br Previous studies revealed that circRNAs can resist RNase digestion
Previous studies revealed that circRNAs can resist RNase digestion, while mRNAs are not (Memczak et al., 2013). Other studies indicated that linear RNA with no 3’ overhang or with only a 4-nucleotide overhang could resist RNase digestion (Barrett and Salzman, 2006). As
shown in Fig. 3, RNase could digest linear mRNA while preserving circRNA. However, ATRNL1 mRNA which has no overhang was not eliminated completely. In addition, a ladder could appear on the agarose gel as a result of the rolling cycle cDNA products (You et al., 2015). We also found that circAC139769.1 rolling cycle cDNA products appeared in A2780 Ethylmalonyl Coenzyme A (Fig. 3F), and this phenomenon could also verify the circular structure indirectly. Besides, circAC139769.1 is
Fig. 6. Knockdown of circHIPK3 promoted cell proliferation and attenuated cell apoptosis. A and B: Cell proliferation of A2780 and SKOV3 cells treated with
circHIPK3 siRNA was examined by cell counting Kit-8 (CCK-8) assay at the indicated timepoint. C: At 48 h following transfection, SKOV3 cells were stained by PE and
Annexin V, and the stained cells were analyzed by flow cytometry. D: The apoptosis cell percentage (percentage of both the upper and lower right quadrant cells) was
compared between the two groups. Data are presented as the mean ± SEM. ***p < 0.001, **p < 0.01. E: CeRNA analysis for circHIPK3 (hsa_circ_0000284). Based on the circRNA, miRNA and mRNA sequencing data, MiRanda and Cytoscape were performed to reveal the network of circHIPK3-miRNA-mRNA. Twelve candidate miRNA and their possible target genes were involved. CircHIPK3 is located in the center, pink ellipses represent miRNA and green rectangles represent mRNA. The association among the nodes is indicated by solid lines.
formed by the longest exon of LncAC139769.1 (1718 bp), the other exons of LncAC139769.1 are quite short (213 bp in total) and are overlapped with part of the RPSAP58 gene. Therefore, we didn’t find the specific primer for the linear RNA. However, further studies about the circAC139769.1 are needed in the future.
To confirm the possible function of circRNAs in EOC, we selected circHIPK3, which is the most abundant circRNA in our data, to explore its eﬀects on ovarian cancer cells. Consistent with the previous study (Zheng et al., 2016), circHIPK3 was expressed principally in the cyto-plasm of A2780 ovarian cancer cells. The previous studies indicated that silencing of circHIPK3 could attenuate retinal endothelial cell and HEK-293 T cell proliferation (Zheng et al., 2016; Shan et al., 2017), whereas over-expression of circHIPK3 decreased invasion and migra-tion of bladder cancer cells (Li et al., 2017). In contrast, circHIPK3 knockdown inhibited colorectal cancer cell migration and invasion and promoted apoptosis (Zeng et al., 2018). These results indicate that circRNA might exhibit diﬀerent eﬀects in diﬀerent cells or tissues. In our study, circHIPK3 was decreased in the EOC group (Fig. 2A), and knockdown of circHIPK3 increased cell proliferation, promoted mi-gration and invasion, and inhibited apoptosis of both the normal ovarian epithelial cells (IOSE80) and ovarian cancer cells (A2780 and SKOV3) in our study (Fig. 5, Fig. 6A–C, Fig. S2B-F), which might be attributed to cell-type-specific expression and function.
Several studies also have revealed that circRNAs might exert a regulatory function via interaction with proteins (Du WW et al., 2017a). Circ-Amotl1 was observed to facilitate c-myc translocation from cyto-plasm to the nucleus, resulting in tumorigenesis of breast cancer (Yang et al., 2017). CircFoxo3 induces cardiac senescence by retaining anti-senescence proteins in the cytoplasm (Du WW et al., 2017b). Herein, we speculated that circHIPK3 might also exert its regulatory eﬀects through interaction with proteins. Further analysis should be conducted to confirm the possible mechanism involved in circHIPK3 regulation.
In conclusion, we analyzed circRNA expression profiling in EOC and NOT using RNA high-throughput sequencing. Six circRNAs were further validated in EOC and NOT by qRT-PCR and subsequently confirmed to International Journal of Biochemistry and Cell Biology 112 (2019) 8–17
resist to RNase digestion. Of these circRNAs, circHIPK3 siRNA could promote proliferation, migration and invasion and inhibit apoptosis of SKOV3 and A2780 ovarian cancer cells as well as IOSE80 cells. The possible mechanism was also predicted using a bioinformatics method. Our investigation indicates that circHIPK3 is an important regulator of ovarian cancer progression.
TF and XJ performed the experimental work and drafted the manuscript. SR and JXM supervised and designed the research. ZM, GYY, LSY, WXS, NJ, QB participated in the experiments, discussion and interpretation of data. SR, ZM, GYY, LSY, WXS, NJ, QB revised the manuscript. All authors have read and approved the final manuscript.