Influence of the Bioactive Diet Components on the Gene Expression Regulation

Diet bioactive components, in the concept of nutrigenetics and nutrigenomics, consist of food constituents, which can transfer information from the external environment and influence gene expression in the cell and thus the function of the whole organism. It is crucial to regard food not only as the source of energy and basic nutriments, crucial for living and organism development, but also as the factor influencing health/disease, biochemical mechanisms, and activation of biochemical pathways. Bioactive components of the diet regulate gene expression through changes in the chromatin structure (including DNA methylation and histone modification), non-coding RNA, activation of transcription factors by signalling cascades, or direct ligand binding to the nuclear receptors. Analysis of interactions between diet components and human genome structure and gene activity is a modern approach that will help to better understand these relations and will allow designing dietary guidances, which can help maintain good health.

saKLK1-374 is More Difficult to Induce KLK1 Expression in Normal Cell Lines Than in Tumor Cell Lines And Inhibits the Growth of Prostate Cancer Cells Not via Induction of KLK1 Expression

Background: RNA activation, as a method of regulating gene expression at the transcriptional level, is far less widely used than RNA interference because of the insufficient understanding of the mechanism and the unstable success rate. It is necessary to analyze the failure cases of RNA activation to promote the application of RNA activation. When we validated the saRNAs designed to induce KLK1 expression, we found that saKLK1-374 can up-regulate KLK1 expression in prostate tumor cell lines, but failed in normal prostate cell lines. In addition, we also found that saKLK1-374 inhibited the growth of prostate cancer cells, which seems to be the opposite of the function of KLK1. This article is about experimental research and analysis of these two issues.Methods: To determine whether the phenomenon that the RNA activation of normal cells is difficult to succeed is only valid when the target gene is KLK1, we used p21WAF1/CIP1 as the target gene to perform RNA activation experiments in normal prostate cells and prostate cancer cells. Next, to determine whether the above phenomenon exists in other tissues, we also performed RNA activation experiments with KLK1 and p21WAF1/CIP1 as target genes in normal cell lines and tumor cell lines derived from the bladder. We have also extended the time from transfection to the detection of target gene expression to evaluate whether a longer saRNA action time can change the phenomenon that saRNA fails to up-regulate target gene expression in normal cells. In terms of mechanism research, we used fluorescently labeled dsRNA to evaluate the transfection efficiency, and also detected the expression of Ago2 and IPO8 proteins. In another issue of saKLK1-374 inhibiting prostate cancer cells, we tested the ROS content and apoptosis levels of prostate cancer cells after saKLK1-374 transfection. We used recombinant KLK1 protein to directly interfere with prostate cancer cells as a positive control for KLK1 function research. In turn, we also used siRNA to inhibit the expression of KLK1 in prostate cancer cells to compare the growth of prostate cancer cells when KLK1 mRNA was up-regulated and reduced.Results: The p21WAF1/CIP1 gene could be significantly upregulated by saRNA in prostate cancer cell lines, but not in normal prostate cell lines. The expression of KLK1 in bladder-derived cell lines was extremely low and could not be induced by saRNA. The p21WAF1/CIP1 gene could be up-regulated by saRNA to a higher extent in bladder cancer cell lines, while it was up-regulated by saRNA in normal urothelial cell line to a lower extent. Prolonging the action time of saRNA could not change that saRNA failed to induce the expression of target genes in normal cell lines. Compared with tumor cell lines, normal cell lines had lower transfection efficiency or lower expression of Ago2 and IPO8. After being transfected with saKLK1-374, prostate cancer cells had increased ROS and increased levels of apoptosis. The recombinant KLK1 protein did not increase ROS in prostate cancer cells, nor did it inhibit their growth. Even though saKLK1-374 up-regulated the expression of KLK1 in prostate cancer cells, siRNA still suppressed the expression of KLK1 below the baseline level, and in this case, the growth of prostate cancer cells was still at a suppressed level.Conclusion: Normal cell lines may be more difficult to be successfully induced target gene expression than tumor cells due to low transfection efficiency or low Ago2 and IPO8 expression. In addition, although saKLK1-374 is designed to up-regulate the expression of KLK1, the reason that it inhibits the proliferation of prostate cancer cells is irrelevant to the up-regulated expression of KLK1.

HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.

Small Activating RNAs: Towards the Development of New Therapeutic Agents and Clinical Treatments

Small double-strand RNA (dsRNA) molecules can activate endogenous genes via an RNA-based promoter targeting mechanism. RNA activation (RNAa) is an evolutionarily conserved mechanism present in diverse eukaryotic organisms ranging from nematodes to humans. Small activating RNAs (saRNAs) involved in RNAa have been successfully used to activate gene expression in cultured cells, and thereby this emergent technique might allow us to develop various biotechnological applications, without the need to synthesize hazardous construct systems harboring exogenous DNA sequences. Accordingly, this thematic issue aims to provide insights into how RNAa cellular machinery can be harnessed to activate gene expression leading to a more effective clinical treatment of various diseases.

Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated. Argonaute 2 (Ago2) is highly expressed in mouse oocyte and early embryos. In this study, we showed that Ago2-dependent degradation involving RNA interference (RNAi) and RNA activation (RNAa) pathways contributes to the decay of over half of the maternal mRNAs in mouse early embryos. We demonstrated that AGO2 guided by endogenous small interfering RNAs (endosiRNAs), generated from double-stranded RNAs (dsRNAs) formed by maternal mRNAs with their complementary long noncoding RNAs (CMR-lncRNAs), could target maternal mRNAs and cooperate with P-bodies to promote MRD. In addition, we also showed that AGO2 may interact with small activating RNAs (saRNAs) to activate Yap1 and Tead4, triggering ZGA-dependent MRD. Thus, Ago2-dependent degradation is required for timely elimination of subgroups of maternal mRNAs and facilitates the transition between developmental states.

Up-regulation of Rb by small activating RNA inhibits cell invasion and migration in gastric cancer cells

Background: Gastric cancer (GC) is a lethal disease that needs further investigation. Recent studies have reported that small activating RNA (saRNA), involved in a process called RNA activation, plays an important role in the development of cancer. Methods: The expression of retinoblastoma (Rb) was detected in human GC tissues and cell lines. We designed three different saRNAs targeting the Rb gene promoter in GC cell lines. Upregulated Rb expression after transfection of the saRNAs was confirmed by PCR and western blotting. And GC cell proliferation, migration, and invasion were detected using CCK8 assays and Transwell assays. Results: Here, we found that Rb displayed lower expression in GC tissues and cells. saRNA-3 significantly increased the expression of Rb in both SGC-7901 and MKN-28 cells. saRNA-3 significantly inhibited the proliferation of both cell types by day 3 after transfection. There was no significant difference between the negative control saRNA and mock transfection groups. saRNA-3 decreased the tumor volume and weight compared with the mock and dsControl groups. In addition, saRNA-3 transfection decreased the migratory ability of SGC-701 and MKN-28 cells.Conclusions: Overall, Rb is a promising novel prognostic biomarker of GC and, due to its role in metastasis, a novel therapeutic target for the clinical management of invasive and metastatic GC.

Up-regulation of Rb by small activating RNA inhibits cell invasion and migration in gastric cancer cells

Gastric cancer (GC) is a lethal disease that needs further investigation. Recent studies have reported that small activating RNA (saRNA), involved in a process called RNA activation, plays an important role in the development of cancer. We designed three different saRNAs targeting the retinoblastoma (Rb) gene promoter in GC cell lines. Upregulated Rb expression after transfection of the saRNAs was confirmed by PCR and western blotting and resulted in inhibition of GC cell proliferation, migration, and invasion compared with the control groups. There was no significant difference between the negative control saRNA and mock transfection groups. Overall, Rb is a promising novel prognostic biomarker of GC and, due to its role in metastasis, a novel therapeutic target for the clinical management of invasive and metastatic GC.