enLanguage
Home / Knowledge / Content

Knowledge

Part2: Downregulation Of Renal Hsa-miR-127-3p Contributes To The Overactivation Of Type I Interferon Signaling Pathway in The Kidney Of Lupus Nephritis

May 09, 2022

For more info. contact tina.xiang@wecistanche.com

The immune system is our body's "guardian" and is the most effective weapon against pathogen invasion. People with low innate immunity are more likely to get sick. The experimental results show that the active ingredients in Cistanche can enhance the body's immunity.

Let's take a closer look.

1. Total glycosides of Cistanche tubulosa 

The total glucosides of Cistanche tubulosa significantly enhanced the antibody production, the number of peripheral blood T lymphocytes, the phagocytic function of peritoneal macrophages, and delayed-type hypersensitivity reactions in Coγ-ray-injured mice. The index, spleen index, and survival rate showed that the total glucosides of Cistanche tubulosa had a strong protective effect on the immune function of radiation-injured mice.

2. Cistanche polysaccharides

Cistanche tubulosa polysaccharides in Cistanche can reduce the content of MDA in the brain, heart, and liver of immunocompromised animal models, increase telomerase activity in the heart and brain tissue, the phagocytic function of peritoneal macrophages, lymphocyte proliferation, spleen T lymphocytes The content of IL-2 and the concentration of calcium ion in thymocytes showed that the polysaccharide of Cistanche deserticola could enhance the immune function of the body.

3. Verbascum 

The active ingredient in Cistanche tubulosa, Xinjiang, caused D-galactose to significantly reduce the content of MDA in the heart, liver, and brain tissue of the subacute aging model mice, and significantly increase the telomerase activity in the heart and brain tissue. The phagocytic function of phagocytes was enhanced, and the proliferation of lymphocytes and the content of IL-2 in peripheral blood were significantly increased. It is shown that Verbascum can enhance the immune function of the body.

Summary: Whether you have low innate immunity or poor acquired immunity, you can eat cistanche-related foods to regulate your own immunity.

effects of cistanche tubulosa adalah improve immunity (25)

Click to know about cistanche tubulosa and where to buy cistanche

RESULTS

Hsa-miR-127-3p ls a Negative Regulator of IFN-I Signaling Pathway

In our previous effort to study renal miRNAs in LN, we compared the miRNA profiles of kidney biopsies from LN patients and a normal control group (described in MATERIALS AND METHODS). There was a distinct miRNA expression pattern in the kidney tissues of LN patients (Figure S1). Among the differentially expressed miRNAs, we selected 9 miRNAs that were downregulated(at least by 50%) and conserved(between human and mouse) and tested their roles in the IFN-I signaling pathway by ISRE-luciferase reporter assay. Considering the limited source of primary human renal cells and they're limited in vitro proliferation ability, we screened and verified the regulatory function of the miRNAs and studied the associated molecular mechanisms using Hela cells. As compared with the other 8 miRNAs, overexpression of hsa-miR-127-3p significantly inhibited ISRE mediated induction of luciferase activity in Hela cells stimulated by IFN-α(Figures 1A and Figure S2). Thus, we chose to further study hsa-miR-127-3p. In addition to IFN-α, IFN-β(another member of IFN-I)stimulated induction of ISRE-luciferase activity could also be inhibited by hsa-miR-127-3p(Figure S3).IFN-I can also induce gene expression mediated by the GAS element (3). As expected, overexpression of hsa-miR-127-3p inhibited GAS-mediated induction of luciferase activity stimulated by IFN-α(Figure 1A). Since miRNAs recognize their targets by complementary base pairing of their seed sequences to the target sites on target mRNAs (6). This inhibitory function of hsa-miR-127-3p on the IFN-I signaling pathway was eliminated by the mutation of its seed sequence(Figures 1A). Consistent with these results, overexpression of hsa-miR-127-3p inhibited IFN-α stimulated phosphorylation of STATl and STAT2, with a much stronger effect on STAT2, in Hela cells(Figures 1B and S4).To prove that this inhibitory effect of hsa-miR-127-3p was not restricted to Hela cells, we further verified that overexpression of hsa-miR-127-3p inhibited IFN-stimulated phosphorylation of STAT2 in two additional cell types(Figure S5).

Tovalidatehsa-miR-127-3p functions as a cell-intrinsic negative regulator of IFN-I signaling pathway in renal resident cells, we performed ISRE-and GAS-luciferase reporter assays using primary HRMCs, which can respond to innate immune stimuli and participate in the pathogenesis of LN as indicated in previous studies(12-15). We found hsa-miR-127-3p antagomir enhanced ISREorGAS mediated induction of luciferase activity stimulated by IFN-α in primary HRMCs(Figure 1C). Further,hsa-miR-127-3p antagomir enhanced the phosphorylation of STAT2 stimulated by IFN-α in primary HRMCs(Figures 1D and S6).

Hsa-miR-127-3p negatively regulates IFN-I signaling pathway. (A) Hela cells transfected with ISRE-luciferase reporter (left) or GAS-luciferase reporter (right) and pRL-TK vectors together with negative control mimics (NC), hsa-miR-127-3p mimics, or hsa-miR-127-3p mutants (mut) were stimulated with universal type I interferon for 8 h. Firefly (F) and renilla (R) luciferase activities were measured. (B) Hela cells transfected with negative control mimics (-) or hsa-miR-127-3p mimics (+) were stimulated with universal type I interferon for 0- or 10-min. Western blot for STAT-1 and STAT-2, and phosphorylated STAT-1(pY701) and STAT-2 (pY690). (C) Primary HRMCs transfected with ISRE-luciferase reporter (left) or GAS-luciferase reporter (right) and pRL-TK vectors together with negative control antagomiR (antagomiR-NC), or hsa-miR-127-3p antagomiR (antagomiR-127-3p) were stimulated with universal type I interferon for 8 h. F and R luciferase activities were measured. (D) Primary HRMCs transfected with negative control antagomiR (-), or hsa-miR-127-3p antagomiR (+) were stimulated with universal type I interferon for 0- or 15-min. Western blot for STAT-2 and STAT-2(pY690). (A, C) The induction fold was calculated by dividing the F/R ratio of a sample in experimental groups by the F/R ratio of an unstimulated control sample. (B, D) Representative pictures from at least 3 independent experiments. (A, C) Data from at least 3 independent experiments are plotted and presented as mean ± SEM. P values were determined by Mann-Whitney U-test

Next, we performed microarray to analyze the inhibitory effect of hsa-miR-127-3p on the expression of genes using RNA samples

from IFN-α treated Hela cells which were previously transfected with hsa-miR-127-3p or various controls (mock transfection, negative control mimics, hsa-miR-127-3p mutant mimics). We calculated the overlapping differentially expressed genes in IFN-o treated Hela cells (upregulated or downregulated by 1.5-fold)between different comparison pairs(hsa-miR-127-3p mimics vs mock, hsa-miR-127-3p mimics vs negative control mimics, and hsa-miR-127-3p mimics vs hsa-miR-127-3p mutant mimics), retrieved all the genes that were inhibited by hsa-miR-127-3p and performed an analysis to identify enriched cis-regulatory motifs of the hsa-miR-127-3p inhibited genes using iRegulon plugin on Cytoscape platform (10). We found that cis-regulatory motif that is used byIFN-I downstream TFs(such as STAT-1, STAT-2, and IRFproteins)dominated the top enriched motifs(9out of the top 10 motifs)(Figure 2A). This further proves that hsa-miR-127-3p mainly inhibits IFN-I downstream signaling. Additionally, from the microarray data, we found overexpression of hsa-miR-127-3p inhibited the induction of many ISGs, including those that constitute the 21-gene IFN signature used for evaluating the efficacy of anifrolumab in blocking IFN-I signaling(8)(Figure 2B). Consistent with previous results, hsa-miR-127-3p mutant mimics could not inhibit those ISGs(Figure 2B). We verified the inhibition of hsa-miR-127-3p on two representatives ISGs(IFIT3 and CXCL10)by additional independent experiments (Figure 2C). While hsa-miR-127-3p antagomir enhanced the induction of IFIT3 and CXCL10 by IFN-α in primary HRMCs(Figure 2D). Therefore, hsa-miR-127-3p functions as a negative regulator of the IFN-I signaling pathway.

Hsa-miR-127-3p inhibits the induction of IFN stimulated genes. (A) Hsa-miR-127-3p inhibited genes as compared with the controls (mock transfection, negative control mimics and hsa-miR-127-3p mutant mimics) in IFN-a treated Hela cells were used for the enrichment analysis of cis-regulatory motif using iRegulon plugin on Cytoscape platform. Big dots with deeper green color represent the motifs with higher normalized enrichment scores. (B) Heatmaps show the expression of the ISGs that constitute a 21-gene IFN signature (YAO score) (left) and additional top 21 IFN induced genes (right) in Hela cells that were transfected and stimulated as indicated in the pictures. Mock transfected Hela cells without IFN stimulation was used as baseline control. Red = upregulated; White = no change; Blue = downregulated. (C) qPCR analysis was performed for IFIT3 and CXCL10 in Hela cells transfected with negative control mimics (NC) or hsa-miR-127-3p mimics and stimulated with universal type I interferon for 8 h. (D) qPCR analysis was performed for IFIT3 and CXCL10 in primary HRMCs transfected with antagomiR-NC or antagomiR-127-3p and stimulated with universal type I interferon for 8 h. (C, D) The induction fold was calculated by dividing the relative expression of IFIT3 (or CXCL10) of a sample in experimental groups by the relative expression of IFIT3 (or CXCL10) of an unstimulated control sample. Data from at least 3 independent experiments are plotted and presented as mean ± SEM. P values were determined by Mann-Whitney U-test.

cistanche tubulosa extract benefits improve immunity

JAK1 Is a Bona Fide Target Gene of Hsa-miR-127-3p

Since hsa-miR-127-3p inhibits the tyrosine phosphorylation of STAT1 and STAT2 and the induction of most ISGs stimulated by IFN-I, we hypothesized that hsa-miR-127-3p may target key upstream signal transduction molecules of IFN-I signaling pathway, such as JAK1 and TYK2. Therefore, we examined the protein levels of JAK1 and TYK2 in the cells transfected with hsa-miR-127-3p or mimic controls. We found overexpression of hsa-miR-127-3p reduced JAK1 expression at both protein level and mRNA level, but not TYK2(Figures 3A and S7, S8). Thus, we decided to test if JAK1 was a target of hsa-miR-127-3p.

By 2 different algorisms(16,17), we predicted 3 potential hsa-miR-127-3pbinding sites onJAK1 mRNA, with one binding site in 3'UTR and the other two in the coding sequence(CDS)(Figure S9). Although weak, hsa-miR-127-3p mimics inhibited the expression of luciferase gene fused with wildtype JAK13'UTR, while mutation of the target sequence in JAK1 3'UTR abolished the inhibition (Figure 3B). Additionally, we cloned a truncated JAK1 coding region containing the 2 hsa-miR-127-3p target sites into the pCDNA3.1 vector. Then, we made a mutant form of JAK1 truncation expression plasmid by disrupting the 2 hsa-miR-127-3p target sites. We found that hsa-miR-127-3p could also inhibit the expression of truncated JAK1 from wild-type plasmid but not the mutant one(Figures 3C and S10). Considering the independent inhibitory effect of hsa-miR-127-3p onJAK1-3'UTRor JAK1-CDS was weak, we propose that hsa-miR-127-3p act on these binding sites together to achieve a significant inhibition of the expression of JAK1. To further verify this targeting relationship between hsa-miR-127-3p andJAK1, we show that JAK1 mRNA was enriched in the Ago2 complex in the cells where hsa-miR-127-3p was overexpressed, but not in the cells overexpressing the mutant form of hsa-miR-127-3p (Figure 3D).

JAK1 is a bona fide target gene of hsa-miR-127-3p. (A) Hela cells were transfected with negative control mimics (NC) or hsa-miR-127-3p mimics and then cultured for 24 h. Whole cell lysates were prepared, and Western blot was performed for JAK1, TYK2, and b-Tubulin. (B) Hela cells were transfected with psiCHECK2, psiCHECK2-JAK1-3’UTR, or psiCHECK2-JAK1-3’UTR-mutant plasmids together with hsa-miR-127-3p mimics and then cultured for 24 h. Cell lysates were prepared, and firefly and renilla luciferase activities were measured. The ratio of renilla to firefly luciferase activity was calculated for each sample. (C) U4A cells were transfected with pcDNA3.1-JAK1t, or pcDNA3.1-JAK1t-mutant plasmids together with negative control mimics (NC) or hsa-miR-127-3p mimics and then cultured for 24 h. Whole cell lysates were prepared, and Western blot was performed for wild type HA-tagged JAK1 truncates (JAK1CDS), mutated HA-tagged JAK1 truncates (JAK1CDSmut), and b-Tubulin. (D) Hela cells were transfected with hsa-miR-127-3p mimics or hsa-miR-127-3p mutant mimics (mut) and then cultured for 12 h. Cell lysates were prepared, and RNA immunoprecipitation was performed. The levels of JAK1 mRNA were quantified for each sample. And the enrichment fold of JAK1 mRNA in anti-Ago2 antibody pull-down products compared to IgG was calculated. (A, C) Representative pictures from at least 3 independent experiments. (B, D) Data from at least 3 independent experiments are plotted and presented as mean ± SEM. P values were determined by MannWhitney U-test.

Further, we found, that the levels of hsa-miR-127-3p in kidney biopsies from LN patients were significantly lower than that of normal controls(Figure 4A), while the levels of JAK1 and IFIT3 in kidney biopsies from LN patients were significantly higher than that from normal controls (Figures 4B, C). Additionally, there was a negative correlation between the levels of hsa-miR-127-3p and JAK1 (or IFIT3)in kidney biopsies from LN patients(Figures 4D, E). Therefore, JAK1 is a bona fide target gene of hsa-miR-127-3p.

Negatve association of the expression levels of JAK1 or IFIT3 with hsa-miR-127-3p in renal tissues of LN patients. Total RNAs were extracted from kidney biopsies of normal controls (n=11) and LN patients (n=14) and subjected to qPCR analysis for hsa-miR-127-3p (A), JAK1 (B) and IFIT3 (C). (D, E) Correlation between the levels of hsa-miR-127-3p and JAK1 (D), or IFIT3 (E) in kidney biopsies from LN patients (n=14). (A-C) Data from each subject of the normal and LN groups are plotted and presented as mean ± SEM. P values were determined by Mann-Whitney U-test. (D, E) Each dot represents individual LN patients. P values were determined by Spearman’s correlation test

effects of cistanche：improve kidney function4

DISCUSSION

In the present study, we continued our previous effort to explore the functions of dysregulated renal miRNAs in LN. Here, we focused on downregulated miRNAs in kidney biopsies from LN patients. We chose 9 of them for the analysis based on 2 criteria: conservation and the degree of downregulation (detailed in RESULTS). Since IFN-I is critical to the development of SLE andLN(3), we screened the 9 miRNAs for potential regulators of the IFN-I signaling pathway. We identified hsa-miR-127-3p as a negative regulator of the IFN-I signaling pathway by showing it could inhibit the phosphorylation of STAT proteins and ISRE(or GAS) mediated induction of gene expression stimulated by IFN-α. We found this inhibitory effect existed in multiple distinct cell types. Further, overexpression of hsa-miR-127-3p could extensively reduce the induction of ISGs. What's more, there is considerable overlap between hsa-miR-127-3p inhibited ISGs and anifrolumab suppressed IFN signature—17 out of the 21 IFN signature genes could be inhibited by hsa-miR-127-3p—which is used to evaluate anifrolumab's inhibitory efficiency (18). Of note, a recent phase 3 clinical trial suggested that monthly administration of nivolumab benefits SLE patients as compared with placebo(2). Thus, our findings indicate that hsa-miR-127-3p may serve as an IFN-I blockade disease (20). This further reinforces the therapeutic potential of hsa-miR-127-3p in treating autoimmune diseases.

There are unsolved questions for future studies. One is that we still don't know the exact mechanism for the downregulation of hsa-miR-127-3p in the kidney of LN, which will help us understand LN pathogenesis. Our unpublished preliminary data indicate that long-term activation of the IFN-I signaling pathway suppressed hsa-miR-127-3p expression. Thus, there may be a feedforward loop formed by the excessive IFN-I and decreased intracellular hsa-miR-127-3p, which amplifies IFN-I signaling. The other is, till now, we could not examine the in vivo inhibitory effects of hsa-miR-127-3p on JAK1 because hsa-miR-127-3p binding sequences of the human JAK1 gene do not exist in mice. In the future, engineered mice expressing the human JAK1 gene could help clarify hsa-miR-127-3p's in vivo efficiency in inhibiting JAK1 and its associated cytokine signaling.

To summarize, we show that JAK1 is a bona fide target of hsa-miR-127-3p and abnormal downregulation of renal hsa-miR-127-3p contributes to LN pathogenesis by enhancing the expression of JAK1 and the subsequent activation of IFN-I signaling pathway in kidney. Since JAK1-associated inflammatory cytokine signaling pathways are critical to the development of many autoimmune diseases(4), future studies are warranted to explore hsa-miR-127-3p's therapeutic effects on LN or other autoimmune disease-related organ damages with abnormal upregulation of JAK1.

effects of herba cistanches：improve kidney function


You Might Also Like