Title: ciRS-7 is a prognostic biomarker and potential gene therapy target for renal cell carcinoma
Journal: Molecular Cancer
Impact factor: 27.401
Renal cell carcinoma (RCC) is the most common urological malignancy after prostate cancer and bladder cancer, and its incidence has continued to rise in the past decade1. Since RCC is not sensitive to radiotherapy and chemotherapy, gene-targeted therapy provides a new direction for its treatment. Some targeted drugs are currently being used in clinical practice2. In addition, RCC has a high tendency of malignant metastasis, and early identification and study of the biological mechanism of RCC metastasis and progression can help to more accurately predict clinical outcomes and develop targeted drugs3.
Figure 1: Stage II - RCC
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Circular RNAs (circRNAs) are a new class of endogenous noncoding RNAs4. According to studies, circRNAs play a key role in the pathogenesis, metastasis and development of various malignant tumors such as RCC5. However, the underlying mechanism of action of ciRS-7 in RCC therapy remains unclear.
This study found that ciRS-7 was highly expressed in RCC tumor tissues and cell lines and could be used as a prognostic marker. Subsequently, it was further confirmed that ciRS-7 acts as a "ceRNA" of miR-139-3p, which can prevent TAGLN degradation and promote the development and metastasis of RCC through the PI3K/AKT signaling pathway. In addition, this study also generated nanocomplexs (PBAE/si-ciRS-7), which can significantly inhibit RCC tumor growth and metastasis in vivo. The research and development of PBAE/si-ciRS-7 will provide new insights for the development of RCC gene therapy-related drugs in the future, which has important practical significance for the clinical treatment of tumors.
Methods and Results
1. ciRS-7 is overexpressed in RCC tumor cells and tissues, which promotes cell proliferation, migration and invasion in vitro.
The researchers first used the GEO database to conduct data analysis and found that ciRS-7 was highly expressed in tumor and metastatic tissues (Figure. 1A-C). Then, agarose gel electrophoresis and Sanger sequencing are used to confirm ciRS-7 PCR product and its sequence, respectively (Figure. 1D). RNase R and actinomycin D assay showed that ciRS-7 was more stable than CDR1 (Figure. 1E, F). The qRT-PCR and FISH showed that ciRS-7 was predominantly localized in the cytoplasm (Figure. 1G, H).
Next, the expression of ciRS-7 in different cell lines and 85 pairs of tissues was examined, and the results showed that ciRS-7 was highly expressed in RCC tumor cells and tissues (Fig. 1J, K). It was further found that the highly expressed ciRS-7 was associated with greater tumor, higher Fuhrman grade, and worse survival were highly correlated (Figure. 1L).
Figure 2: ciRS-7 is overexpressed in RCC tissues and cells and promotes RCC cell proliferation, migration and invasion in vitro
The results of FISH assay showed that the silencing of ciRS-7 indeed inhibited the expression of ciRS-7 in the cytoplasm of 786-O and ACHN cells; while overexpression of ciRS-7 increased its expression in the cytoplasm. CCK-8, colony formation, and EdU assays showed that inhibition of ciRS-7 could significantly attenuate the proliferation of 786-O and ACHN cells; the opposite was true for ciRS-7 overexpression (Figure 1N, Q). After a series of functional experiments, it was found that overexpression of ciRS-7 promoted RCC cell migration and invasion in vitro (Figure. 1R, S).
2. ciRS-7 acts as a sponge of miR-139-3p in RCC cells and miR-139-3p inhibits RCC cell proliferation, migration, and invasion in vitro
The researchers predicted potential miRNAs molecules that may interact with ciRS-7 by using software such as circBank, and found two potential target miRNAs (miR-7-5p and miR-139-3p) (Figure. 2A). Then, the interaction between ciRS-7 and miR-139-3p was demonstrated by RNA pull-down assay and dual luciferase assay. In addition, the researchers found that miR-139-3p expression was lower in RCC tumor tissue among the 85 pairs of clinical samples tested (Figure. 2B).
Subsequently, the researchers predicted potential miRNAs that may interact with ciRS-7 by using software such as circBank, miRanda, circAtlas, and RNAhybrid software, and found two potential target miRNAs (miR-7-5p and miR-139- 3p) (Figure. 3A). Then, the interaction between ciRS-7 and miR-139-3p was demonstrated by RNA pull-down assay and dual luciferase assay. In addition, we found that miR-139-3p expression was lower in RCC tumor tissues among the 85 pairs of clinical samples tested (Figure. 3B). The qRT-PCR results showed that both miR-139-3p-Mimic and miR-139-3p plastids increased the expression level of miR-139-3p (Figure. 3C).
Figure 3: ciRS-7 regulates the miR-139-3p/TAGLN axis, activates the PI3K/AKT signaling pathway, and promotes RCC cell proliferation, migration, and invasion
Further functional experiments demonstrated that ciRS-7 acts as a sponge for miR-139-3p in RCC cells, and miR-139-3p inhibits RCC cell proliferation, migration and invasion in vitro. Transcriptome sequencing and protein-level analysis of sh-ciRS-7 and sh-NC 786-O stably transduced cell lines revealed that only TAGLN was downregulated at both the transcriptional and protein levels, and dual-luciferase assays also indicated that miR-139-3p can bind to TAGLN (Figure. 3D). In addition, KEGG enrichment analysis showed that the PI3K/AKT signaling pathway was significantly enriched. At the protein level, we found that miR-139-3p-Mimic could partially counteract the increased expression of TAGLN, p-AKT and p-PI3K caused by OE-ciRS-7 (Figure. 3E); miR-139-3p-Inhibitor could partially rescue the reduced expression of TAGLN, p-AKT and p-PI3K inhibited by sh-ciRS-7 (Figure. 3F). Further functional studies revealed the molecular mechanism: ciRS-7 could act as the "ceRNA" of miR-139-3p, activate the PI3K/AKT signaling pathway to prevent the degradation of TAGLN, and promote the progression and metastasis of renal cell carcinoma.
3. PBAE/si-ciRS-7 nanocomplexes significantly inhibits RCC progression and metastasis
The researchers prepared nine different ratios of nanocomplexes according to the weight ratio of PBAE and si-ciRS-7, and finally found that when the weight ratio of PBAE and si-ciRS-7 exceeded 40, relatively homogeneous nanoparticles were formed. Excellent performance and high load efficiency (98%) were obtained with a PBAE/si-ciRS-7 of 80 (Fig. 4A, B). In addition, both TEM and particle size potentiometer showed that the particle size of the PBAE/si-ciRS-7 nanocomposite was about 160 nm (Fig. 4C, D).
Furthermore, compared with PBAE and si-ciRS-7 alone, PBAE/siciRS-7 nanocomposite had a stronger inhibitory effect on the proliferation of 786-O and ACHN cells by CCK-8 assay (Fig. 4E, F). Using subcutaneous xenograft tumor and lung metastasis models, we found that sh-ciRS-7 enhanced RCC tumor cell growth and metastasis in vivo, while PBAE/si-ciRS-7 nanocomplexes significantly inhibited these effects.
Figure 4: ciRS-7 enhances RCC tumor growth and metastasis in vivo and PBAE/si-ciRS-7 nanocomplexes inhibits RCC growth and metastasis in vivo.
This study revealed a potential mechanism of action of ciRS-7 in RCC development and metastasis (Figure. 5). ciRS-7 could promote RCC progression and metastasis through PI3K/AKT signaling pathway by binding to miR-139-3p and blocking its inhibitory effect on TAGLN. In addition, this study also developed a nanocomposite targeted drug-PBAE/si-ciRS-7, which could significantly inhibit renal cell carcinoma tumor development and metastasis in vivo. This could provide new insights for RCC gene therapy and related drug development in the future.
Figure 5. ciRS-7 promotes RCC progression and metastasis via miR139-3p/TAGLN/PI3K/AKT
Vazyme’s Product Support
Vazyme's HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Cat. No. R312) and AceQ qPCR SYBR Green Master Mix (High ROX Premixed) (Cat. No. Q141) facilitate this study. The researchers used R312 and Q141 products to detect the relative expression of circRNAs and verify the interaction between ciRS-7 and miR-139-3p, further revealing that ciRS-7 plays a role in RCC cell proliferation, migration and in vitro. The promotion of invasion and the inhibition of miR-139-3p help to identify the potential mechanism of action of ciRS-7 in RCC therapy.
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Product name: HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper)
Cat. No. #: R312
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