Our promise to you:
Guaranteed product quality, expert customer support.
24x7 CUSTOMER SERVICE
CONTACT US TO ORDER
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to the 3' untranslated regions (3' UTRs) of target mRNAs, leading to translational repression or degradation. Due to their critical roles in development, homeostasis, and disease, miRNAs are a major focus in both basic and applied research. While methods like antagomirs, CRISPR/Cas9, and target site mutagenesis are commonly used to disrupt miRNA function, they can be transient, inefficient, or unsuitable for studying miRNA families or achieving long-term knockdown.
MicroRNA sponge technology offers a stable and effective alternative. Engineered with multiple tandem binding sites for specific miRNAs or families, sponges competitively sequester miRNAs, freeing their natural targets. Delivered via plasmids or viral vectors, they allow for sustained inhibition in vitro and in vivo—making them ideal for long-term functional studies.
The core mechanism of a miRNA sponge is based on competitive inhibition. The sponge mRNA contains tandem repeats of miRNA target sites—often located in the 3' UTR region—designed to bind the miRNA seed region (typically positions 2–8) without triggering RNA-induced silencing complex (RISC)-mediated cleavage. This is often achieved by introducing bulges in the binding sites, which prevent perfect complementarity and degradation.
Figure 1. Mechanistic Basis of miRNA Sponge. (Nigussie A, et al., 2018)
Methods of miRNA Inhibition and Their Pros and Cons:
| Method | Advantages | Disadvantages | Tissue-Specific Expression | qPCR Detectable |
| Anti-sense Oligonucleotide | Easy to synthesize and design | Cannot knock down miRNAs rich in consecutive Ts | ✕ | ✕ |
| CRISPR/Cas9 Targeting miRNA | Directly targets miRNA genes; suitable for short miRNAs | sgRNA design is limited; not ideal for tissue-specific expression | ✕ | ✓ |
| miRNA Sponge | Long-term expression; can inhibit multiple miRNAs; suitable for tissue-specific expression | Does not degrade miRNA directly; effectiveness depends on miRNA abundance | ✓ | ✕ |
Creative Biogene offers comprehensive and fully customizable miRNA sponge construction services based on years of technical expertise. From initial sequence design and vector construction to lentiviral packaging and functional validation, we deliver turnkey solutions to suit a wide range of research needs.
Our miRNA sponge constructs typically carry 5–8 tandem binding sites that competitively inhibit target miRNAs by mimicking natural binding motifs. These constructs can target individual miRNAs or entire miRNA families based on conserved seed sequences. More uniquely, we can design a single vector capable of simultaneously binding multiple, even unrelated, miRNAs by incorporating distinct binding sites—providing unmatched flexibility for complex experimental models.
We provide multiple expression options tailored to your experimental design:
Clients can customize their miRNA sponge vectors using combinations of the following components:
| Promoters | Fluorescent Reporters | Antibiotic Markers |
| CMV | EEGFP | Puromycin (Puro) |
| CAG | EYFP | Neomycin (Neo) |
| hEF1α | EBFP | Hygromycin (Hyg) |
| TRE | tRFP | Blasticidin (Bla) |
We also support dual-function vectors combining miRNA sponge sequences with fluorescent markers or luciferase for real-time tracking or functional assays.
Our miRNA sponge service follows a streamlined yet customizable workflow:
All delivered vectors are fully sequence-verified. Viral products are accompanied by titer reports and are ready for direct use in cell or animal systems. Our project managers work closely with clients to ensure technical precision and scientific relevance throughout the process.
miRNA sponge constructs have broad applications across biological disciplines:
Functional studies
Clarify miRNA-target interactions, regulatory networks, and signaling pathways
Disease modeling
Investigate the role of miRNAs in cancer, cardiovascular, or neurological disorders
Family-wide inhibition
Dissect redundancy among miRNA family members with shared seed sequences
Therapeutic target validation
Evaluate the impact of miRNA inhibition in preclinical models
Whether used for mechanistic research or translational development, miRNA sponges provide a stable, reversible, and tunable approach to miRNA inhibition.
Case Study 1
In a study using two primary human breast cancer xenografts (CRCM494 and CRCM168), Creative Biogene's miR-600 sponge lentivirus was employed to explore the role of miR-600 in regulating breast cancer stem cell (bCSC) self-renewal and differentiation. Silencing miR-600 led to bCSC expansion, while its overexpression inhibited WNT signaling, promoting bCSC differentiation and reducing tumorigenicity in vivo. This research revealed that low miR-600 levels are linked to active WNT signaling and poor prognosis in breast cancer. Creative Biogene's miRNA sponge technology offers a powerful tool for regulating miRNA function, providing valuable insights into cancer biology and therapeutic strategies.
Figure 2. miR-600 controls self-renewal and differentiation of breast cancer stem cells by targeting SCD1 and reducing active WNT production. (El Helou R, et al., 2017)
Case Study 2
Melanoma is one of the most aggressive and highly resistant tumors, and cell plasticity is a key factor behind its metastatic capabilities. However, the detailed molecular mechanisms controlling melanoma plasticity are still not fully understood. In this study, we combine mathematical models of phenotypic switching with experiments on IgR39 human melanoma cells to identify potential key targets for impairing phenotypic switching. Our mathematical model suggests that a cancer stem cell subpopulation within the tumor prevents phenotypic switching of other cancer cells. Experiments reveal that hsa-miR-222 is a critical factor enabling this process. To further explore this, we used sponge lentiviral plasmids provided by Creative Biogene, which were constructed with 8 inhibitor sequences targeting hsa-miR-222-5p (pEGFP–Sponge-mir222-5p, cod. PSE2774). A control sponge plasmid, with 8 inhibitor sequences for a scramble control sequence (pEGFP–scramble, cod. PSE2775), was also constructed. These findings shed new light on melanoma plasticity and provide a potential target and guidance for therapeutic studies.
Figure 3. CXCR6 expression levels in IgR39 cells transfected with hsa-miR-222-5p inhibitor (sh-mir222) or scramble sequence (scramble) as control, analyzed by flow cytometry with PE-conjugated anti-human CXCR6 antibody. (Lionetti MC, et al., 2020)
At Creative Biogene, we combine advanced cloning platforms with RNA biology expertise to deliver high-quality miRNA sponge systems tailored to your specific needs. Our experience includes:
Whether you're designing a basic research experiment or building a complex therapeutic model, our team ensures scientific rigor and technical excellence from start to finish.
Q1: How does miRNA sponge technology compare to CRISPR/Cas9 for miRNA inhibition?
A: CRISPR/Cas9 targets miRNA genes at the DNA level, which may lead to permanent loss and potential off-target edits. miRNA sponges allow for stable yet reversible inhibition, especially for miRNA families, and are better suited for temporal or conditional studies.
Q2: Can miRNA sponges be expressed in a tissue-specific manner in animal models?
A: Yes. We offer tissue-specific promoter options, such as Albumin for liver or CAG for neurons, enabling spatially restricted miRNA knockdown in vivo.
Q3: Will miRNA sponge expression cause cytotoxicity or unintended effects?
A: When properly designed with an optimal number of binding sites (5–8), sponges exhibit minimal cytotoxicity. We also offer inducible systems to fine-tune expression and avoid over-inhibition.
Q4: Is it possible to target more than one unrelated miRNA with a single sponge?
A: Absolutely. We can design hybrid sponges with tandem binding sites for different miRNAs, ensuring compatibility within a single expression vector.
Q5: Do you offer stable cell line generation or animal model development as a follow-up?
A: Yes. Beyond vector construction and packaging, we offer downstream services such as stable cell line screening and in vivo delivery assistance to support your entire experimental pipeline.
References:
Copyright © Creative Biogene. All rights reserved.