Changhong Sun

Real-Time Polymerase Chain Reaction MicroRNA Detection Based on Enzymatic Stem-Loop Probes Ligation

MiRNAs (microRNAs) are a group of endogenous, small noncoding RNA with the length of 18-25 nucleotides, which have recently been demonstrated to play important roles in a wide range of biological processes. In this work, we developed a simple, sensitive, specific, and inexpensive assay through the combination of enzymatic probe ligation and real-time PCR amplification for the measurement of mature miRNAs. A couple of novel DNA probes with a stem-loop structure were implemented to reduce nonspecific ligation by at least 100-fold. The assay has several remarkable features including wide dynamic range, low total RNA input (0.02-0.2 ng), distinct anti-interference from precursor miRNAs (signal-to-noise ratio > 500), and single-base mismatch discrimination among miRNA sequences. In addition, a one-tube assay could be accomplished by designing a couple of universal probes, which makes it feasible to examine the expression of a whole family of miRNA (such as let-7) at one time. Finally, we validated the method for quantifying the expression of four mature miRNAs including miR-122, miR-1, miR-34a, and let-7a across 10 mouse tissues, where U6 snRNA could be simultaneously examined as an endogenous control. Thus, this method revealed a great potential for miRNA quantitation in ordinary laboratory studies and clinical diagnoses.

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

miRNA globally deregulates human carcinoma. A critical open question is how many miRNAs functionally participate in cancer development, particularly in metastasis. We systematically evaluate the capability of all known human miRNAs to regulate certain metastasis-relevant cell behaviours. To perform the high-throughput screen of miRNAs, which regulate cell migration, we developed a novel self-assembled cell microarray. Here we show that over 20% of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. MiR-23b, which is downregulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including tumour growth, invasion and angiogenesis in vivo. It regulates a cohort of prometastatic targets, including FZD7 or MAP3k1. These findings provide new insight into the physiological and potential therapeutic importance of miRNAs as a new class of functional modulators.

Quantitative analysis of zeptomole microRNAs based on isothermal ramification amplification

To date, approximately 700 microRNA (miRNA) molecules have been identified in humans. Accurate and sensitive quantification of miRNA levels will help unveil their biological functions. Here, we extend the isothermal ramification amplification (RAM) approach to a sensitive and specific real-time assay for quantitative analysis of miRNA. This RAM miRNA assay is based on the threshold cycle (C(T)) principle similar to that of real-time PCR. It has a dynamic range of at least seven orders of magnitude, allowing for the quantification of miRNA input from 10(3) to 10(10) copies per reaction (10 nM to 1 fM). The capabilities of discriminating single-base mismatch and distinguishing mature miRNAs from their precursors are achieved by coupling the reverse-transcription of miRNA to the generation of a closed C-probe, rather than using expensive detection probes like in real-time PCR. Quantitative measurement of 5 miRNAs (mir-1, miR-122, mir-150, mir-143, and let-7a) across 12 mouse tissues is validated in total RNA samples without further purification. U6 snRNA, snoRNA 135, and miRNA-191 could be simultaneously quantified as endogenous controls. These results suggest that our RAM miRNA assay might provide a universal tool for miRNA detection and functional studies to meet the needs for bench examination, clinical diagnosis, and on-site detection.

An Integrated Chip for the High‐Throughput Synthesis of Transcription Activator‐like Effectors

A TALE of two assays: Transcription activator-like effectors (TALEs) are programmable proteins that can specifically recognize a DNA sequence. Previous strategies for the synthesis of TALEs were complicated and time-consuming. The solid-phase synthesis strategy demonstrated here allows quick and simple purification of the ligation product.

miR-19b promotes tumor growth and metastasis via targeting TP53.

Tumor suppressor TP53 (or p53) is one of the most important regulators in numerous physiological and pathological processes. Recently, the miRNA-mediated post-transcription regulation of p53 has been studied. However, systematic studies of miRNA targeting sites within the p53 gene are still a challenging task. Here, we developed a dual-color assay capable of identifying miRNA targeting sites in a certain gene, specifically p53, in a simple, direct, and robust manner. Results showed that p53 was a direct and critical target of miR-19b, but not miR-19a, regardless of sequence similarity. Overexpression of miR-19b observed in human cancer cells can diminish p53 protein levels and, subsequently, downstream components such as Bax and p21. This miR-19b-mediated p53 reduction was shown to promote cell cycle, cell migration or invasion, and repress senescence and apoptosis in vitro. Further investigation revealed that miR-19b controls tumor growth and metastasis in vivo. Therefore, it is possible that miR-19b antagomirs or sponges could be developed as therapeutic agents against tumor development.

The synergistic regulation of VEGF-mediated angiogenesis through miR-190 and target genes

VEGF is a major contributor to angiogenesis, a vital process in normal growth and development and tumor transition. However, the current clinical efficacy of VEGF inhibitors is limited, and the molecular mechanism underlying VEGF regulation remains to be elucidated. Here we show that miR-190 directly targets a group of angiogenic effectors besides VEGF per se. Noted, these effectors can transcriptionally regulate VEGF expression in an intracellular or intercellular manner, thus demonstrating that miR-190 modulates the VEGF-mediated tumor angiogenesis at three levels. The synergistic effect of miR-190 and its target genes demonstrates a complex but apparently more stable system, allowing for the tight control of the level of VEGF. Finally, we showed that miR-190 significantly suppresses tumor metastasis, especially angiogenesis. Together, these results indicate that miR-190 is a promising antitumor target in clinical applications.

Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis.

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver function. Here we show a functional conservation of miR-122 in the TGFβ pathway: miR-122 target site is present in the mouse but not human TGFβR1, whereas a noncanonical target site is present in the TGFβ1 5′UTR in humans and other primates. Experimental switch of the miR-122 target between the receptor TGFβR1 and the ligand TGFβ1 changes the metastatic properties of mouse and human liver cancer cells. High expression of TGFβ1 in human primary liver tumours is associated with poor survival. We identify over 50 other miRNAs orthogonally targeting ligand/receptor pairs in humans and mice, suggesting that these are evolutionarily common events. These results reveal an evolutionary mechanism for miRNA-mediated gene regulation underlying species-specific physiological or pathological phenotype and provide a potentially valuable strategy for treating liver-associated diseases.

Engineered TAL Effector modulators for the large-scale gain-of-function screening

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE–DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE–TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation.

An integrative approach for the large-scale identification of human genome kinases regulating cancer metastasis.

UNLABELLED Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. FROM THE CLINICAL EDITOR This team of authors have utilized a self-assembled cell microarray to screen genes that regulate cancer cell migration using a human genome siRNA library of kinases. They validated previously known genes and identified novel ones that may serve as therapeutic targets.

A Magnetic Bead-Integrated Chip for the Large Scale Manufacture of Normalized esiRNAs

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.