M6A-mediated upregulation of LINC00958 increases lipogenesis and acts as a nanotherapeutic target in hepatocellular carcinoma

Fresh tumor tissues and paired adjacent non-tumor samples were collected from 80 HCC patients who underwent surgical resection from January 2012 to December 2014 in the First Affiliated Hospital of Nanjing Medical University. The tissue samples were preserved in liquid nitrogen. All patients did not receive preoperative chemotherapy or radiotherapy and signed the written informed consents. This study was approved by the ethical review board of the First Affiliated Hospital of Nanjing Medical University.

Specific FISH probes to LINC00958 and miR-3619-5p were designed and synthesized by Servicebio (Wuhan, China). The hybridization was performed in HCC cells and tissues as previously reported [19]. All images were analyzed on a confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany). The FISH probe sequences are shown as follows: LINC00958: 5′-TCCTCCCATGTTTTTGTCTTCCCTACCACC-3′; miR-3619-5p: 5′-GCTGCACCAGCCTGCCTGCTGA-3′.

We purchased lentivirus overexpressing LINC00958 or HDGF, and small hairpin RNA (shRNA) targeting LINC00958 or METTL3 from Genechem (Shanghai, China). Lentiviruses were transfected into HCC cells with 5 mg/ml polybrene for 48 h. Stable cell clones were selected for 1 week using puromycin (5 μg/ml). The overexpression or knockdown efficiency was detected by RT-qPCR. The sequences used are provided as follows: sh1-LINC00958: 5′-GTACCCAAGTTATTCAGGATT-3′, sh2-LINC00958: 5′-GTGACTAGCTTAAACTAAATT-3′, sh3-LINC00958: 5′-GAGGTACCCAATAGTTTCATT-3′; sh-METTL3: 5′-GCCAAGGAACAATCCATTGTT-3′.

RIP assay was performed using a Magna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) in accordance with the manufacturer’s protocol. Cells were isolated and lysed by RIP lysis buffer and incubated with antibodies against AGO2 (Abcam, Cambridge, MA, USA), or m⁶A (Synaptic Systems, Goettingen, German) at 4 °C overnight. IgG was used as negative control. The immunoprecipitated RNAs were eluted and analyzed by RT-qPCR.

Cells lysates were harvested 48 h after transfecting with 50 nM of biotin-labeled miRNAs (GeneCreate, Wuhan, China). Streptavidin-coupled Dynabeads (Invitrogen) were washed and resuspended in the buffer. Then an equal volume of the biotin-labeled miRNAs was added in the buffer. After incubating at room temperature for 10 min, the coated beads were separated with a magnet for 2 min and washed three times. The isolated RNAs were then subjected to RT-qPCR analysis.

Total RNA was isolated from sh-NC (n = 3) and sh-LINC00958 (n = 3) HCCLM3 cells. RNA samples were analyzed by RNA sequencing (BGI, Shenzhen, China) based on the manufacturer’s protocols. Briefly, BGISEQ-500 platform was used to sequence the samples for subsequent generation of raw data. Genes significantly differentially expressed between sh-NC and sh-LINC00958 cells were selected based on fold change ≥ 2.0 and P ≤ 0.001 using the DEGseq method. Functional pathway analysis was conducted using KEGG pathway enrichment analysis.

HCC cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized in 60% isopropanol for 10 s. Subsequently, cells were stained with Oil Red O working solution for 30 min at room temperature, washed three times with PBS, and photographed under a microscope. Oil Red O staining in frozen sections of HCC tissues were similarly performed.

NOD/SCID and BALB/c mice were used for the establishment of the HCC PDX model. Briefly, we collected the primary HCC tissues from two patients after surgical resection and kept the specimens in iced culture medium supplemented with 1% penicillin/streptomycin. Then, the tissues were diced into 2–3-mm³ pieces and subcutaneously implanted into the flanks of NOD/SCID mice. When the xenografted tumors grew up to 1–2 cm³, we harvested the tissues from the mice bearing PDX tumors and cut the tissues into pieces. The tumor fragments were further implanted into BALB/c nude mice for the serial transplantation. When the tumor volume reached 50 mm³, we intratumorally injected recombinant lentivirus vectors into tumor tissues continuously for 20 days. Tumor weight and volume were recorded.

We used the double emulsion solvent diffusion method for NP preparation as previously described [20]. si-LINC00958 was reconstituted in DEPC water and then mixed with spermidine (Sigma-Aldrich) at the N/P ratio (the ratio of polyamine amine groups to siRNA phosphate groups) of 8:1. The resultant mixture was incubated for 15 min at room temperature to form si-LINC00958/spermidine complex. PLGA-PEG-COOH (10 mg; DaiGang Biomaterial Co. Ltd., Jinan, China) was dissolved in 500 μl of dichloromethane (Aladdin Industrial Corp., Shanghai, China). Then, the above dichloromethane solution was added dropwise to si-LINC00958/spermidine complex with a probe sonicator (VCX 130; Sonics & Materials, Inc., Newtown, CT, USA) in an ice bath. The resultant primary emulsion was further added dropwise to 4 ml of an aqueous phase containing 2.5% polyvinyl alcohol (Aladdin Industrial Corp.) and emulsified using probe sonication for 1 min. The second emulsion was then stirred at room temperature for 4 h to evaporate the organic solvent. Subsequently, the NPs were collected by centrifugation for 15 min and washed twice with DEPC water.

We used dynamic light scattering (DLS) with a Nano Particle Analyzer (Zetasizer Nano ZSE, Malvern Instruments Ltd., UK) to investigate the size, zeta potential, and polydispersity index (PDI) of the NPs. A drop of the sample was placed onto a copper mesh and dried in room temperature to obtain transmission electron microscopy (TEM) images of the NPs. The siRNA encapsulated in PLGA was measured using UV spectrophotometry to determine the encapsulation efficiency as previously described [21].

To investigate the suppressive effect of PLGA-PEG(si-LINC00958) NPs on HCC cell growth in vivo, PDX tumor models were created as described above. When the tumors developed to 50 mm³, PLGA-PEG(si-LINC00958) NPs or PLGA-PEG(siRNA control) NPs at a dose of 200 mg/kg were injected into the mice (n = 14 in each group) through the tail vein twice weekly. Treatment continued until 4 weeks later, at which point four mice in each group were sacrificed. Tumor weight and volume were recorded immediately. Tumors were subjected to subsequent RT-qPCR and western blotting analyses. The major organs, including the liver, kidney, lung, spleen, and heart, were harvested and fixed with 4% paraformaldehyde for further hematoxylin-eosin (H&E) examination. Blood alanine transaminase (ALT), aspartate transaminase (AST), creatinine (Cr), and blood urea nitrogen (BUN) were also analyzed. The remaining ten mice in each group were monitored for survival analysis with 10 weeks as the cutoff.

SPSS 24.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) were used to perform the statistical analysis. Data are shown as mean ± SEM of the mean. Two-sided Student’s t test was used to analyze the differences between groups. The differences of LINC00958 and HDGF expression levels between tumor and non-tumor specimens were evaluated by paired t test. Chi-square test was adopted to analyze the association of LINC00958 and METTL3 expression with clinicopathological features. Kaplan-Meier curve with log-rank test was used to compare the survival outcome, and Cox proportional hazards model was employed for multivariate survival analysis. Pearson’s correlation was performed to analyze the correlation between LINC00958, miR-3619-5p, METTL3, and HDGF levels. P value less than 0.05 was considered statistically significant.

Supplementary methods are described in Additional file 1.

With a stringent filter of logFC > 2.0 and P value < 0.005, we established the profile of differentially expressed lncRNAs in HCC based on TCGA data. As demonstrated in Additional file 2: Figure S1A, the expression levels of 441 lncRNAs were significantly altered in HCC tumor samples. LINC00958 was suggested to be upregulated in HCC (logFC = 4.092782 and P value = 4.44 × 10^− 7). We then used the data retrieved from starBase platform and found a markedly higher expression of LINC00958 in HCC (Additional file 2: Figure S1B). To verify the bioinformatics results, we performed RT-qPCR to quantify the expression levels of LINC00958 in normal human liver cell line QSG-7701 and six HCC cell lines. As shown in Fig. 1a, LINC00958 was highly expressed in HCC cell lines. Furthermore, we examined LINC00958 expression in 80 paired HCC tissues and non-tumor specimens. LINC00958 was remarkably overexpressed in HCC tissues (Fig. 1b), especially in those with moderate/poor differentiation, microvascular invasion, and TNM III/IV stage (Fig. 1c–e). FISH assay verified the overexpression of LINC00958 in HCC tissues compared to the non-tumor samples (Fig. 1f).

To investigate the clinical significance of LINC00958 in HCC, we classified the enrolled 80 patients into LINC00958^high and LINC00958^low groups based on the median expression value (2^−△△Ct = 0.134). As indicated in Additional file 3: Table S1, high LINC00958 expression was associated with tumor differentiation (P = 0.019), tumor size (P = 0.025), microvascular invasion (P = 0.014), and TNM stage (P = 0.013). Bioinformatics prediction implied a correlation between LINC00958 expression status and patient survival (P = 0.014; Additional file 2: Figure S1C), and we confirmed that patients with high LINC00958 expression had poorer overall survival than those with low LINC00958 expression (P = 0.003; Fig. 1g). Multivariate Cox regression analysis showed that LINC00958 was an independent prognostic factor for HCC patients [hazard ratio (HR) 2.153, 95% confidence interval (CI) 1.105–4.195, P = 0.024; Fig. 1h and Additional file 4: Table S2].

We performed RT-qPCR and FISH assays and showed that LINC00958 was predominantly located in cytoplasm (Fig. 2a, b). To evaluate the function of LINC00958 in HCC, we stably knocked down the expression of LINC00958 by three shRNAs (sh1-LINC00958, sh2-LINC00958, and sh3-LINC00958) in HCCLM3 and Focus cells. As shown in Fig. 2c, sh2-LINC00958 exhibited the most evident knockdown effect and was chosen for the subsequent experiments. CCK-8 assays demonstrated that silencing LINC00958 significantly reduced the proliferative capabilities of HCCLM3 and Focus cells (Fig. 2d). The inhibitory effects of LINC00958 knockdown on HCC cell proliferation were further confirmed by colony formation and EdU assays (Fig. 2e, f). Transwell assays showed that HCC cells transfected with sh-LINC00958 presented a markedly decreased migration and invasion abilities (Fig. 2g). In addition, we overexpressed LINC00958 via lentivirus in Hep3B and HepG2 cells (Additional file 5: Figure S2A). Based on the results from CCK-8 assays, we observed increased cell growth rates in HCC cells with LINC00958 overexpression (Additional file 5: Figure S2B). We also performed colony formation and EdU assays and showed that LINC00958 overexpression significantly elevated the proliferation of Hep3B and HepG2 cells (Additional file 5: Figure S2C-D). LINC00958 overexpression greatly promoted cell migration and invasion abilities in HCC (Additional file 5: Figure S2E). Collectively, these data indicated that LINC00958 facilitates HCC proliferation and migration in vitro.

Given the cytoplasmic distribution of LINC00958, we hypothesized that LINC00958 might exert its effects via targeting miRNAs. As demonstrated in Fig. 3a, the results from RIP assays using an anti-AGO2 antibody showed that endogenous LINC00958 was preferentially enriched in the AGO2 IP pellet compared to control IgG IP pellet. To explore the underlying regulatory mechanism of LINC00958, we used starBase and miRDB databases and found six miRNAs with potential complementary binding sequences (Fig. 3b and Additional file 6: Supplementary Material 1). AGO2-RIP assays showed that miR-3619-5p was the highest enriched miRNA in the LINC00958-overexpressed group compared to the negative control (NC) group (Fig. 3c) and LINC00958 enrichment was much higher in the miR-3619-5p mimics group compared to the miR-NC group (Fig. 3d). These data suggested that LINC00958 and miR-3619-5p existed in the same RNA-induced silencing complex. Furthermore, we generated a mutant sequence of LINC00958 that could not bind miR-3619-5p for the subsequent luciferase reporter assays (Fig. 3e). As demonstrated in Fig. 3f, miR-3619-5p mimics significantly decreased the luciferase activity in HCC cells transfected with the wildtype LINC00958 sequence, whereas the luciferase activity was not obviously altered in HCC cells transfected with the mutant LINC00958. Subsequently, biotin-labeled miRNA pull-down assays showed significantly increased LINC00958 interaction in the HCC cells transfected with biotin-labeled miR-3619-5p compared to that in the control (Fig. 3g). FISH assays revealed the colocalization of LINC00958 and miR-3619-5p in the cytoplasm in HCC cells (Fig. 3h).

To confirm that LINC00958 exerted the tumor-promoting effects via miR-3619-5p, we upregulated the expression of miR-3619-5p in LINC00958-overexpressed Hep3B cells (Additional file 7: Figure S3A) and conducted rescue experiments. As indicated in Additional file 7: Figure S3B-C, the elevated proliferation ability in LINC00958-overexpressed Hep3B cells was counteracted by miR-3619-5p mimics. Similarly, miR-3619-5p overexpression could rescue the increased cell migration and invasion in Hep3B cells overexpressing LINC00958 (Additional file 7: Figure S3D).

To investigate the targets of miR-3619-5p regulated by LINC00958, we performed bioinformatics analysis using four different algorithms including PicTar, TargetScan, miRDB, and RNA22 (Additional file 8: Supplementary Material 2). Figure 4a shows the overlapping target genes of miR-3619-5p. Expression analysis yielded that only hepatoma-derived growth factor (HDGF) was downregulated in HCCLM3 cells upon LINC00958 knockdown and upregulated in Hep3B cells upon LINC00958 overexpression (Fig. 4b). Furthermore, RNA sequencing was performed to identify the differentially expressed genes in LINC00958-silenced HCC cells. Using DEGseq method, we found that 429 genes were downregulated and 134 genes were upregulated after LINC00958 knockdown (Fig. 4c). Enriched KEGG pathway analysis on RNA sequencing data was shown in Fig. 4d. Notably, HDGF was found downregulated by 2.63-fold after LINC00958 knockdown. We then mutated the miR-3619-5p binding site of the 3′-UTR of HDGF to construct the pmirGLO-HDGF 3′-UTR-MUT vector (Fig. 4e). Luciferase reporter assays indicated that miR-3619-5p mimics significantly decreased the luciferase activity of pmirGLO-HDGF 3′-UTR-WT, but had no effect on the activity of pmirGLO-HDGF 3′-UTR-MUT (Fig. 4f). We then investigated the expression levels of HDGF in HCC cells transfected with miR-3619-5p mimics or inhibitor. In HCCLM3 cells, miR-3619-5p mimics led to a decreased expression level of HDGF. Compared with the control cells, Hep3B cells transfected with miR-3619-5p inhibitor presented a higher level of HDGF expression (Fig. 4g). As demonstrated in both TCGA data (Fig. 4h) and our RT-qPCR results (Fig. 4i), HDGF was remarkably upregulated in HCC tissues. Correlation analysis suggested a negative correlation between the level of miR-3619-5p and HDGF expression level in HCC tissue specimens (Fig. 4j). In addition, the expression level of LINC00958 was positively correlated with HDGF expression level (Fig. 4k).

To confirm that HDGF was the downstream target of LINC00958-mediated HCC progression, we performed the subsequent functional rescue assays. We first overexpressed HDGF in HCCLM3 sh-LINC00958 cells and verified the overexpression efficiency by RT-qPCR and western blotting (Additional file 9: Figure S4A-B). Based on the CCK-8 and EdU assays, we found that HDGF overexpression could rescue the suppressed proliferative capability in HCCLM3 sh-LINC00958 cells (Additional file 9: Figure S4C-D). Transwell assays showed that the inhibited cell motility was restored after overexpressing HDGF in HCCLM3 sh-LINC00958 cells (Additional file 9: Figure S4E). Together, these data indicated the tumor-promoting role of the LINC00958/miR-3619-5p/HDGF axis in HCC.

Mounting evidence indicates that abnormal lipid metabolism plays crucial parts in HCC development [22‐24], and HDGF was recently reported to be a lipogenesis-associated gene in tumorigenesis [25]. We started to explore whether LINC00958 could affect lipogenesis in HCC cells. Pathway enrichment results also revealed that fatty acid metabolism was among the top canonical pathways (Fig. 4d). As shown in Fig. 5a, b, HCCLM3 and Focus cells with LINC00958 knockdown exhibited reduced cellular levels of cholesterol and triglyceride, whereas Hep3B and HepG2 cells overexpressing LINC00958 presented higher cholesterol and triglyceride levels compared with the control cells. HCCLM3 and Focus cells with LINC00958 knockdown showed decreased mRNA levels of several key enzymes in lipogenesis, including SREBP1, FASN, SCD1, and ACC1 (Fig. 5c). In contrast, Hep3B and HepG2 cells with LINC00958 overexpression showed increased SREBP1, FASN, SCD1, and ACC1 mRNA levels (Fig. 5d). Western blotting results confirmed that LIN00958 was positively correlated with the protein levels of SREBP1, FASN, SCD1, and ACC1 in HCC cells (Fig. 5e). To further examine such positive correlation of LINC00959 with lipogenesis in HCC cells, we performed Oil Red O staining in HCC cells with LINC00958 knockdown or overexpression. Lipid droplets comprising mainly triglycerides and sterol esters, as indicated by Oil Red O staining, were less abundant in HCCLM3 cells with LINC00958 knockdown than in the control cells. Conversely, more lipid droplets were observed in Hep3B cells with LINC00958 overexpression than in the control cells (Fig. 5f). Furthermore, more lipid droplets were detected by Oil Red O staining in HCC patient samples with high LINC00958 level compared to those with low LINC00958 expression level (Fig. 5g, h).

To verify that LINC00958 promoted lipogenesis through miR-3619-5p, we performed the subsequent rescue experiments. As demonstrated in Additional file 10: Figure S5A-B, the elevated cholesterol and triglyceride levels in LINC00958-overexpressed Hep3B cells were ameliorated by miR-3619-5p mimics. High SREBP1 mRNA and protein levels in LINC00958-overexpressed Hep3B cells were counteracted by transfecting miR-3619-5p mimics (Additional file 10: Figure S5C-D). Oil Red O staining results suggested that miR-3619-5p overexpression restored the elevated lipid droplets in LINC00958-overexpressed Hep3B cells (Additional file 10: Figure S5E). Likewise, we investigated the effects of HDGF overexpression on lipogenesis in LINC00958-silenced HCCLM3 cells. As shown in Additional file 11: Figure S6A-E, the inhibited cellular cholesterol and triglyceride levels, SREBP1 levels, and lipid droplet levels in LINC00958-silenced HCCLM3 cells were rescued by HDGF overexpression, suggesting that HDGF was the downstream effector of LINC00958-mediated lipogenesis. Together, these data supported that LINC00958 promoted lipogenesis in HCC through the miR-3619-5p/HDGF signaling pathway.

PDX mouse models were utilized to investigate the effects of LINC00958 on HCC growth in vivo. We intratumorally injected recombinant sh-NC, sh-LINC00958, LINC00958, and NC on four groups of PDX mice, respectively (Fig. 6a). Clinical characterization of the donor patients is presented in Fig. 6b. We histopathologically analyzed the engrafted tumors using H&E staining (Fig. 6c). As indicated in Fig. 6d–f, we found that LINC00958 knockdown resulted in a blunted tumor growth in terms of tumor weight and volume, whereas LINC00958 overexpression accelerated tumor growth. We then determined the expression levels of LINC00958 in the four groups of PDX tumors by FISH and RT-qPCR. As shown in Fig. 6g, h, decreased LINC00958 levels were detected in the sh-LINC00958 group, whereas elevated LINC00958 levels were observed in the LINC00958 group. The expression level of HDGF was found downregulated in the sh-LINC00958 group and upregulated in the LINC00958 group (Fig. 6i). The expression levels of HDGF, SERBP1, and Ki67 in the PDX tumors were then examined by immunohistochemistry. As presented, downregulated HDGF, SERBP1, and Ki67 expression levels were detected in the sh-LINC00958 group, while upregulated levels of HDGF, SERBP1, and Ki67 were found in the LINC00958 group (Fig. 6j).

Recent advancements in tumor epigenetic regulation have shed light on the involvement of m⁶A modification in lncRNA [26, 27]. We then wondered whether m⁶A was associated with LINC00958 upregulation in HCC. According to the results from an online bioinformatics database m6Avar [28], we found four RRACU m⁶A sequence motifs in the exon region (at ch11:13001568, 13002361, 13002410, and 13011005). m⁶A RIP-qPCR analysis showed that m⁶A was highly enriched within LINC00958 in Hep3B, HepG2, HCCLM3, and Focus cells (Fig. 7a). METTL3 is a crucial m⁶A methyltransferase and has been reported to be involved in HCC development [29]. RT-qPCR results indicated that METTL3 expression level was positively correlated with the level of LINC00958 in 50 HCC tissues (Additional file 12: Figure S7A). As indicated in Additional file 13: Table S3 and Additional file 12: Figure S7B, high METTL3 expression was associated with tumor differentiation (P = 0.002), tumor size (P = 0.018), microvascular invasion (P = 0.023), TNM stage (P = 0.001), and unfavorable overall survival. Patients in the LINC00958^highMETTL3^high group had the worst survival outcomes compared with other groups, indicating its prognostic value in HCC (Additional file 12: Figure S7C).

To explore the effects of METTL3 on LINC00958 upregulation in HCC, we knocked down the expression of METTL3 using lentivirus in HCCLM3 and Focus cells. As shown in Fig. 7b, c, RT-qPCR and western blotting assays verified the knockdown efficiency. Compared to the control group, the m⁶A level of LINC00958 was lower in METTL3-silenced HCC cells (Fig. 7d). We found that METTL3 downregulation was associated with decreased LINC00958 expression level (Fig. 7e). We then treated HCC cells with actinomycin D to block transcription and found that METTL3 knockdown significantly decreased the half-life of LINC00958 in HCCLM3 and Focus cells (Fig. 7f, g). These data suggested that METTL3-mediated m⁶A is associated with the upregulation of LINC00958 in HCC, probably by regulating the stability of its transcript. In addition, we investigated whether DNA methylation or histone modification could affect LINC00958 in HCC, whereas no significant results were observed (Fig. 7h, i). A graphic illustration of the tumor-promoting role of LINC00958 in HCC is depicted in Fig. 7j.

To investigate the potential utility of LINC00958 as a therapeutic target for HCC, we developed a novel PLGA-based nanoplatform encapsulating si-LINC00958. The double emulsion solvent diffusion method was used to prepare the PEGylated PLGA NPs loaded with si-LINC00958 and spermidine, named as PLGA-PEG(si-LINC00958) NPs hereafter. PEGylation of PLGA improves the stability of NPs in physiological environment by decreasing their interactions with serum proteins [30]. Spermidine can neutralize the charge of the anionic siRNA, turning it less hydrophilic and more likely to be encapsulated into hydrophobic PLGA [16]. As shown in representative TEM image (Fig. 8a), PLGA-PEG(si-LINC00958) NPs were spherical in shape and presented narrow size distributions. DLS were used to measure their size and zeta potential. The average diameter of PLGA-PEG(si-LINC00958) NPs was 170.49 ± 4.45 nm, with a PDI of 0.15 ± 0.01 (Fig. 8b and Additional file 14: Figure S8A). The zeta potential was − 4.85 ± 0.02 mV (Additional file 14: Figure S8B). A negative surface charge has been previously reported to be optimal to achieve a long-lasting in vivo circulation time [31]. The encapsulation efficiency of the PLGA-PEG(si-LINC00958) NPs was 40.8 ± 1.1%.

We then evaluated the in vitro release behavior of si-LINC00958 from PLGA-PEG(si-LINC00958) NPs (Additional file 14: Figure S8C). In the first 24 h, the cumulative release amount of siRNA from free si-LINC00958 and PLGA-PEG(si-LINC00958) NPs was 80.3% and 42.7%, respectively. As time went by, the si-LINC00958 entrapped in NPs was gradually released, and the sustained release continued for approximately 1 week. The data suggested that PLGA-PEG(si-LINC00958) NPs provided controlled release.

The cellular uptake of NPs into HCCLM3 cells was examined by incubating the cells with Courmarin-6 labeled NPs. Fluorescence microscopy revealed that Courmarin-6 NPs exhibited strong fluorescent signal inside the cells compared to the control (Fig. 8c), indicating that NPs increased the cellular drug uptake.

Then, we evaluated the targeting properties of NPs in vivo. Free 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) or DiR-NP was intravenously injected via the tail vein. The fluorescent intensity of the DiR-NP group in tumor was significantly stronger than that of the free DiR group (Additional file 15: Figure S9A-B). The data demonstrated the in vivo tumor-targeting capacity of the NPs.

As shown in Additional file 14: Figure S8D, we verified the knockdown efficiency of PLGA-PEG(si-LINC00958) NPs in HCCLM3 cells. We then performed CCK-8 assays to assess the in vitro antitumor capability of PLGA-PEG(si-LINC00958) NPs. The results showed that PLGA-PEG(si-LINC00958) NPs could decrease the proliferative ability of HCC cells (Additional file 14: Figure S8E).

We treated the HCC PDX model by injecting PLGA-PEG(siRNA control) NPs or PLGA-PEG(si-LINC00958) NPs via the tail vein. Tumor growth was significantly inhibited following treatment with PLGA-PEG(si-LINC00958) NPs compared with PLGA-PEG(siRNA control) NPs (Fig. 8d). The xenograft tumor weight and volume were markedly reduced in mice injected with PLGA-PEG(si-LINC00958) NPs (Fig. 8e, f). RT-qPCR results confirmed the consistent knockdown of LINC00958 in xenografts derived from mice treated with PLGA-PEG(si-LINC00958) NPs (Fig. 8g). Downregulated expression levels of HDGF and SREBP1 in xenograft tumors were verified by western blotting (Fig. 8h). Survival analysis showed that systemic administration of PLGA-PEG(si-LINC00958) NPs remarkably prolonged the mouse overall survival (Fig. 8i).

We evaluated systemic toxicity by H&E staining and showed that intravenous administration of PLGA-PEG(si-LINC00958) NPs exhibited no significant toxicity to major organs including the liver, kidney, lung, spleen, and heart (Fig. 8j). In addition, we performed blood index analyses of ALT, AST, Cr, and BUN and confirmed the absence of significant hepatotoxicity and renotoxicity (Fig. 8k–n).