CircPAK1 promotes the progression of hepatocellular carcinoma via modulation of YAP nucleus localization by interacting with 14-3-3ζ

CircPAK1 is highly expressed in HCC

To identify circRNAs involved in HCC progression, high-throughput sequencing was performed on 3 paired tissues of HCC and adjacent tissues. Among the 351 differential expressed circRNAs (fold change≥2 and q value≤0.001),a total of 211 were upregulated and 140 were downregulated in the HCC tissues than their paired normal tissues. As shown in Fig. 1A, from the circular RNA sequencing results, 44 differentially expressed genes (DEGs) with fold change ≥15 were identified, all higher expressed in tumor tissues than those in normal tissues. The 44 upregulated circRNAs were listed in Table S6. Usually, the levels of circRNAs are in accordance with their respective mRNA levels [16]. Therefore, 25 DEGs whose host genes are significantly upregulated in HCC tissues were selected from these 44 DEGs by using The Cancer Genome Atlas (TCGA) database. We designed specific primers targeting the junction site of these 25 DEGs, respectively, and only 11 circRNAs could be successfully amplified by qRT-PCR. Then, we examined the expression of these 11 circRNAs by qRT-PCR in 20 HCC tissues and matched adjacent normal tissues, and only 5 DEGs were statistically significant. As the P value of paired comparison of circPAK1 is the most significant, so circPAK1 was eventually selected for the subsequent study (Fig. S1A).

Fig. 1figure 1

Identification of circPAK1. A Flow diagram of circRNAs screening in 3 pairs HCC tissue. B Illustration of the annotated genomic region of circPAK1 Sanger sequencing was conducted to confirm the ‘head-to-tail’ splicing of circPAK1. C The divergent primers detected circPAK1 in cDNA but not in gDNA, GAPDH was used as a negative control. D Relative RNA level of circPAK1 and linear PAK1 in different time point. E and F showed the relative RNA level of circPAK1, linear PAK1 and GAPDH treated with RNase R. G The nuclear mass separation assay and H FISH showed that the sub-cellular distribution of circPAK1 was mostly present in the cytoplasm. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

CircPAK1 is derived from chromosome 11: 77085372–77,103,586 and consists of 4 adjacent exons in the PAK1 gene, sanger sequencing confirmed the junction site of circPAK1 (Fig. 1B). To further identify the ring structure, convergent and divergent primers were designed, and the PCR product was determined by agarose gel electrophoresis (Fig. 1C). Additionally, the RNase R digestion and actinomycin D RNA stability assays confirmed the stability of circPAK1 compared with PAK1 (Fig. 1E, F). The FISH and qRT-PCR results suggested that the localization of circPAK1 was mostly in cytoplasm (Fig. 1G, H).

The levels of circPAK1 in 60 pairs of HCC tissues and adjacent non-tumor tissues were determined by qRT-PCR. The results showed that circPAK1 was significantly overexpressed in HCC tissues than normal liver tissues (Fig. 2A, B). Besides, circPAK1 was also overexpressed in HCC cell lines compared with normal hepatocytes (L02) (Fig. 2C). For the subsequent study, we chose LM3 and Focus cells to establish circPAK1 stable knockdown cell lines by infection with shRNA specially targeting the junction sites of circPAK1, while Hep-3B to infect with a lentiviral vector to establish circPAK1 stable overexpression cell lines. As shown in Fig. 2D, stable circPAK1-knockdown and circPAK1-overexpression cell lines were successfully established. To evaluate the clinical value of circPAK1, the 60 HCC patients were divided into two groups based on the median expression of circPAK1 (Table 1). Our data suggested that higher expression of circPAK1 was more likely to develop larger tumor size, higher risks of LN metastasis, advanced TNM stage and microvascular invasion (MVI) (all P values<0.05); however, no statistical significance for age, gender, AFP, PIVKA-II, HBsAg status, tumor multiplicity, differentiation and vascular invasion was observed. Importantly, patients in the high expression group showed poor overall survival and disease-free survival than the patients in the low expression group (Fig. 2E). Taken together, these results demonstrate that circPAK1 is abundant and upregulated in HCC tissues and cell lines, and it could serve as a valuable molecular biomarker for HCC.

Fig. 2figure 2

Characteristics of circPAK1 in HCC. A Differential expression of circPAK1 in human HCC tissues and adjacent nontumor tissues of 60 patients. B Relative expression of circPAK1 in 60 pairs of HCC tissues and matched normal tissues, shown as log2 (Tumor/Normal). C Relative expression of circPAK1 in HCC cell lines was assessed by qRT-PCR. D LM3 and Focus cells were transfected with circPAK1 shRNA (sh-circPAK1) or control shRNA (sh-NC); Hep-3B cells were infected with circPAK1 overexpression lentivirus (Lv-circPAK1) or control lentivirus (Lv-NC), the transfection efficiency was assessed by qRT-PCR. E Elevated expression of circPAK1 was negatively correlated with the overall survival (OS) and disease-free survival (DFS) of HCC patients. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

Table 1 The clinicopathological relevance analysis of circPAK1 expression in HCC patientsCircPAK1 promotes HCC proliferation, migration and invasion

To explore the effect of circPAK1 on cell proliferation, CCK-8, EdU and colony formation assays were performed, and the results showed that circPAK1 knockdown significantly attenuated the growth of LM3 and Focus cells, while converse results were observed in circPAK1 overexpression cell lines (Fig. 3A, B and C). To further investigate the effect of circPAK1 on HCC cell migration and invasion, wound-healing and transwell assays were performed. The results revealed that circPAK1 inhibition could significantly reduce the migration and invasion ability of LM3 and Focus cells, while circPAK1 overexpression markedly increased these abilities (Fig. 3D, E). Taken together, these results indicated that circPAK1 enhances HCC cell proliferation and motility.

Fig. 3figure 3

CircPAK1 promotes HCC proliferation, migration and invasion. A The proliferation of sh-circPAK1 or Lv-circPAK1 cells was determined by CCK8 assay. B EdU incorporation of sh-circPAK1 or Lv-circPAK1 cells was decided, scale bar, 50 μm. C The colony formation of sh-circPAK1 or Lv-circPAK1 cells was determined. D Cell migration ability was evaluated by wound healing assay, scale bar, 100 μm. E Invasive or migrated cells were measured by transwell assay with or without matrix, scale bar, 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

CircPAK1 inhibits HCC apoptosis

We also explored whether circPAK1 could regulate the cell cycle and apoptosis. We found that the intervention of circPAK1 did not affect the cell cycle (Fig. S1B). However, it is worth noting that circPAK1 knockdown promoted cell apoptosis in both LM3 and Focus cells (Fig. 4A). These results suggest that the promotion ability of circPAK1 on HCC cell growth may depend on inhibiting tumor apoptosis.

Fig. 4figure 4

CircPAK1 promotes the apoptosis and angiogenesis of HCC cells. A The percentage of apoptotic cells of STK39 stable knockdown or overexpression cells was analyzed by Annexin V-FITC/PI staining assay. B HUVEC tube formation, migration and invasion were evaluated using the conditioned medium (CM) from LM3 (circPAK1 knockdown) and Hep-3B (circPAK1 overexpression) cells, scale bar, 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

CircPAK1 promotes angiogenesis of HCC in vitro

Tumor cells can promote angiogenesis by modifying the tumor microenvironment, contributing to early progression, invasion, postoperative recurrence, and metastasis of HCC [17, 18]. Therefore, the ability to promote angiogenesis is also one of the important indicators to measure tumor invasiveness. Then, we performed an angiogenesis experiment to study whether circPAK1 has a promoting effect on tumor angiogenesis.

HUVEC tube formation, migration and invasion were evaluated using the conditioned medium (CM) from LM3 (circPAK1 knockdown) and Hep-3B (circPAK1 overexpression) cells. As shown in Fig. 4B, CM from circPAK1 knockdown cells significantly inhibit the migration and invasion of HUVEC cells. In contrast, the opposite results were found in circPAK1 overexpression cells. As envisioned, the tube formation assay also showed that the number of branch sites was decreased in HUVECs treated with CM from circPAK1 knockdown cells compared with that from control cells. The above abilities of HUVECs were enhanced after incubation with CM from circPAK1 overexpression cells. Taken together, these findings suggest that circPAK1 could induce angiogenesis of HCC in vitro.

CircPAK1 enhances in vivo HCC tumor growth and metastasis and CS/si-circPAK1 nanocomplexes could effectively inhibit these abilities

To assess the in vivo effect of circPAK1 on HCC growth and metastasis, we then generated tumor xenograft and lung metastasis models. The xenograft tumor model showed that no matter the tumor size or the tumor weight was significantly decreased in the sh-circPAK1 group, while increasing in the Lv-circPAK1 group (Fig. 5A). The results of IHC staining also showed that the level of Ki-67 was lower in the sh-circPAK1 group and higher in the Lv-circPAK1 group (Fig. 5B). Additionally, the lung metastasis model showed a similar trend as the results of xenograft model, for there were less metastasis foci in the sh-circPAK1 group, and more in the Lv-circPAK1 group (Fig. 5C). These results indicate that circPAK1 could promote tumor growth and metastasis of HCC.

Fig. 5figure 5

CircPAK1 enhances HCC tumor growth and metastasis in vivo and CS/si-circPAK1 nanocomplexes inhibits HCC growth and metastasis in vivo. A Nude mice were subcutaneously injected with circPAK1 stable knockdown or overexpression cells. The tumor volume and average weight were determined. B IHC analysis of Ki-67 in the tumors derived from mice, scale bar, 50 μm. C CircPAK1 stable knockdown or overexpression cells were injected into the tail vein of nude mice to induce lung metastasis, and gross lung tissues were obtained; liver metastatic nodules were subjected to HE staining as indicated, scale bar, 200 μm. D The loading efficiency of CS. E TEM images of CS/si-circPAK1 nanocomplexes (CS/ si-circPAK1 = 50/1). F The flow diagram showed the scheme of intratumorally/intravenously with saline, si-circPAK1 or CS/si-circPAK1 into mice. G and H Weight volume and weight change after 20 days of treatment with saline, si-circPAK1 or CS/si-circPAK1 for xenograft tumors or lung metastasis models. The tumor volume and average weight were determined; gross lung tissues were obtained and liver metastatic nodules were subjected to HE staining as indicated, scale bar, 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

Based on the new understanding of tumor pathogenesis, gene targeting therapy in cancer has attracted a wide range of attention and proved to be a rational approach in cancer therapy. Chitosan (CS)-based materials have a long history as drug delivery vehicles due to their characteristics of non-toxicity, biodegradability, high drug carrying capability, high plasma membrane permeability, including transport across mucosal membranes, the pH-dependent release of therapeutic agents, multi-functionality, and suitable circulation time [19,20,21]. We established three nanocomplexes in different proportions based on the weight ratios of CS and si-circPAK1. The loading efficiency of chitosan to si-circPAK1 was more than 99% under these three ratios (Fig. 5D). Therefore, to ensure the loading efficiency, we choose the largest chitosan utilization ratio-CS/si-circPAK1 of 50. The TEM (Fig. 5E) and particle size potentiometer (Fig. S2A) revealed that the particle size of CS/si-circPAK1 nanocomplexes was on an average of 101.9 nm. The zeta potential was − 21.4 and + 31.6 mV (Fig. S2B). By simulating the temperature and pH of human body to detect the release of siRNA from CS, we found that siRNA can be released persistently and efficiently (Fig. S2C).

To study the effect of CS/si-circPAK1 nanocomplexes on the proliferation and metastasis of HCC in vivo, we generated several groups of subcutaneous xenograft tumor and lung metastasis models. Figure 5F shows the injection scheme with saline, si-circPAK1 or CS/si-circPAK1 nanocomplexes into mice. We noticed that no matter the tumor size or the weight in the CS/si-circPAK1 nanocomplexes treated group was much smaller and lighter than the saline-treated group, even better than the si-circPAK1 group (Fig. 5G). IHC staining of tumors from CS/si-circPAK1 nanocomplexes treated group also revealed a significant decrease of Ki67 (Fig. S2D). Furthermore, the lung metastasis models from CS/si-circPAK1 nanocomplexes treated group showed a better inhibition effect on lung metastasis lesions (Fig. 5H). Collectively, these findings suggest that circPAK1 could promote the in vivo growth and metastasis of HCC, and the CS/si-circPAK1 nanocomplexes could effectively inhibit these abilities.

CircPAK1 facilitates the progression of HCC through hippo signaling pathway by promoting nucleus transport of YAP

To explore the molecular mechanisms of circPAK1 involved in HCC development, we performed RNA-sequencing. By listing the top 10 of pathway enrichment, a strong correlation between the Hippo signaling pathway and the downstream of circPAK1 is shown (Fig. 6A). The hippo signaling pathway is highly conserved in evolution, and it can regulate organ size by regulating cell proliferation and apoptosis [22, 23]. The dysregulation of Hippo signaling pathway will cause uncontrolled proliferation of cells, thus leading to the overgrowth of tissues and organs [24]. Several studies have pointed out that the inactivation of Hippo signaling pathway was closely correlated with the progression of HCC [25,26,27].

Fig. 6figure 6

CircPAK1 mediates oncogenic effects on HCC through inactivating Hippo signaling pathway. A Pathway enrichment analysis of differentially expressed genes in RNA-sequence data. B Levels of p-YAP (ser127), LATS1/2 and p-LATS1/2 were examined by western blotting in circPAK1 stable knockdown or overexpression cells. C The expression of YAP1 protein in nuclear and cytoplasmic component of circPAK1 stable knockdown or overexpression cells. D IF assay in stable circPAK1-knockdown LM3 cells or stable circPAK1-overexpressed Hep-3B cells, scale bar, 20 μm. E The level of downstream target gene of YAP, CTGF and CYR61, were determined by qRT-PCR. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

Combined with the above tumor-promoting phenotype of circPAK1 and the result of RNA-seq, we, therefore, explored the effect of circPAK1 on Hippo signaling pathway. We first performed a western blot to analyze the changes in proteins related to Hippo signaling pathway. We found that neither the knockdown nor the overexpression of circPAK1 did not affect the level of LATS1/2 or its phosphorylation level (Fig. 6B); however, p-YAP was increased in the circPAK1 knockdown group, while decreased in the circPAK1 overexpression group. Cytoplasmic retention of YAP plays a vital role in the Hippo pathway-mediated control of cell proliferation and apoptosis. It is well established that non-phosphorylated YAP can be transported into the nucleus and interact with TEAD1–4 to activate multiple downstream target genes, which is very important to enhance tumor proliferation and apoptosis inhibition [28,29,30]. However, YAP (p-YAP) phosphorylation by p-LATS1/2 will be sequestrated in the cytoplasm and eventually degraded [31].

Based on the above perceptions, we speculate that circPAK1 may promote the nucleus transport of YAP and then affect the proliferation, invasion and metastasis of HCC. Subsequently, we conducted the cytoplasm and nucleus fractionation assay to observe the effect of circPAK1 on the localization of YAP. As shown in Fig. 6C, overexpression of circPAK1 promoted the nucleus localization of YAP, while reducing the p-YAP (ser-127) level. The opposite results were observed under the condition of circPAK1 knockdown. Meanwhile, immunofluorescence also confirmed this observation (Fig. 6D). These findings suggest that circPAK1 could guide the nucleus transportation of YAP. Besides, the main downstream target gene of YAP, CTGF and CYR61, were downregulated when circPAK1 knockdown, but upregulated when circPAK1 overexpression (Fig. 6E).

We further explored whether the tumor-promoting effect of circPAK1 is dependent on YAP by treating the circPAK1 overexpression Hep-3B cells with YAP siRNA. Fig. S3A shows the efficiency of YAP knockdown. As envisioned, the tumor promoting effect of circPAK1 on HCC was significantly weakened (Fig. S3B-F). Taken together, our data revealed that circPAK1 enhances HCC progression by accelerating YAP nucleus transport, which leads to the inactivation of Hippo signaling pathway.

CircPAK1 competitively binds 14–3-3ζ with YAP thus promoting the nucleus transportation of YAP

As a transcription coactivator, the nucleus transportation of YAP will directly determine whether it can interact with TEAD1–4 to activate multiple downstream target genes [28,29,30]. There have been many studies on downstream signals of YAP after its nucleus implantation, but little is known about by what mechanism YAP is regulated before its transportation to the nucleus.

Serving as miRNA sponge is one of the most common biological functions of circRNA, while binding with argonaute 2 (AGO2) RNA is the vital basis for circRNA to act as competitive endogenous RNA (ceRNA). However, RIP-qPCR assay showed that circPAK1 couldn’t be enriched in AGO2 antibody complexes compared with anti-IgG, so acting as ceRNA of circPAK1 was excluded (Fig. S4A).

It has been reported that circRNA can facilitate YAP nucleus transport by binding YAP protein directly, thus promoting the metastasis of colorectal cancer [32]. To verify whether circPAK1 can directly bind to YAP, we also performed RIP-qPCR and found that the result was not consistent with our hypothesis (Fig. 7A). So, how does circPAK1 affect the cellular spatial localization of YAP? We speculate that another mediator protein mediates the association between circPAK1 and YAP. Next, we performed an RNA-pulldown assay and mass spectrometry analysis to identify potential circPAK1-interacting proteins in LM3 cells. Surprisingly, 14–3-3ζ protein was identified as the main protein using silver staining (Fig. 7B) and liquid chromatography mass spectrometry (Fig. S4B). WB (Fig. 7C) and RIP were also performed and confirmed this specific binding (Fig. 7D).

Fig. 7figure 7

CircPAK1 competitively binds 14–3-3ζ with YAP thus reduces the phosphorylation of YAP by p-LATs. A RIP-qPCR was performed to explore the association between circPAK1 and YAP. B Silver-stained SDS-PAGE gel-containing proteins derived from RNA pulldown by circPAK1 probe and negative control. The red rectangle was used for mass spectrometric analysis. C Western blot analysis showed the specific association of circPAK1 with 14–3-3ζ. D qRT-PCR analysis of the RNAs derived from the RIP assays. E A proposed model for 14–3-3ζ mediation of p-LATS-YAP interactions. F Western blot analysis identified 14–3-3ζ protein expression level in LM3 transfected sh-circPAK1. G CircPAK1 stable knockdown LM3 cells were subjected to IP using 14–3-3ζ antibody or control IgG, followed by IB with 14–3-3ζ, p-LATS1/2 and YAP antibody, to confirm and determine the 14–3-3ζ- p-LATS-YAP protein complex. H CircPAK1 stable knockdown LM3 cells were subjected to IP using YAP antibody or control IgG, followed by IB with 14–3-3ζ, p-LATS1/2 and YAP antibody. *p < 0.05; **p < 0.01; ***p < 0.001. Data were shown as mean ± SEM

Nucleus, import/export of YAP, is strictly regulated by 14–3-3ζ, as evidenced by the fact that 14–3-3ζ assembles with YAP, isolates it in the cytoplasm and prevents it from further signal amplification [33]. Therefore, the direct or indirect change of 14–3-3ζ level will affect the spatial localization of YAP in a cell. It has been proved that 14–3-3ζ can recruit p-LATS and YAP to form a complex, which can induce the phosphorylation of YAP and lead to its cytoplasmic retention, and 14–3-3ζ is the key regulatory factor of this complex (Fig. 7E) [34]. In addition, 14–3-3ζ can be dissociated from non-phosphorylated YAP under the condition of hypoxia, thus leading to the nucleus transport of YAP [35]. These studies showed that the cytoplasmic fixation of 14–3-3ζ on YAP is based on the non-phosphorylation of YAP. We hypothesized that circPAK1 could directly or indirectly affect the level of 14–3-3ζ by interacting with 14–3-3ζ, thus affecting the recruitment of P-LATs and YAP, then further inhibiting the phosphorylation of YAP, and finally, exerting the tumor promoting effect by facilitating the nucleus transport of YAP. Interestingly, we found that circPAK1 did not affect the 14–3-3ζlevel, suggesting that the level of 14–3-3ζmay be affected indirectly (Fig. 7F). To approach this, we first performed immunoprecipitation assay in circPAK1-knockdown LM3 cells. The existence of 14–3-3ζ- p-LATS-YAP protein complex was confirmed, and the interaction between p-LATS and YAP in the circPAK1-knockdown group was significantly increased compared with the sh-NC group, while significantly decreased in the circPAK1-overexpression group (Fig. 7G, Fig. S4D). Next, we silenced 14–3-3ζ in circPAK1 stable knockdown cell lines to explore whether the interaction between p-LATS and YAP can still be influenced by the change of circPAK1 level. The silencing efficiency of si-14-3-3ζ was shown in Fig. S4C. Surprisingly, we found that the increased interaction between p-LATS and YAP was significantly reversed after 14–3-3ζ knockdown (Fig. 7H). The immunofluorescence also showed that the nucleus location of YAP was restored after the knockdown of 14–3-3ζ (Fig. S4E). Taken together, the promotion of YAP nucleus transport mediated by circPAK1 is through binding with 14–3-3ζ, which weakens the recruitment of p-LATs and YAP, and then reduces the phosphorylation of YAP by p-LATs.

CircPAK1 is upregulated in lenvatinib-resistant HCC cells and exosome-mediated circPAK1 transfer and transmission of lenvatinib resistance

To examine whether circPAK1 is involved in mediating HCC lenvatinib resistance, we generated two lenvatinib-resistant HCC cell lines (LM3-LR and Hep-3B-LR). The IC50 of the two lenvatinib-resistant cell lines and their parental cell lines (LM3-P and Hep-3B-P) were shown in Fig. S4F. Next, we performed a qRT-PCR analysis and confirmed that circPAK1 was consistently increased in the two lenvatinib-resistant cell lines than in their parental cell lines (LM3-P and Hep-3B-P) (Fig. 8A). To explore the potential correlations between circPAK1 and lenvatinib resistance in HCC, we first silenced circPAK1 in LM3-LR and Hep-3B-LR cells and confirmed the knockdown efficiency (Fig. 8B). We found that the depletion of circPAK1 remarkably increased the lenvatinib sensitivity of the two lenvatinib-resistant HCC cell lines as determined by cell viability assays (Fig. 8C). Collectively, circPAK1 is critical for maintaining lenvatinib resistance.

Fig. 8figure 8

CircPAK1 transmits lenvatinib resistance by exosomes. A The level of circPAK1 in the two lenvatinib-resistant HCC cell lines was assessed by qRT-PCR. B The knockdown efficiency of circPAK1 in the two lenvatinib-resistant HCC cell lines was assessed by qRT-PCR. C The effect of circPAK1 knockdown on lenvatinib resistance was assessed by CCK8 assay. D TEM and NTA of exosomes isolated from LM3-LR and Hep-3B-LR culture media, scale bar, 100 nm. E Exosomal protein positive markers (Alix, Tsg101 and CD9) and negative marker (Calnexin) detection by western blot from purified exosomes and exosome-depleted cell extracts. F qRT-PCR analysis showed that exosomal circPAK1 was upregulated in lenvatinib-resistant cells in contrast to parental cells. G Intercellular trafficking of exosomes among different cell lines by isolated exosomes labeled with PKH67 dye, scale bar, 100 nm. H Treatment with exosomes derived from LM3-LR and Hep-3B-LR cells increased circPAK1level, however this effect was abrogated when LM3-LR and Hep-3B-LR cells were silenced with circPAK1. I cell viability in exosome-treated parental cells

It has been reported that circRNAs are abundant in exosomes and can be transferred from cell to cell via exosomes [36,37,38]. Other reports demonstrated that exosomes could mediate drug resistance by exosomes [12, 13, 39, 40]. Therefore, the subsequent research was mainly focused on whether circPAK1 could confer lenvatinib resistance by integrating into exosomes. We then isolated exosomes from the CM of LM3-LR and Hep-3B-LR cells and their parental cell lines. Exosomes isolated from the CM of LM3-LR and Hep-3B-LR cells were identified by TEM, NTA and WB. The representative micrograph and video were taken by TEM; NTA analysis revealed that the average diameter of exosomes was 100 nm (Fig. 8D). The typical positive exosome biomarkers (Calnexin) and negative exosome biomarkers (Alix, Tsg101 and CD9) were detected by WB (Fig. 8E). In addition, qRT-PCR indicated that exosomes isolated from LM3-LR and Hep-3B-LR CM contained more circPAK1 than those from their parental cell lines, suggesting that exosomes may have the ability to transmit circPAK1 (Fig. 8F).

We used three prolonged stages to explore whether exosome could mediate the transfer of circPAK1 disseminates lenvatinib resistance. Firstly, we labeled exosomes isolated from LM3-LR and Hep-3B-LR cells with PKH67 followed by incubation with their parental cells, respectively, for 48 h. As shown in Fig. 8G, a strong green signal was observed in LM3-P and Hep-3B-P cells, indicating that the exosomes were taken up by recipient parental cells. Next, we determined the level of circPAK1 in recipient parental cells and found that after incubation with exosomes isolated from the lenvatinib-resistant cells, higher levels of circPAK1 were determined, but exosomes from circPAK1 knockdown lenvatinib-resistant cells failed to raise the level of circPAK1 in recipient cells (Fig. 8H). The results of these two stages indicated that exosomes could transport circPAK1 from lenvatinib-resistant cell lines to their parental cell lines. Lastly, we explored whether exosome-transferred circPAK1 could induce the resistance of recipient cells to lenvatinib. Surprisingly, when we treated the parental cells with exosomes isolated from their lenvatinib-resistant cell lines, their sensitivity to lenvatinib was significantly decreased (Fig. 8I). Taken together, our data demonstrated that circPAK1 could be transported by exosomes from lenvatinib-resistant HCC cells to recipient parental cells and confer the resistant phenotype to recipient cells.

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