CircFAM73A is upregulated in GC and high circFAM73A predicts poor prognosis
To assess the circRNA involved in GC, we searched the circRNA datasets established for gastric cancer in the GEO DataSets. Six datasets were found, and GSE83521, was chosen for the subsequent analysis, as it contains the highest number of samples. Several differentially expressed circRNAs were calculated and then filtered according to their log Fold Change ≥ 2 and adjusted P value ≤ 0.01. Four circRNAs, hsa_circ_0001789, hsa_circ_0007376, hsa_circ_0052001 and hsa_circ_0002570 were screened out. Next, we verified their expression by qRT-PCR analysis in our 60 paired cancer tissues and their matched adjacent non-cancer tissues from GC patients. As shown in Fig. 1A, the most significant expression change was observed for hsa_circ_0002570, thus prompting us to further investigate its role in GC malignancy.
Hsa_circ_0002570 (circFAM73A) originates from exon 3,4,5,6,7 of the FAM73A genome (UCSC data in NCBI) (Fig. 1B). We validated the head-to-tail back splicing in RT–PCR product of circFAM73A using Sanger sequencing (Fig. 1C). qRT-PCR results showed the highest circFAM73A abundance in BGC823 cells and the lowest in SGC7901 cells (Fig. 1D). We therefore selected BGC823 and SGC7901 cell lines for subsequent studies. As shown in Fig. 1E, circFAM73A was resistant to the digestion of RNase R exonuclease compared to the linear form of FAM73A in BGC823 and SGC7901. In order to avoid the possibilities of trans-splicing or genomic rearrangements, we conducted several universal circRNA detection experiment[23]. We first designed divergent primers to amplify circFAM73A and convergent primers to amplify FAM73A mRNA. Using cDNA (complementary DNA) and gDNA (genomic DNA) from BGC823 and SGC7901 and two randomly GC tissues as templates, circFAM73A was only amplified from cDNA by divergent primers, whereas no amplification product was obtained when using gDNA (Fig. 1F). Actinomycin D, an inhibitor of transcription, was then used to measure the half-life of circFAM73A and FAM73A in BGC823 and SGC7901. The results indicated that circFAM73A was more stable than FAM73A mRNA (Fig. 1G and Figure S1A). These findings clearly showed that the circular characteristics of circFAM73A.
Next, we examined the relative expression levels of circFAM73A in the cytoplasm and nuclear compartments of BGC823 and SGC7901. qRT-PCR demonstrated that circFAM73A preferentially localized in the cytoplasm (Fig. 1H and Figure S1B), which was confirmed using fluorescence in situ hybridization (FISH) against circFAM73A (Fig. 1I and Figure S1C).
High expression of circFAM73A was then authenticated in an additional 100 paired GC tissue samples (Fig. 1J) using qRT-PCR. 63% samples (n = 63) exhibited higher expression levels in the cancer tissue samples than in matched noncancerous tissue samples (Figure S1D and S1E). Our analysis of clinicopathological characteristics showed that circFAM73A expression significantly correlates with TMN stage and tumor size (Table 1). The expression of circFAM73A in tumor tissues at III stage was notably higher than that at I-II stage and in matched adjacent tissues, whereas abundance in tumors at I-II stage showed no difference from that in noncancerous tissues (Fig. 1L). Similarly, we also found that the expression in tumors larger than 3 cm was considerably higher than that in tumors smaller than 3 cm and in adjacent tissues (Fig. 1M). In contrast, other clinicopathological characteristics including age, gender, tumor site, lymph node metastasis or blood vessel invasion (Figure S1F-1J) showed no correlation. Moreover, GC patients with higher circFAM73A expression had significantly shorter overall survival than those with the lower circFAM73A expression by Kaplan–Meier survival analysis (Fig. 1K). Further Cox multivariate survival analysis revealed high circFAM73A expression as an independent prognostic factor for poor survival of GC patients (hazard ratio [HR] = 2.171, 95% confidence interval [CI] = 1.015–4.645, p = 0.046) (Table 2). In contrast, no obvious change of linear FAM73A mRNA was found in our GC samples (Figure S1K), and FAM73A showed no correlation with the prognosis of GC patients both in our samples and TCGA database (Figure S1L and S1M).
Table 1
Correlation between circFAM73A expression and the clinicopathologic parameter of 100 GC patients. * p < 0.05, ** p < 0.01.
Clinicopathologic parameter | Number | Number of patients | p value |
circFAM73Alow | circFAM73Ahigh |
Age | < 60 years | 36 | 16 | 20 | 0.532 |
| ≥ 60 years | 64 | 34 | 30 | |
Gender | Male | 75 | 39 | 36 | 0.645 |
| Female | 25 | 11 | 14 | |
Tumor size | < 3 cm | 26 | 19 | 7 | 0.011* |
| ≥ 3 cm | 74 | 31 | 43 | |
Tumor site | Proximal | 44 | 24 | 20 | 0.546 |
| Non-proximal | 56 | 26 | 30 | |
Lymph node metastasis | N0 | 38 | 24 | 14 | 0.063 |
| N1-N3 | 62 | 26 | 36 | |
TMN stage | I-II | 44 | 28 | 16 | 0.026* |
| III | 56 | 22 | 34 | |
Blood vessel invasion | Negative | 73 | 40 | 33 | 0.176 |
| Positive | 27 | 10 | 17 | |
Table 2
Univariate and Multivariate Cox regression analysis of overall survival in 100 GC patients, * p < 0.05, ** p < 0.01, *** p < 0.001.
Clinicopathologic parameter | Overall Survival | | | | |
| Univariate analysis | | | Multivariate analysis | |
| HR (95% CI) | p value | | HR (95% CI) | p value |
Age (≥ 60 years vs < 60 years) | 0.850 (0.412–1.751) | 0.659 | | | |
Gender (Female vs Male) | 1.659 (0.781–3.524) | 0.188 | | | |
Tumor size (≥ 3 cm vs < 3 cm) | 1.292 (0.557–2.999) | 0.551 | | | |
Tumor site (Proximal vs Non-proximal) | 1.284 (0.623–2.646) | 0.497 | | | |
Lymph node metastasis (N1-N3 vs N0) | 3.196 (1.310–7.799) | 0.011* | | 2.806 (1.140–6.902) | 0.025* |
TMN stage (III vs I-II) | 2.860 (1.278–6.402) | 0.011* | | | |
Blood vessel invasion (Positive vs Negative) | 2.004 (0.972–4.132) | 0.060 | | | |
circFAM73A expression (High vs Low) | 2.518 (1.185–5.349) | 0.016* | | 2.171 (1.015–4.645) | 0.046* |
Taken together, these data proved the upregulation of circFAM73A in GC and its clinical significance in GC patients.
CircFAM73A promotes the proliferation, migration and facilitates cisplatin resistance of GC in vitro
To disentangle the biological function of circFAM73A, two siRNAs were designed to specifically target the back-splice junction (Figure S2A). Si-circ-1 successfully suppressed circFAM73A expression with no effects on the levels of linear FAM73A in BGC823 and SGC7901 (Figure S2B). In addition, we also transfected overexpression vectors into both cell lines and the efficiency was verified via qRT-PCR (Figure S2C).
We first performed colony formation and CCK8 assay to examine the effects of circFAM73A on cell proliferation. The results showed that transfecting with siRNA dramatically suppressed the proliferation of BGC823 and SGC7901. In contrast, exogenous expression of circFAM73A exerted the opposite effects (Fig. 2A and 2B). Next, 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay also demonstrated that circFAM73A increased the rate of EdU incorporating cells (Fig. 2C and Figure S2D). A 3D GC organoid model was then established to further test the proliferation ability. We found that silencing of circFAM73A significantly decreased the diameter of organoids and the opposite findings were acquired in circFAM73A reconstitution groups (Fig. 2D and Figure S2E). To analyze whether circFAM73A interfered with cell cycle, we then measured cell-cycle distribution using flow cytometry. As shown in Fig. 2E, interference of circFAM73A expression distinctly increased the percentage of G0/G1 phase cells and diminished the S phase cells, while over-expressing circFAM73A showed the opposite trend. These observations indicated that interference of circFAM73A induced cell-cycle arrest in the G0/G1 phase, thereby constrained the proliferation of GC cells. Moreover, cell migration was impeded by circFAM73A depletion and fortified by overexpressing circFAM73A in both BGC823 and SGC7901 based on Transwell assay (Fig. 2F).
For advanced or metastatic GC patients, chemotherapy based on cis-dichlorodiammine platinum (CDDP) /cisplatin is currently recommended as first-line therapy. We therefore assessed the effects of circFAM73A on the chemo-sensitivity of GC cells. BGC823 and SGC7901 cells resistant to CDDP (referred to as BGC823CDDP and SGC7901CDDP cells) were established as described before[24]. As shown in Figure S3A, circFAM73A expression was considerably increased in BGC823CDDP and SGC7901CDDP cells compared with the corresponding sensitive cells by qRT-PCR. To test whether circFAM73A modulated the sensitivity of cisplatin treatment in GC cells, we treated cells with various concentrations of CDDP. circFAM73A over-expression reinforced cell viability and elevated the IC50 in BGC823 and SGC7901 cells (Figures S3B and S3C), whereas reduction of circFAM73A in BGC823CDDP and SGC7901CDDP cells showed the reverse effects (Figures S3D and S3E). Our colony formation assay demonstrated that up-regulation circFAM73A enhanced the long-term viability of CDDP-sensitive cells. In comparison, down-regulation circFAM73A displayed the opposite results in CDDP- resistant cells (Figures S3F). Moreover, results of flow cytometry assay exhibited that circFAM73A reduced the apoptosis rates of GC cells treated with CDDP (Figures S3G).
Collectively, these findings demonstrated that circFAM73A promotes GC cell proliferation, migration and facilitates cisplatin resistance of in vitro.
CircFAM73A enhances the stem cell-like property in GC cells
A growing number of studies suggest that the acquisition of cancer stem cell-like properties is crucial for the initiation and maintenance of the malignancy process of GC[3, 4]. Given the facilitating role of circFAM73A on cell proliferation and cisplatin resistance, we asked whether circFAM73A enhances the stem cell-like properties of GC cells.
We first explored the role of circFAM73A on the ability of GC cells self-renew. To this end, we performed a sphere formation assay. As shown in Fig. 3A and 3B, Overexpression of circFAM73A promoted the generation and cell content of tumor spheres of BGC823 and SGC7901 cultured in suspension. Conversely, circFAM73A-silenced cells formed fewer spheres with lower cell content. The induction of circFAM73A in the self-renewal ability was also confirmed using limiting dilution assay (Fig. 3C).
In addition, augmentation of circFAM73A increased, while downregulation of circFAM73A reduced the proportion of CD44 (GC stem cell-like marker) positive cells in BGC823 and SGC7901 (Fig. 3D). By qRT-PCR and Western blot showed that circFAM73A significantly increase expression of CD44 and the stemness-associated transcriptional factors including SOX-2, OCT-4 and Nanog. The opposite results were acquired in circFAM73A-depressed cells (Fig. 3E and 3F).
Together, these findings supported that circFAM73A exhibits a positive effect on CSC-like properties in GC cells.
CircFAM73A acts as a sponge of miR-490-3p, HMGA2 is the direct downstream target of miR-490-3p
CircRNAs function primarily as miRNA sponges by sequestering specific miRNAs, resulting in the changes of specific genes expression. We therefore investigated the potential miRNAs associated with circFAM73A.
In consideration of the elevated expression and the promotive effects of circFAM73A in GC, we screened the predicted miRNA targets obtained from TargetScan and RNAhybrid together with miRNAs that were significantly downregulated in GC samples according to TCGA database (fold change > 2, p < 0.05) (Fig. 4A). We identified 8 candidate miRNAs that matched these criteria (Figure S4A). A biotin-labeled circFAM73A probe was then designed to examine the potentially miRNAs that interacted with circFAM73A. The probe efficiency was verified in GC cells while circFAM73A overexpression further enhanced the pull-down efficiency (Figure S4B). As shown in Fig. 4B, qRT-PCR revealed that miR-490-3p was the only one that was pulled down by circFAM73A probe in both BGC823 and SGC7901. To further verify the direct binding of circFAM73A and miR-490-3p, luciferase reporter assays were then carried out, which demonstrated that overexpression of miR-490-3p considerably decreased the luciferase activity of the reporter containing the wild type circFAM73A sequence, but had no effects on the reporter containing circFAM73A with mutant miR-490-3p-binding site in BGC823 and SGC7901 (Fig. 4C). Furthermore, in comparison with the mutant biotin-labeled miR-490-3p, wild-type miR-490-3p captured more circFAM73A in GC cells with circFAM73A overexpression (Fig. 4D). FISH assay showed the co-location in cytoplasm between circFAM73A and miR-490-3p (Fig. 4E). The reduction of miR-490-3p in GC tissues was also found in our samples (Figure S4C). These results suggested that circFAM73A exerts its function by sponging miR-490-3p.
To identify miR-490-3p target genes, we screened TCGA for genes that were significantly upregulated in GC (fold change > 2, P < 0.05), combined with the predicted targets from TargetScan (Fig. 4F). Using this strategy, we identified 14 genes. We then analyzed the correlations between miR-490-3p expression and these 14 candidate targets in TCGA GC database. Four genes (AURKA, ONECUT2, RNF207 and HMGA2) showed clear negative correlation with miR-490-3p (Fig. 4G and Figure S4D), and were therefore chosen for further experiments.
qRT-PCR verified the upregulation of AURKA, ONECUT2 and HMGA2 in our 100 paired GC tissues compared with adjacent tissues, whereas no significant changes in RNF207 expression were observed (Fig. 4H). Moreover, ONECUT2 and HMGA2 expression levels exhibited a clear negative correlation with miR-490-3p, while we found no such correlation for AURKA and RNF207 (Fig. 4I). As shown in Fig. 4J and S4E, overexpression of miR-490-3p reduced the expression of ONECUT2 and HMGA2, in comparison, inhibition of miR-490-3p increased the expression of ONECUT2 and HMGA2. However, expression of AURKA and RNF207 showed no change after miR-490-3p overexpression or inhibition. Next, siRNAs specific to these four genes were transfected into GC cells. Only interference sequence of AURKA and HMGA2 suppressed the viability by CCK-8 assay in BGC823 and SGC7901 cells (Figure S4F and S4G).
On the basis of these results, we chose HMGA2, a potential tumor promotor and target of miR-490-3p in GC, for further studies. Luciferase reporter assays confirmed that HMGA2 was a direct downstream target of miR-490-3p (Figure S4H and S4I). Importantly, Kaplan–Meier survival analysis in our 100 GC patients and TCGA database showed that higher HMGA2 expression correlates with poor overall survival in GC patients (Fig. 4K and 4L).
CircFAM73A regulates HMGA2 expression by miR-490-3p
High Mobility Group A2 (HMGA2) is a stem cell factor primarily expressed during embryogenesis, with low abundance identified in adult human tissues[25]. HMGA2 is aberrantly expressed in several types of cancer, with high levels of HMGA2 associated with a highly malignant phenotype, as it relates with increased tumor proliferation, invasiveness, stemness and reduced survival[26–28]. As shown in Fig. 5A, a positive correlation between mRNA levels of circFAM73A and HMGA2 was detected in GC tissues, implying the potential regulation of circFAM73A on HMGA2 in GC.
To assess whether circFAM73A regulates HMGA2 expression by miR-490-3p, we reduced miR-490-3p expression in circFAM73A-repressing cells and elevated miR-490-3p in circFAM73A-overexpressing cells. Our Western Blot and qRT-PCR data showed that circFAM73A increased the expression of HMGA2 while the miR-490-3p mimic attenuated this effect. In contrast, reduction of HMGA2 caused by circFAM73A inhibition also reversed by miR-490-3p knocking-down both in BGC823 and SGC7901 (Fig. 5B, Figure S5A and S5B). Moreover, flow cytometry demonstrated that circFAM73A exogenous expression increased the proportion of CD44 positive cell and this effect was reversed by miR-490-3p mimic. In comparison, circFAM73A suppression led to the decline of CD44 positive cells which was also overturned by miR-490-3p inhibition (Fig. 5C and Figure S5C). The same results were also acquired when we examined the effects of circFAM73A on other stemness-associated transcriptional factors (SOX-2, OCT-4 and Nanog) by Western Blot (Fig. 5D and Figure S5D).
In addition, FISH assay showed the co-localization between circFAM73A and miR-490-3p in GC tissues. FISH scores confirmed that expression of circFAM73A was higher in GC tissues than in matched noncancerous tissues, whereas miR-490-3p expression showed the adverse change (Fig. 5E). Similarly, IHC staining also exhibited the increase of HMGA2 protein levels in GC tissues (Fig. 5F and5G). Importantly, we found that circFAM73A, miR-490-3p and HMGA2 correlated well in these GC tissues (Fig. 5H).
These observations supported that circFAM73A regulates the HMGA2 expression and stemness-related pathway by sponging miR-490-3p.
CircFAM73A promotes stem cell-like property and cell malignancy in GC cells by upregulating HMGA2 expression
Based on the results above, we hypothesized that circFAM73A improves stem cell-like property and cell malignancy in GC by sponging miR-490-3p, thus upregulating HMGA2 expression. To test this hypothesis, we either repressed or reconstituted HMGA2 expression in circFAM73A overexpressed or silenced cells, respectively.
Using a sphere formation assay, we found that the induction of circFAM73A on cell self-renewal ability was counteracted by HMGA2 downregulation. While HMGA2 reconstitution reversed the inhibitory function of circFAM73A repression (Fig. 6A and Figure S6A). Similarly, HMGA2 downregulation also impaired the capability on cell proliferation and migration caused by exogenous circFAM73A expression and ectopic HMGA2 expression overturn the inhibitory effects of circFAM73A suppression based on the results of further experiments including colony formation assay (Fig. 6B and Figure S6B), EdU assay (Fig. 6C and Figure S6C), flow cytometry (Fig. 6D and Figure S6D) and Transwell assay (Fig. 6E and Figure S6E).
Together, these results confirmed that circFAM73A promotes the cancer stem cell-like properties and cell malignancy in GC cells by upregulating HMGA2 expression.
CircFAM73A regulates HMGA2 expression level to promote GC growth and metastasis in vivo
To delineate the roles of circFAM73A and HMGA2 in vivo, we first generated xenografts tumors in nude mice. Xenograft tumors generated from circFAM73A-overexpressing BGC823 cells showed considerably faster growth, whereas circFAM73A-suppressed xenografts were smaller in volume than those formed from control cells. Moreover, repressing HMGA2 expression reversed the positive effect of circFAM73A on xenograft tumors formation, while exogenous expression of HMGA2 attenuated the effects caused by cirFAM73A disruption (Fig. 7A-7C, Figure S7A and S7B). Our qRT-PCR results showed that circFAM73A increased the mRNA levels of HMGA2 and CD44 in xenograft tumors samples, an effect that was reversed by HMGA2 depletion (Figure S7C). In contrast, exogenous HMGA2 expression reversed the reduction caused by cirFAM73A suppressing (Figure S7D). Immunohistochemistry staining of HMGA2 and CD44 in xenograft tumors demonstrated the same effects (Fig. 7G and 7H).
To examine the metastasis abilities of circFAM73A and HMGA2, we established two in vivo models. In lung metastasis model, respective tumor cells were injected into the caudal veins of mice and metastasis were monitored by an IVIS Imaging system. After 6 weeks, mice were euthanized. The lung tissues were obtained for HE staining (Fig. 7D) and lung metastatic foci were quantified (Fig. 7E and 7F). In liver metastasis model, tumor cells were injected into the portal veins of mice. Livers were harvested after 6 weeks and stained with HE (Fig. 7I). The number of nodes were counted (Figure S7E and S7F) and the liver indexes (liver weight/ body weight) were calculated (Fig. 7J). These two models clearly showed that circFMA73A enhanced the metastasis of GC in vivo, which was also mediated by HMGA2
Together, these data confirmed that circFAM73A promotes both GC growth and metastasis in vivo via regulating HMGA2 expression levels.
HMGA2 enhances the transcriptional activation of FAM73A by E2F1 and elevates the efficiency of cirFAM73A circularization by HNRNPL, which in turn elevates circFAM73A expression
HMGA2 functions as an architectural transcription modulator and facilitate the function of several transcription factors, including E2F1. Previous studies revealed that the interaction between HMGA2 and pRB enhances the activation of E2F1 in pituitary adenomas[26]. We then investigated whether HMGA2 facilitate the function of E2F1 in GC.
Firstly, the immunoprecipitation assay confirmed the interactions between HMGA2 and pRB in GC cells (Fig. 8A). We then examined the effects of HMGA2 on E2F1 activity by measuring the expression of several classical E2F1 responsive effectors (CDC2, CCNE1 and TK1). Western blot and qRT-PCR demonstrated that both protein and mRNA levels of these effectors increased by ectopic expression of HMGA2 and declined by HMGA2 repressing (Fig. 8B, Figure S8A and S8B). However, no change of E2F1 expression was detected upon HMGA2 alteration, suggesting that HMGA2 might simply increase the activity of E2F1, rather than its expression in GC.
Intriguingly, two putative binding sites of E2F1 were found in the FAM73A promoter region using JASPAR (#1 ATGGCGGGAGC, -77 to -67, #2 GGCCCGCCAAA, -696 to -686) (Fig. 8C). We therefore investigated whether E2F1 promotes the transcription of FAM73A and results in the increase of FAM73A mRNA or circFAM73A. qRT-PCR by specific primers (Figure S8C) showed that pre-FAM73A, FAM73A and circFAM73A levels were increased following E2F1 overexpression, while being suppressed upon E2F1 depletion (Fig. 8D and Figure S8D). To examined the binding of E2F1 on FAM73A promoter, luciferase reporter gene assay was performed. As shown in Fig. 8E and Figure S8E, E2F1, rather than vector plasmid, elevated the luminescence of the luciferase reporter containing FAM73A promoter region. ChIP-PCR analysis using specific antibody against E2F1 confirmed the occupancy of E2F1 on binding site #1 in FAM73A promoter region. Moreover, this effect could be promoted by HMGA2 ectopic expression (Fig. 8F and 8G, Figure S8F and S8G), indicating that HMGA2 enhanced the transcription of FAM73A by E2F1. As expected, HMGA2 increased both pre-FAM73A and circFAM73A production. However, elevated pre-FAM73A caused by HMGA2 induction only resulted in the increase of circFAM73A, but not linear FAM73A mRNA (Fig. 8H and Figure S8H).
The uncorrelated levels of circular RNAs and linear mRNAs indicated that HMGA2 might also affect the post-transcriptional processing and increase the back-splicing efficiency in pre-FAM73A, making circular RNAs the preferred gene output than linear RNAs. Mounting evidence suggests that splicing factors contribute to circRNA biogenesis post-transcriptionally by targeting specific sequence within flanking introns and drawing back-splicing exons ends into close proximity[20, 29, 30]. We then utilized the MEME Suite[31] to analyze the known RBPs binding motifs in flanking intron-2 and intron-7 of circFAM73A (p < 0.001, q < 0.5). Several RBPs motifs were found and among them, HNRNPL was predicted to harbor binding sites on both intron-2 and intron-7 (Figure S8I) and was previously confirmed to promote circRNA circularization[32].
To identify the direct HNRNPL binding sites in the flanking intron of circFAM73A, we performed RNA-immunoprecipitation (RIP) assays, using qRT-PCR to quantify HNRNPL occupancy within the introns adjacent to circFAM73A-forming exons. Three pairs of primers were designed according to the potential binding sequences. Primer 1 contains the three predicted HNRNPL binding sites in intron-2. Primer 2 targets the first predicted binding sites in intron-7 and the 2 to 6 predicted binding sites in intron-7 are covered in the amplification sequence of Primer 3 (Fig. 8I). We found that HNRNPL bound to Primer 1 region in intron-2 and Primer 3 region in intron-7, rather than Primer 2 region (Fig. 8J and Figure S8J). Furthermore, knock down of HNRNPL resulted in a significant reduction of circFAM73A but not pre-FAM73A. In addition, we also observed a small but significant increase of FAM73A mRNA (Fig. 8K and Figure S8K). Together, these findings demonstrated that HNRNPL binds to the flanking intron region and elevates the circFAM73A formation.
We then investigated whether HMGA2 promotes the circularization of circFAM73A by HNRNPL. The ratio of circRNA expression to mRNA expression was calculated to reflect the circularization efficiency. As shown in Fig. 8L and Figure S8L, exogenous HMGA2 expression resulted in the upregulation of circularization efficiencies, which was reduced when HNRNPL was additionally suppressed, suggesting that HMGA2 enhances the efficiency of cirFAM73A circularization by HNRNPL.
In addition, we also found that both E2F1 and HNRNPL correlated well with HMGA2 in TCGA database (Fig. 8M and 8N), which indicated the regulation of E2F1 and HNRNPL on circFAM73A and its downstream target, HMGA2.
Collectively, these results demonstrated that HMGA2 plays a dichotomous role on regulating circFAM73A expression, i.e. HMGA2 facilitates the transcriptional activation of FAM73A by E2F1 and elevated the efficiency of cirFAM73A circularization by HNRNPL.
CircFAM73A directly interacts with HNRNPK and facilitates β-catenin stabilization
To further explore the mechanism of circFAM73A, an RNA pulldown assay was conducted using a specific biotin-labeled circFAM73A probe. Coomassie blue staining showed several bands of proteins compared with antisense probe (Fig. 9A) while mass spectrometry analysis identified HNRNPK (Fig. 9B), one of the major pre-mRNA-binding proteins. The RIP assay confirmed the direct interaction between circFAM73A and HNRNPK (Fig. 9C and Figure S9A). However, the expression of HNRNPK showed no obviously change upon circFAM73A overexpression or knocking-down by Western Blot (Fig. 9D and Figure S9B) in BGC823 and SGC7901, indicating that circFAM73A might regulate the activity rather than the expression of HNRNPK.
It has been reported that several lncRNA interacts with HNRNPK and facilitates the HNRNPK-mediated stability of β-catenin. Co-IP analysis was performed and indicated the endogenous interaction between HNRNPK and β-catenin in GC cells (Fig. 9E and Figure S9C). We therefore investigated whether circFAM73A regulates the expression of β-catenin by HNRNPK. Western Blot demonstrated that ectopic expression or knockdown of circFAM73A increased or decreased β-catenin levels (Fig. 9F and Figure S9D). Moreover, the reduction of β-catenin caused by circFAM73A repression was abolished by MG132 treatment (Fig. 9G and Figure S9E). Besides, after treatment of cycloheximide, Western Blot assay showed that circFAM73A knocking-down shortens the half‐life of β-catenin (Fig. 9H and Figure S9F), indicating that circFAM73A stabilizes β-catenin by reducing the degradation of β-catenin protein. We then investigated the roles of circFAM73A in HNRNPK-mediated β-catenin stability, and found that ectopic expression or knockdown of circFAM73A promoted and weakened the interaction between HNRNPK and β-catenin in GC cells (Fig. 9I and Figure S9G), respectively. Moreover, overexpression or knockdown of circFAM73A also resulted in increase and decrease of nuclear translocation and of β-catenin (Fig. 9J and Figure S9H).
Furthermore, functional experiment showed that the promoting effects of circFMA73A were reversed upon HNRNPK interfering (Fig. 9K-9M and Figure S9I-S9K), demonstrating HNRNPK as the downstream of circFAM73A.
Taken together, our results indicated that circFAM73A directly interacts with HNRNPK and facilitates the stabilization of β-catenin.