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A daphnane diterpenoid isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity.

Khong A, Forestieri R, Williams DE, Patrick BO, Olmstead A, Svinti V, Schaeffer E, Jean F, Roberge M, Andersen RJ, Jan E - PLoS ONE (2012)

Bottom Line: GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay.Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells.In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21-23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3'untranslated region (3'UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

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Related in: MedlinePlus

Structure of (A) genkwanine M (GENK), (B) diacetyl GENK, and (C) genkwanine P.(B) Diacetyl GENK was prepared from GENK by treatment with acetic anhydride and DMAP in anhydrous THF.
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pone-0039621-g002: Structure of (A) genkwanine M (GENK), (B) diacetyl GENK, and (C) genkwanine P.(B) Diacetyl GENK was prepared from GENK by treatment with acetic anhydride and DMAP in anhydrous THF.

Mentions: To identify compounds that interfere with miRNA and siRNA regulation and/or processing, we screened natural products using a cell-based reporter system that monitors miRNA and siRNA activity. A stably transfected clonal HeLa cell line expressing a reporter RNA encoding the eGFP gene and a sequence that bears perfect complementarity to the let-7a miRNA within the 3′UTR (CMV-GFP-let-7) was generated (Figure 1A). Under basal conditions, endogenously-expressed let-7a binds to the complementary sequence leading to Ago2-mediated cleavage of the reporter RNA resulting in low GFP expression (Figure 1B). By contrast, transfection of an antisense 2′-O-methyl let-7 (AS-let-7) but not a randomized antisense RNA (AS-Ran) resulted in GFP expression, indicating that GFP expression is under the control of let-7a (Figure 1B). Using this cell-based reporter system, a library of crude marine and plant extracts (∼12,000) was screened and four crude extracts were identified that led to an increase in GFP expression (Figure 1B). Specifically, we searched for extracts that led to a >3 fold increase GFP expression over background which is similar to that observed when cells were transfected with the AS-let-7. Guided by the reporter GFP system, sequential fractionation of one of the extracts led to the isolation of genkwanine M (GENK) (Figure 2A). Unfortunately, the other three extracts did not yield a pure compound. As a result, we focused on the characterization of GENK and its cellular effects.


A daphnane diterpenoid isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity.

Khong A, Forestieri R, Williams DE, Patrick BO, Olmstead A, Svinti V, Schaeffer E, Jean F, Roberge M, Andersen RJ, Jan E - PLoS ONE (2012)

Structure of (A) genkwanine M (GENK), (B) diacetyl GENK, and (C) genkwanine P.(B) Diacetyl GENK was prepared from GENK by treatment with acetic anhydride and DMAP in anhydrous THF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383676&req=5

pone-0039621-g002: Structure of (A) genkwanine M (GENK), (B) diacetyl GENK, and (C) genkwanine P.(B) Diacetyl GENK was prepared from GENK by treatment with acetic anhydride and DMAP in anhydrous THF.
Mentions: To identify compounds that interfere with miRNA and siRNA regulation and/or processing, we screened natural products using a cell-based reporter system that monitors miRNA and siRNA activity. A stably transfected clonal HeLa cell line expressing a reporter RNA encoding the eGFP gene and a sequence that bears perfect complementarity to the let-7a miRNA within the 3′UTR (CMV-GFP-let-7) was generated (Figure 1A). Under basal conditions, endogenously-expressed let-7a binds to the complementary sequence leading to Ago2-mediated cleavage of the reporter RNA resulting in low GFP expression (Figure 1B). By contrast, transfection of an antisense 2′-O-methyl let-7 (AS-let-7) but not a randomized antisense RNA (AS-Ran) resulted in GFP expression, indicating that GFP expression is under the control of let-7a (Figure 1B). Using this cell-based reporter system, a library of crude marine and plant extracts (∼12,000) was screened and four crude extracts were identified that led to an increase in GFP expression (Figure 1B). Specifically, we searched for extracts that led to a >3 fold increase GFP expression over background which is similar to that observed when cells were transfected with the AS-let-7. Guided by the reporter GFP system, sequential fractionation of one of the extracts led to the isolation of genkwanine M (GENK) (Figure 2A). Unfortunately, the other three extracts did not yield a pure compound. As a result, we focused on the characterization of GENK and its cellular effects.

Bottom Line: GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay.Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells.In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21-23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3'untranslated region (3'UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

Show MeSH
Related in: MedlinePlus