Characterization and expression patterns of hsa_circ_0001846 in PC.
To differentiate it from the linear RNA, excluding its effects on the experiments, we have analyzed and verified the structure and properties of hsa_circ_0001846 by querying the circBase software, found that hsa_circ_0001846 originated from the exons 11,12,13, and 14 of ubiquitin-associated protein 2 (UBAP2) gene (chr 9:33944362–33956144). The mature sequence was 747 bp and its reverse shear site (TAAGCTTT) was identified by Sanger sequencing (Fig. 1A). Next, we designed qRT-PCR primers and convergent and divergent primers based on the gene sequence of hsa_circ_0001846. The agarose gel electrophoresis experiment showed that successful amplification of hsa_circ_0001846 was only possible by using divergent primers in the cDNA libraries (Fig. 1B). The qRT-PCR results showed that hsa_circ_0001846 was significantly upregulated in various PC cell lines compared to HPDE cells (Fig. 1C). At the same time, we collected 25 pairs of PC tissues, and qRT-PCR results showed that hsa_circ_0001846 had higher expression in cancer tissues than the matched adjacent tissues (Fig. 1D). Moreover, the FISH results indicated that hsa_circ_0001846 was mainly distributed in the cytoplasm of PC cells (Fig. 1E). The above results demonstrated that the UBAP2 gene-derived hsa_circ_0001846 was significantly enriched and stably presented in PC tissues and cytoplasm.
Hsa_circ_0001846 promotes tumor invasion, proliferation and migration in PC.
Combining the above results, we suspected that hsa_circ_0001846 may play a key role in the malignant progression of PC. Therefore, we investigated the effects in vitro and vivo of hsa_circ_0001846 on migration, proliferation and invasion of PC cells. Firstly, we designed and synthesized three small interfering RNAs (siRNAs) targeting the reverse shear site of hsa_circ_0001846, and verified their silencing efficiency in PANC-1 and AsPC-1 cells. The qRT-PCR results indicated that three siRNAs partially silenced hsa_circ_0001846, but siRNA-3 was most efficiently silenced in PANC-1 and AsPC-1 cells, so we chose siRNA-3 for the following studys (Fig. 2A, B). We silenced hsa_circ_0001846 and performed functional studies in two PC cell lines (PANC-1 and AsPC-1). First, scratch experiments showed that silencing hsa_circ_0001846 caused a significant weakeness in the migration ability of PC cells compared to the NC group (Fig. 2C, D). CCK-8 and EdU experiments also demonstrated that the proliferation of PC cells was inhibited after transfecting si-hsa_circ_0001846 (Fig. 2E-H). We also simulated the cell invasion process by transwell experiment with matrix glue to proved that silencing hsa_circ_0001846 reduced the number of cells passing through the chamber micropores and decreased the invasion ability of PANC-1 and AsPC-1 cells (Fig. 2I, J).
On the contrary, to more strongly prove the role of hsa_circ_0001846 on the biological functions of PC cell lines, we constructed the overexpression vector pCE-RB-Mam-EGFP-hsa_circ_0001846 and verified the overexpression efficiency with qRT-PCR (Fig. 3A). The scratch assay showed that the overexpression of hsa_circ_000184 promoted the migration of PC cell lines (Fig. 3B, C). Meanwhile, CCK-8, EdU and Transwell experiments also demonstrated that overexpression of hsa_circ_0001846 promoted cell proliferation and invasion (Fig. 3D-I). Next, PANC-1 cell line with stable knockout of hsa_circ_0001846 was constructed in order to explore whether hsa_circ_0001846 affected the growth of PC cells in vivo (Fig. 3J). We resuspended sh-PANC-1 and sh-NC cells with matrix gel and injected them into the subcutaneous tissue of nude mice to construct nude mouse xenograft models (Fig. 3L). Four weeks later, nude mice were sacrificed, meanwhile we collected and measured the tumor tissues. We observed that the volume and weight of xenografts from sh-hsa_circ_0001846 cells were significantly smaller and lighter compared with sh-NC (Fig. 3K, M, N).
Hsa_circ_0001846 is a sponge for miR-204-3p.
Through the above in vivo and vitro functional experiments, we determined that hsa_circ_0001846 has a promoting effect on PC deterioration. To further clarify how hsa_circ_0001846 regulates PC progression, we performed mechanistic studies. Previous more studies have reported that circRNA mainly acts as a sponge of miRNAs to regulate the expression of the corresponding downstream target proteins. The miRNAs target prediction softwares, including Circinteractome, Starbase and Circbank, were used to predict the potential targeting miRNAs of hsa_circ_0001846. We also performed the RNA pulldown assay by designing a specific biotin probe for hsa_circ_0001846. The regulatory effect of hsa_circ_0001846 on the candidate miRNAs was explored. The results showed that miR-204-3p was significantly accessible to be catched by hsa_circ_0001846 than the other candidate miRNAs (Fig. 4A, B). Subsequently, we mutated the site of hsa_circ_0001846 bound to miR-204-3p to performed dual luciferase reporter assay and found that miR-204-3p mimic significantly reduced the luciferase activity of WT-hsa_circ_0001846 vector, while the luciferase activity of Mut-hsa_circ_0001846 vector did not change (Fig. 4C, D). Meanwhile, qRT-PCR results indicated that silencing hsa_circ_0001846 upregulated the expression level of miR-204-3p in PANC-1 (Fig. 4E). The above experimental results proved that there are binding and regulatory relationships between hsa_circ_0001846 and miR-204-3p.
The effect of miR-204-3p on the biological function of PC cells is the opposite of hsa_circ_0001846.
Since the role of miR-204-3p in PC has been rarely explored, its functions were further investigated in the PANC-1 and AsPC-1 cell lines. Wound healing assay showed that transfection of miR-204-3p mimic in cells inhibited the migratory capacity of PC cells (Fig. 5A), while an inhibitor of miR-204-3p promoted cell migration (Fig. 5B). The same results were shown in other functional experiments, CCK-8 and EdU experiments showed that miR-204-3p mimic inhibited the proliferation capacity of cells, while miR-204-3p inhibitor promoted cell proliferation (Fig. 5C-F). Transwell experiment demonstrated that treatment of cells with miR-204-3p mimic inhibited cell invasion, while the miR-204-3p inhibitor promoted cell invasion (Fig. 5G, H). These results indicated that in contrast to hsa_circ_0001846, miR-204-3p inhibited the proliferation, migration and invasion of PC cells.
MiR-204-3p inhibitor reverses the effects of si-hsa_circ_0001846 on proliferation, migration, and invasion in PC cells.
To further demonstrate that hsa_circ_0001846 regulated PC progression by sponging miR-204-3p, we co-transfected si-hsa_circ_0001846 and miR-204-3p inhibitor in PANC-1 cells. Functional experiments showed that compared with the NC group, silencing hsa_circ_0001846 attenuated the migration, proliferation and invasion abilities of PANC-1 cells, while miR-204-3p inhibitor enhanced these abilities. At the same time, compared with si-hsa_circ_0001846 group, the migration, proliferation and invasion abilities of PANC-1 cells were saved by transfecting miR-204-3p inhibitor (Fig. 6A-D). The above results indicated that miR-204-3p is a key acting molecule downstream of hsa_circ_0001846.
The hsa_circ_0001846 exerts its carcinogenesis effects by indirectly regulating the expression level of KRAS by miR-204-3p.
A large number of previous studies have confirmed that KRAS is one of the oncogenes involved in PC progression. We used the bioinformatics websites of miRDB, miRDIP, miRWALK and TargetScan to analyze the sequence of miR-204-3p and KRAS and found a binding relationship between them (Fig. 7A). Therefore, we mutated the binding site of miR-204-3p and KRAS, constructed the Mut-KRAS/WT-KRAS plasmid and performed dual-luciferase experiment, which showed that HEK293T cells with co-transfected WT-KRAS and miR-204-3p mimic significantly reduced the luciferase activity, while Mut-KRAS and miR-204-3p mimic did not significantly change the luciferase activity in the cells (Fig. 7B, C). Although we found that miR-204-3p inhibitor did not significantly increase the mRNA level of KRAS, the western blot results showed that it was able to increase the protein expression level of KRAS, suggesting that miR-204-3p may primarily affect the post-transcriptional level of KRAS (Fig. 7D, E). Simultaneously, silencing hsa_circ_0001846 inhibited KRAS expression, and miR-204-3p inhibitor antagonized this effect (Fig. 7F-H). Functionality experiments also showed that silencing KRAS can reverse the cancer-promoting effect caused by overexpression of hsa_circ_0001846 (Fig. 8A-D).
The above results indicated that KRAS is a downstream target of miR-204-3p, and hsa_circ_0001846 indirectly regulated KRAS expression by sponging miR-204-3p, thus exerting its cancer-promoting effect.