Plant miRNAs are present in human and animal sera and organs. (A) The levels (Solexa reads) of 10 plant miRNAs detected by Solexa sequencing in sera from healthy Chinese men and women, and eight pooled samples (each pooled from 10 healthy Chinese subjects). For normalization, the sequencing frequency of each plant miRNA was normalized to the total amount of mammalian miRNAs. (B) The absolute levels of plant miRNAs in the sera of various mammals detected by qRT-PCR (n = 6). Endogenous animal miRNAs, miR-16, and miR-25 serve as controls (insert). (C) Semi-quantitative RT-PCR analysis of the indicated miRNAs in the serum from human, mouse, rat, calf, horse, and sheep. Accurate amplification of each miRNA was confirmed by Sanger-based method to sequence the PCR products. (D) Northern blotting analysis of the expression levels of MIR168a, MIR156a, and miR-16 in human serum (100 ml) and calf serum (40 ml). Synthetic MIR168a, MIR156a, and miR-16 (1 pmol) served as positive controls. (E) Equal amount of total small RNAs (each from 100 ml of human serum) were treated with/without sodium periodate. After the reactions, the RNAs were purified and then subjected to Solexa sequencing. Solexa reads of the plant miRNAs in oxidized and unoxidized groups were compared. Total and individual mammalian miRNAs were compared to serve as controls (insert). The absolute Solexa reads of miRNAs are indicated. (F) Equal amount of synthetic plant miRNAs (without 2′-O-methylated 3′ ends) and total small RNAs isolated from rice and human serum were treated with/without sodium periodate. After the reactions, the RNAs were subjected to qRT-PCR with the miScript PCR system. Synthetic miR-16 and total small RNAs isolated from mouse liver and human serum were treated as above. MiR-16 expression levels with/without oxidation were compared to serve as controls (insert). (G) The levels of plant miRNAs detected by qRT-PCR in MVs isolated from C57BL/6J mouse plasma (n = 4). (H) The levels of plant miRNAs detected by Solexa sequencing in various organs of C57BL/6J mice. (I) The levels of plant miRNAs detected by qRT-PCR in various organs of C57BL/6J mice (normalized to U6; n = 6). As before, miR-16 and miR-25 serve as controls (insert). (J, K) The levels of plant miRNAs detected by Solexa sequencing (J) and qRT-PCR (K) in mouse liver after oxidation. Similarly, mammalian miRNAs serve as controls (insert). The absolute Solexa reads of miRNAs are indicated.

The exogenous mature plant miRNAs in food can pass through gastrointestinal (GI) tract and enter the sera and organs. (A) The levels of MIR168a, MIR156a, and MIR166a detected by qRT-PCR in fresh rice and chow diet (n = 6). Two endogenous animal miRNAs, miR-16, and miR-150, served as controls (insert). UD, undetectable. (B, C) The levels of MIR168a in mouse serum (B) and liver (C) after feeding with fresh rice or chow diet for 0.5 h, 3 h, or 6 h (n = 8). The control group (named 0 h) was euthanized after a 12-h of fasting. (D, E) The levels of MIR168a in mouse serum (D) and liver (E) following gavage feeding with RNA extracted from fresh rice (n = 8). After 0.5 h, 3 h, or 6 h, MIR168a levels were detected by qRT-PCR. The control group (0 h) was euthanized after a 12-h of fasting. (F, G) The levels of MIR168a in mouse serum (F) and liver (G) following gavage feeding with synthetic MIR168a and synthetic methylated MIR168a (n = 8). The control group was gavage fed with ncRNA. ^*P < 0.05; ^**P < 0.01.

Plant MIR168a binds to exon 4 of mammalian LDLRAP1 and decreases LDLRAP1 protein level in vitro. (A) Schematic description of the hypothesized duplexes formed by interactions between the exon 4 of LDLRAP1 and MIR168a. Paired bases are indicated by a black oval and G:U pairs are indicated by two dots. The predicted free energy of the hybrid is indicated. Note that the potential binding site of MIR168a to LDLRAP1 mRNA is highly conserved across species. (B) qRT-PCR analysis of MIR168a levels in pre-MIR168a-transfected HepG2 cells (n = 9). (C) Western blot analysis of LDLRAP1 protein levels in pre-MIR168a-transfected HepG2 cells. (D) The quantification of the LDLRAP1 protein expression in C (n = 9). (E, F) Semi-quantitative RT-PCR (E) and qRT-PCR (F) analysis of LDLRAP1 mRNA levels in pre-MIR168a-transfected HepG2 cells (n = 5). (G) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached with the wild-type (WT) or mutant (MUT) MIR168a complementary site (CS), WT, or MUT LDLRAP1 BS, LDLRAP1 exon 4, and the LDLRAP1 CDS. (H) Luciferase activities in HepG2 cells co-transfected with luciferase reporters described in G and pre-MIR168a or pre-ncRNA (n = 9). (I) Constructs for the expression of the WT or MUT ORFs of LDLRAP1. (J) LDLRAP1 level in 293T cells co-transfected with WT or MUT LDLRAP1 ORFs and pre-MIR168a or pre-ncRNA. (K) The quantification of the LDLRAP1 protein expression in J (n = 3). (L) The LDLRAP1 ORF tagged with GFP at its carboxyl terminus. (M) 293T cells were co-transfected with the GFP-tagged LDLRAP1 ORF (top) and pre-MIR168a or pre-ncRNA. GFP-positive cells were analyzed by fluorescence microscopy at 24 h of post-transfection. (N) The percentage of GFP-positive cells in M (n = 6). ^*P < 0.05; ^**P < 0.01.

AGO2-associated mature MIR168a in Caco-2 cell-derived MVs sufficiently reduces mammalian LDLRAP1 protein level in recipient HepG2 cells. (A) A flow chart of the experimental design. (B) The elevation of MIR168a in Caco-2 MVs after transfection with mature MIR168a (left) and in HepG2 cells after treatment with Caco-2 MVs (right; n = 9). Caco-2 MVs were harvested after transfecting the cells with mature MIR168a or ncRNA. (C) Western blot analysis of LDLRAP1 protein levels in HepG2 cells treated with or without Caco-2 MVs. Caco-2 MVs were harvested after transfecting the cells with mature MIR168a or ncRNA. (D) The quantification of the LDLRAP1 protein expression in C (n = 6). (E, F) Semi-quantitative RT-PCR (E) and qRT-PCR (F) analysis of the LDLRAP1 mRNA levels in HepG2 cells treated with or without Caco-2 MVs (n = 5). (G, H) The levels of MIR168a (G) and LDLRAP1 protein (H) in HepG2 cells treated with Caco-2 MVs derived from Caco-2 cells transfected with different doses (10, 50, or 100 pmol/10⁵ cells) of mature MIR168a or ncRNA. (I) The quantification of the LDLRAP1 level in H (n = 3). (J) The luciferase activities in luciferase reporter-transfected HepG2 cells treated with or without Caco-2 MVs (n = 9). (K) LDLRAP1 protein level in 293T cells transfected with WT or MUT LDLRAP1 ORF and then treated with Caco-2 MVs. (L) The quantification of the LDLRAP1 protein expression in K (n = 3). (M, N) The association of MIR168a with AGO2 in Caco-2 MVs (M) and HepG2 cells treated with Caco-2 MVs (N). The levels of MIR168a and miR-16 (control) in anti-AGO2 immunoprecipitated products detected by qRT-PCR (n = 9). (O) The association of LDLRAP1 mRNA with AGO2 in Caco-2 MV-treated HepG2 cells or Caco-2 MVs. The LDLRAP1 mRNA in anti-AGO2 immunoprecipitated products from HepG2 cells (lanes 1 and 2) and Caco-2 MVs (lanes 3 and 4) was detected by semi-quantitative RT-PCR with 25-30 cycles. ^*P < 0.05; ^**P < 0.01.

Anti-MIR168a ASO reverses rice feeding-induced reduction of mouse liver LDLRAP1 protein at 6 h feeding. (A, B) The levels of MIR168a (A) and LDLRAP1 protein (B) in mouse liver after feeding with chow diet, fresh rice, or fresh rice accompanying an intravenous injection of anti-MIR168a ASO or anti-ncRNA for 6 h (n = 8). (C) The quantification of the LDLRAP1 level in B (n = 8). ^*P < 0.05; ^**P < 0.01.

Exogenous MIR168a inhibits mouse liver LDLRAP1 expression and elevates plasma LDL-cholesterol level at 3 days after food intake. (A) The weight changes of mice fed with chow diet or fresh rice (n = 8). (B) The food intake of chow diet or fresh rice (n = 8). (C, D) The levels of MIR168a in mouse sera (C) and livers (D) after chow diet or fresh rice feeding (n = 8). (E) The LDLRAP1 protein levels in mouse livers after chow diet or fresh rice feeding. (F) The quantification of LDLRAP1 protein expression in E (n = 8). (G) The levels of LDL-cholesterol in mouse plasma after chow diet or fresh rice feeding (n = 8). (H, I) The levels of MIR168a (H) and LDLRAP1 protein (I) in mouse livers after 3 days of feeding with chow diet, fresh rice, or fresh rice accompanying an injection of anti-MIR168a ASO or anti-ncRNA (n = 8). (J) The quantification of LDLRAP1 protein expression in I (n = 8). (K) The levels of LDL-cholesterol in mouse plasma after 3 days of feeding with chow diet, fresh rice, or fresh rice accompanying an injection of anti-MIR168a ASO or anti-ncRNA (n = 8). (L) The association of MIR168a with AGO2 in mouse livers after 3 days of feeding with chow diet, fresh rice, or fresh rice plus injection of anti-MIR168a ASO or anti-ncRNA. The levels of MIR168a and miR-16 (control) in anti-AGO2 immunoprecipitated products were detected by qRT-PCR. (M) The association of LDLRAP1 mRNA with AGO2 in mouse livers after 3-day feeding. The LDLRAP1 mRNA in anti-AGO2 immunoprecipitated products was detected by semi-quantitative RT-PCR. (N, O) The levels of MIR168a (N) and LDLRAP1 protein (O) in mouse livers after 3 days of feeding with chow diet and mature MIR168a or ncRNA (n = 8). (P) The quantification of LDLRAP1 protein expression in O (n = 8). (Q) The levels of LDL-cholesterol in mouse plasma after 3 days of feeding with chow diet and mature MIR168a or ncRNA (n = 8). ^*P < 0.05; ^**P < 0.01.