ciRS-7 is a prognostic biomarker and potential gene therapy target for renal cell carcinoma

Circular RNAs are a new class of non-coding RNAs that have been shown to play critical roles in the development and progression of renal cell carcinoma (RCC). However, little is known about the functional mechanisms and therapeutic role of ciRS-7 in RCC. A series of in vitro and in vivo experiments were performed to investigate the functional mechanism and therapeutic role of ciRS-7, such as real-time quantitative PCR, CCK-8, wound healing, transwell, colony formation, Edu, tumor xenograft and lung metastasis in NSG mice. RNA pull-down, dual luciferase reporter, fluorescence in situ hybridization (FISH) and rescue assays were used to determine the relationship between ciRS-7, miR-139-3p and TAGLN. In addition, we constructed PBAE/si-ciRS-7 nanocomplexes with PBAE material to evaluate the therapeutic effect of the nanocomplexes on tumor in vivo. ciRS-7 was highly expressed in RCC tumor tissues and cell lines, and high ciRS-7 expression correlated with tumor size, high Fuhrman grade and poor survival. Depletion of ciRS-7 significantly inhibited RCC cell proliferation, invasion, tumor growth and metastasis in vivo, while overexpression of ciRS-7 had the opposite effect. Mechanistically, ciRS-7 acts as a "ceRNA" for miR-139-3p to prevent TAGLN degradation and promoting RCC progression and metastasis via the PI3K/AKT signaling pathway. In addition, miR-139-3p mimics or inhibitor could reverse the altered malignant tumor behavior caused by ciRS-7 overexpression or silencing. Furthermore, the PBAE/siciRS-7 nanocomplexes could significantly inhibit RCC tumor progression and metastasis in vivo. ciRS-7 acts as a tumor promoter by regulating the miR-139-3p/TAGLN axis and activating the PI3K/AKT signaling pathway to promote RCC progression and metastasis. Drug development of PBAE/si-ciRS-7 nanocomplexes targeting ciRS-7 may represent a promising gene therapeutic strategy for RCC.


Background
Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system, only second to prostate and bladder cancers in incidence, which has been on a continuous rise in the last decade [1][2][3]. Since RCC is insensitive to radiotherapy and chemotherapy, gene-targeted therapy has provided a promising new direction for its treatment; several targeted drugs are now being used in clinical practice [4]. Circular RNAs (circRNAs) are a newly discovered type of non-coding RNAs (ncRNAs). Accumulating evidence suggests that circRNAs may play a crucial role in the pathogenesis of various malignancies, Open Access *Correspondence: njxb1982@126.com; mingchenseu@126.com; liweitongji@163.com; pengbo6908@163.com including RCC, bladder cancer, breast cancer, and hepatocellular carcinoma [5][6][7]. However, the functional mechanisms underlying the therapeutic roles of ciRS-7 in RCC remain unknown.

ciRS-7 is overexpressed in RCC cells and tissues and it promotes in vitro cell proliferation, migration and invasion
To identify circRNAs involved in RCC progression and metastasis, we obtained three GSE datasets (GSE100186, GSE108735, and GSE137836) from the GEO database. The Venn diagram showed that only two circRNAs (hsa_circ_0001946 and hsa_circ_0002484) were detected in three datasets and these were highly expressed in the tumor and metastatic tissues (Fig. 1A-C and Fig. S1A). For this study, we selected ciRS-7 (hsa_circ_0001946). ciRS-7 is formed due to the reverse shearing of CDR1. The ciRS-7 PCR product and its sequence were confirmed by agarose gel electrophoresis and Sanger sequencing, respectively ( Fig. 1D and Fig. S1B). RNase R and actinomycin D assay showed that ciRS-7 was more stable than CDR1 (Fig. 1E, F). qRT-PCR and FISH showed that ciRS-7 was predominantly localized in the cytoplasm (Fig. 1G, H). We examined the expression of ciRS-7 in different cell lines and 85 pairs of tissues. The results showed that ciRS-7 was highly expressed in RCC tumor cell lines and tumor tissues (Fig. 1J, K). In addition, higher ciRS-7 expression was associated with greater tumor size, high Fuhrman grade, and poorer survival ( Fig. 1L and Table S4, S5).

TAGLN is a target of ciRS-7, and ciRS-7 regulates the miR-139-3p/TAGLN axis, activates the PI3K/AKT signaling pathway, and promotes RCC cell proliferation, migration, and invasion
The sh-ciRS-7 and sh-NC 786-O stable transduction cell lines were sequenced and we found that only TAGLN was downregulated at both the transcriptional and protein levels (Fig. S5A-C and Fig. S6A and Excel S3). The dual-luciferase assay also showed that miR-139-3p could bind to TAGLN (Fig. S5D). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that PI3K/AKT signaling pathway was significantly enriched (Fig. S5E-G, Fig. S6B, C and Excel S5, S6).

ciRS-7 enhances in vivo RCC tumor growth and metastasis and PBAE/si-ciRS-7 nanocomplexes can inhibit these effects
Based on the new insights into tumor pathogenesis, gene targeting therapy has gained attention as a potential approach for cancer treatment. Poly(β-amino ester) s (PBAEs) is an excellent candidate for gene delivery due to its low toxicity and high transfection efficiency [4,8,9]. The 1 HNMR spectrum of PBAE is shown in Fig. S8A. We prepared nine nanocomplexes in different proportions according to the weight ratios of PBAE and si-ciRS-7. We found that relatively homogeneous nanoparticles were formed when the weight ratio of PBAE and si-ciRS-7 exceeded 40. Excellent performance and high loading efficiency (98%) were obtained for PBAE/si-ciRS-7 of 80 ( Fig. 2A, B). Further both TEM and particle size potentiometer showed that the particle size of PBAE/si-ciRS-7 nanocomplexes was approximately 160 nm (Fig. 2C,  D). In addition, the CCK-8 assay showed that PBAE/si-ciRS-7 nanocomplexes had a stronger inhibition of the proliferation of 786-O and ACHN cells as compared to PBAE and si-ciRS-7 alone (Fig. S8B, C).
To assess the effect of ciRS-7 on in vivo RCC growth and metastasis, we generated subcutaneous xenograft tumor and lung metastasis models (Fig. 2E). The xenograft tumor model showed that the tumor size and weight of mice in the OE-ciRS-7 group were significantly higher as compared to the control group, while the tumor size and weight of mice in the sh-ciRS-7 group were significantly lower (Fig. 2F-H). IHC staining showed that the expressions of Ki67 and TAGLN were significantly upregulated in the OE-ciRS-7 group and downregulated in the sh-ciRS-7 group (Fig. 2I).
In addition, the lung metastasis model showed that the OE-ciRS-7 group had more metastatic foci in the lungs of mice, followed by the control group, and the sh-ciRS-7 group had the least numbers. Micro-metastases were detected by H&E (Fig. 2J).
We further examined the effect of PBAE/si-ciRS-7 nanocomplexes on the in vivo RCC growth and metastasis (Fig. 2K). We found that the tumor size and weight of the mice in the PBAE/si-ciRS-7 nanocomplexes treated group was significantly reduced (Fig. 2L-N); IHC staining also showed a significant decrease in the expression of Ki67 and TAGLN (Fig. 2O). Moreover, IVIS imaging showed that PBAE/si-ciRS-7 nanocomplex injection minimized the size of renal in situ implantation tumors (Fig. 2P). In addition, in vivo lung metastasis models with PBAE/si-ciRS-7 nanocomplex treatment had better inhibition of lung metastasis (Fig. 2Q). The PBAE/si-ciRS-7 nanocomplexes could better inhibit the growth and metastasis of RCC tumors as compared to the animalgrade si-ciRS-7.

Conclusion
As shown in Fig. 2R, this study showed that ciRS-7 could function as an oncogenic circRNA for the progression of RCC. ciRS-7 could promote RCC progression and metastasis through a novel regulatory pathway by binding miR-139-3p and blocking its inhibitory effect on TAGLN, which could promote RCC progression and metastasis through PI3K/AKT signaling pathway. In addition, we generated PBAE/si-ciRS-7 nanocomplexes, which, in the future, could provide novel insights for the development of RCC gene therapy-related drugs.