between the expression of the target genes and the survival rate of breast cancer patients was analyzed using the Kaplan-Meier Plotter platform. Results: STXBP5-AS1 and RFX3-AS1 exhibited anti- and pro-proliferation effects, respectively, in the cancer cells, and the effects of Rg3 and KRG extract on apoptosis and cell proliferation were weakened after deregulating the lncRNAs. Of the genes located close to STXBP5-AS1 and RFX3-AS1 on the chromosome, STXBP5, GRM1, RFX3, and SLC1A1 were regulated by the lncRNAs on the RNA and protein level. Breast cancer patients that exhibited a higher expression of the target genes of the lncRNAs had a higher metastasis-free survival rate. Conclusion: The current study is the first to identify lncRNAs that are regulated by the presence of Rg3 and KRG extract and that subsequently contribute to inhibiting the proliferation of cancer cells.
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문제 정의
This study aimed to identify lncRNAs that were affected by Rg3 and KRG extract treatment and to understand the molecular mechanisms employed by these lncRNAs during cancer cell proliferation. Our initial methylation array analysis identified seven lncRNAs whose methylation levels were significantly affected by Rg3 treatment.
제안 방법
The methylation and expression of RFX3-AS1 (A) and STXBP5-AS1 (B) after treatment with Rg3 in MCF-7 cells. Changes in the methylation levels of the two lncRNAs were identified using microarray analysis and they were examined further using methylation-specific PCR and qPCR. All of the experiments were performed in triplicate and the values are presented as the mean ± standard error (SE).
Gene expression levels were calculated using the 2-ΔΔCt method and normalized using GAPDH.
One of the regulatory mechanisms of lncRNAs is the cisregulation of neighboring genes on the chromosome. To identify genes that could be potentially regulated by RFX3-AS1 and STXBP5- AS1, those located close to the target lncRNAs were screened and their expression levels examined at the RNA and/or protein level. Four genes were found within 900 kb of RFX3-AS1 (Fig.
To investigate the role of STXBP5-AS1 and RFX3-AS1 in cancer cells, the lncRNAs were deregulated by transiently transfecting the MCF-7 cells with siRNAs or overexpression recombinant plasmids and then monitoring cellular activity, such as apoptosis and proliferation. We decided to overexpress RFX3-AS1 because it was hypermethylated and downregulated by Rg3 (Supplementary Fig.
대상 데이터
The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% FBS (Capricorn, Ebsdorfergrund, Germany) and 1% penicillin-streptomycin.
이론/모형
05 was considered statistically significant. The association between gene expression levels and the overall survival rate of breast cancer patients was evaluated using the Kaplan-Meier Plotter (http://kmplot.com/analysis) and databases from GEO (https://www.ncbi.nlm.nih.gov/geo, Affymetrix microarrays only), EGA (https://www.ebi.ac.uk/ega), and TCGA (https://cancergenome.nih.gov).
Representative images are shown. The effect of the siRNA-driven downregulation of STXBP5-AS1 on cell proliferation was examined using colony formation analysis (B) and CCK-8 assays (C). Representative images are shown for flow cytometry and colony formation.
성능/효과
Both of the lncRNAs investigated in this study, RFX3-AS1 and STXBP5-AS1, affected a set of nearby genes, suggesting that they operate in cis-acting mode. Cis-acting lncRNAs can differ in terms of their origin; they may arise from promoters, enhancers, or the antisense transcripts of protein-coding genes [25].
In conclusion, two lncRNAs, RFX3-AS1 and STXBP5-AS1, were identified as being regulated by Rg3 and KRG extract via the alteration of methylation levels at the promoter. These lncRNAs play a role in the apoptosis and proliferation of MCF-7 cancer cells, regulating the expression of both their partner gene encoded by the opposite DNA strand and a number of cis genes, which then mediated the regulatory activity of the lncRNAs.
Rg3 has been shown to regulate a large number of protein-coding genes, thus participating in important cellular activities, but little is known about its relationship with lncRNAs. In this study, two lncRNAs, RFX3-AS1 and STXBP5-AS1, whose promoter methylation levels were affected by Rg3 in the breast cancer cell line MCF-7, were identified. The expression of these lncRNAs and their effect on cancer cell proliferation and apoptosis were examined at both the molecular and cellular level.
Some targets of miR-190b, such as ERG, STK38L, and FNDC3A, are positively associated with STXBP5- AS1 as the result of this competition. The results of our cell proliferation and apoptosis assays suggest that RFX3-AS1 is a noncoding RNA with pro-proliferation activity, while STXBP5-AS1 is characterized by anti-proliferation activity.
후속연구
15; Table 1). After searching the PubMed database, two lncRNAs whose partner coding gene encoded by the DNA sense strand is related to cancer development were selected for further investigation. The first, the 1109-base RFX3-AS1, is the anti-coding strand for the RFX-coding gene.
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