Our study utilized two sets of HCC samples. The first set comprised 92 paired HCC tissues, which were analyzed for LINC01607 expression, and the patient characteristics of the LINC01607-high and LINC01607-low groups are detailed in Table 1. The second set included freshly collected surgical specimens used to establish organoid cultures for in vitro assessment of various interventions. Six independent HCC patient-derived organoid (PDO) lines were successfully generated and included in the present study. Baseline LINC01607 expression was measured in each PDO line by quantitative reverse transcription polymerase chain reaction (qRT-PCR) before the intervention experiments. All procedures involving human participants were approved by the Ethics Committee of Tongji Hospital (approval No. TJ-IRB20230863) and were conducted in accordance with the Declaration of Helsinki. For the retrospective analysis of archived clinical specimens and de-identified clinical data, a waiver of informed consent was granted by the ethics committee. For prospectively collected fresh clinical specimens used to establish PDO models, written informed consent was obtained from the corresponding patients. All animal experiments were approved by the Institutional Ethics Committee of Tongji Hospital (approval No. TJH-202212001) and were conducted in accordance with institutional animal welfare and ethical guidelines.

Hep3B and Huh7 cells were used for in vitro functional and mechanistic experiments. LINC01607 knockdown or overexpression, SQSTM1/p62 overexpression, and Nrf2 knockdown were performed as indicated. qRT-PCR, fluorescence in situ hybridization (FISH), western blotting, lipid ROS measurement, C11-BODIPY staining, intracellular Fe²⁺ measurement, and GSH assays were conducted to evaluate LINC01607 expression, pathway alterations, and ferroptosis-associated changes. Detailed experimental procedures, reagent information, and siRNA/shRNA sequences are provided in Supplementary Methods and Supplementary Tables 1 and 2.

Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation, colony formation, Transwell, and wound-healing assays were performed to evaluate cell proliferation, migration, and invasion. Drug-sensitivity assays were used to assess the effects of LINC01607 modulation on responses to sorafenib, RSL3, and ferroptosis inhibitors. Detailed procedures are described in Supplementary Methods.

Male BALB/c nude mice were used to evaluate tumor growth and metastatic potential in vivo. Hep3B cells with LINC01607 overexpression, knockdown, or corresponding controls were used to establish subcutaneous, orthotopic, and metastasis models as indicated. Tumor growth, tumor weight, bioluminescence signals, and histological changes were analyzed. All animal experiments were approved by the Institutional Ethics Committee of Tongji Hospital and performed in accordance with the ARRIVE guidelines (TJH-202212001). Detailed procedures are provided in Supplementary Methods.

Fresh HCC tissues were used to establish PDO models. Organoid identity was evaluated by histological and immunohistochemical analyses, and organoid viability was assessed after LINC01607 silencing, sorafenib treatment, or combined intervention. Detailed culture conditions and medium components are provided in Supplementary Methods and Supplementary Table 3.

Total RNA was isolated from Hep3B cells transfected with siLINC01607 or negative control siRNA. Three independent biological replicates were included in each group. RNA sequencing and downstream bioinformatic analyses were performed to identify differentially expressed genes and enriched pathways. Detailed sequencing procedures are provided in Supplementary Methods.

Statistical analyses were performed using SPSS 22.0. Data are presented as the mean ± standard deviation (SD). Comparisons between groups were performed using Student’s t-test, Mann–Whitney U test, chi-square test, or repeated-measures analysis of variance (ANOVA), as appropriate. Survival analyses were performed using Kaplan–Meier analysis, log-rank testing, and Cox proportional hazards regression models. Statistical significance was set at P < 0.05.

Based on our previous gene expression profiling data, we further evaluated LINC01607 expression in paired HCC and adjacent peritumor liver tissues by qRT-PCR and FISH. LINC01607 levels were substantially higher in HCC specimens than in matched adjacent nontumor tissues. Elevated LINC01607 expression predicted shorter overall survival in both the Tongji cohort and The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset [Figure 1A-C]. Further clinicopathological analysis showed that high LINC01607 expression was associated with poor differentiation, microvascular invasion, and advanced Tumor–Node–Metastasis (TNM) stage [Figure 1D]. Cox proportional hazards analysis further revealed that elevated LINC01607 levels, in addition to these clinical factors, were independent predictors of reduced overall survival [Figure 1E]. Overall, these results identify LINC01607 as an upregulated lncRNA in HCC that is associated with unfavorable clinicopathological and survival outcomes.

To investigate the functional role of LINC01607 in HCC, we performed silencing and overexpression experiments in the Hep3B and Huh7 cell lines. Successful transfection was confirmed by qRT-PCR [Supplementary Figure 1A and B]. Cell growth was assessed using CCK-8, colony formation, and EdU assays. LINC01607 overexpression increased proliferative capacity, whereas LINC01607 silencing suppressed cell growth [Figure 2A-C]. In Transwell and wound-healing assays, cells with elevated LINC01607 displayed stronger migration and invasion, while LINC01607 knockdown weakened these phenotypes [Figure 3A and B].

In nude mouse models, stable overexpression of LINC01607 in Hep3B cells resulted in larger tumors, with significantly increased tumor volume and weight compared with control groups [Figure 2D and E]. Immunohistochemistry (IHC) revealed increased Ki-67 expression in tumors with LINC01607 overexpression [Figure 2F]. Moreover, a lung metastasis model using luciferase-labeled cells confirmed that LINC01607 overexpression markedly enhanced metastatic potential [Figure 3C]. Together, these findings suggest that LINC01607 contributes to HCC cell proliferation and metastatic capacity.

To investigate transcriptional changes associated with LINC01607 depletion, we conducted RNA-seq analysis in control and LINC01607-knockdown Hep3B cells. This analysis identified 253 upregulated and 233 downregulated genes (|log2 fold change| > 1 and P < 0.05) [Figure 4A]. Functional enrichment analysis showed changes in antioxidant-associated biological processes and highlighted ferroptosis-related pathway enrichment in LINC01607-knockdown cells [Figure 4B].

Functional rescue assays showed that ferrostatin-1 and liproxstatin-1, but not Z-VAD-FMK, necrostatin-1, or Baf-A1, alleviated the growth-suppressive effect of LINC01607 knockdown [Figure 4C and Supplementary Figure 2A]. LINC01607 silencing also increased relative Fe²⁺ accumulation under erastin treatment compared with control cells [Figure 4D]. Therefore, we hypothesized that ferroptosis might be a significant determinant of LINC01607 knockdown-induced cell death, and we measured several ferroptotic events. We observed increased GSH depletion [Figure 4E] and lipid peroxidation [Figure 4F and Supplementary Figure 2B] upon LINC01607 knockdown. Transmission electron microscopy (TEM) of LINC01607-depleted cells revealed characteristic ferroptotic features, such as shrunken mitochondria with increased membrane density [Figure 4G]. Using C11-BODIPY staining, we observed a red-to-green fluorescence shift, which is indicative of lipid peroxidation in LINC01607-silenced cells [Figure 4H and Supplementary Figure 2C].

Ferroptosis is a complex process involving the modulation of multiple genes. To elucidate the downstream targets of LINC01607 that affect the cellular phenotype of HCC and ferroptosis, Western blot analysis showed that LINC01607 knockdown was associated with reduced expression of key ferroptosis-resistance proteins, including SQSTM1/p62, NRF2, NQO1, HO-1, GPX4, and SLC7A11. Conversely, LINC01607 overexpression increased the expression of these proteins [Figure 4I].

To determine whether ferroptosis was involved in the effects of LINC01607 depletion, ferrostatin-1 rescue experiments were performed. Ferrostatin-1 partially attenuated the inhibitory effects of LINC01607 knockdown on cell proliferation and metastatic phenotypes, as shown by CCK-8 and Transwell assays [Figure 4J and Supplementary Figure 2D; Figure 4K and Supplementary Figure 2E]. These findings suggest that LINC01607 depletion promotes a ferroptosis-associated phenotype in HCC cells.

To identify downstream pathways associated with LINC01607-mediated ferroptosis resistance in HCC cells, we further analyzed RNA-sequencing data from LINC01607-knockdown cells and reported that SQSTM1 (p62), a selective autophagy receptor, was among the downregulated genes [Figure 5A]. Compared with adjacent normal tissues, HCC specimens showed elevated p62 expression [Figure 5B]. SQSTM1/p62 has been reported to influence Nrf2 signaling by promoting Keap1 turnover, thereby facilitating the expression of antioxidant and ferroptosis-resistance genes such as SLC7A11, GPX4, HO-1, and NQO1^[23]. Through this antioxidant program, Nrf2 may help limit lipid peroxidation and ferroptosis. We next overexpressed p62 in LINC01607-downregulated HCC cells. Western blot analysis confirmed that LINC01607 knockdown was accompanied by increased Keap1 levels and reduced NRF2, NQO1, HO-1, GPX4, and SLC7A11 levels, whereas p62 overexpression partially reversed these changes. Consequently, the expression of downstream antioxidant and ferroptosis resistance proteins decreased again after Nrf2 depletion [Figure 5C]. In addition, SQSTM1/p62 mRNA expression changed in parallel with p62 protein levels following LINC01607 perturbation. Consistent with the protein data, p62 overexpression reversed both the decrease in GSH levels [Figure 5D] and the increase in lipid ROS levels detected with BODIPY 581/591 C11 [Figure 5E]; both effects were reversed when Nrf2 was subsequently silenced.

These findings support a functional connection between LINC01607 and the p62–Keap1–Nrf2 pathway but do not establish a direct molecular mechanism by which LINC01607 regulates SQSTM1/p62 expression.

Given that LINC01607 depletion promoted ferroptosis-associated phenotypes, we next asked whether this increase in ferroptosis vulnerability translated to altered drug sensitivity. We first examined the response to sorafenib, a multikinase inhibitor with ferroptosis-related activity, and then assessed the sensitivity to RSL3, a canonical ferroptosis inducer. Evaluation of sorafenib sensitivity by cell viability and IC50 analysis revealed that cells with elevated LINC01607 expression were less sensitive to sorafenib, whereas LINC01607 depletion increased sorafenib sensitivity [Figure 6A]. To test whether the effect of LINC01607 was not restricted to sorafenib alone, we next examined the response to RSL3. LINC01607 depletion significantly increased sensitivity to RSL3, as reflected by reduced cell viability. These data support that LINC01607 modulates ferroptosis-related drug sensitivity beyond sorafenib [Figure 6B]. To determine whether this phenotype was linked to ferroptosis, we performed rescue experiments under sorafenib treatment using ferrostatin-1. The ferroptosis inhibitor partially reversed the enhanced sorafenib sensitivity induced by LINC01607 depletion, indicating that ferroptosis contributes, at least in part, to this phenotype [Figure 6C]. In vivo, xenograft tumors derived from LINC01607-depleted cells showed an enhanced response to sorafenib [Figure 6D]. Hematoxylin and eosin (H&E) staining and IHC analysis were used to evaluate p62, KEAP1, NRF2, GPX4, and HO-1 expression in subcutaneous tumor tissues. LINC01607 knockdown and sorafenib treatment were associated with reduced p62, NRF2, GPX4, and HO-1 expression and increased KEAP1 expression. Moreover, the combination of silencing LINC01607 and sorafenib further changed the indicator levels [Figure 6E]. Under sorafenib treatment, viability of PDOs positively correlated with baseline LINC01607 expression levels across PDO lines [Figure 6F]. After LINC01607 was depleted, the organoid diameter decreased, and the cell survival rate decreased; combined treatment with sorafenib further decreased the organoid diameter and cell survival rate [Figure 6G]. Together, these findings indicate that LINC01607 depletion increases sorafenib sensitivity in association with increased ferroptosis vulnerability.