Zingiberensis New Saponin Inhibits LncRNA TCONS-00026762/AKR1C1 Pathway, Revealing Unique Insights into Cellular Processes and Drug Response in Hepatocellular Carcinoma

Zingiberensis New Saponin Inhibits LncRNA TCONS-00026762/AKR1C1 Pathway, Revealing Unique Insights into Cellular Processes and Drug Response in Hepatocellular Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zingiberensis New Saponin Inhibits LncRNA TCONS-00026762/AKR1C1 Pathway, Revealing Unique Insights into Cellular Processes and Drug Response in Hepatocellular Carcinoma Liang Luo, Keqing He, Pingsheng Zhou, Xing Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4315084/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Long non-coding RNAs (lncRNAs) are often involved in regulating various cellular processes during cancer progression. This study aimed to investigate the role of Zingiberensis new saponin (ZnS) in hepatocellular carcinoma (HCC) cells through the lncRNA TCONS-00026762/AKR1C1 pathway. Methods Bioinformatics analysis was initially used to assess the prognostic significance of AKR1C1 in TCGA liver cancer data. Huh7 and Huh7-SR cells were either transfected with sh-TCONS-0026762 and oe-AKR1C1 or treated with ZnS and oe-TCONS-00026762. The expression of TCONS-00026762 and AKR1C1 was quantified using quantitative real-time PCR. The effects of either TCONS-00026762 knockdown or ZnS treatment on autophagy, ferroptosis, and drug sensitivity were investigated using a combination of immunofluorescence staining, western blot, and CCK-8 assays. Results Bioinformatics analysis revealed that AKR1C1 is a prognostic marker for HCC and is association with autophagy, ferroptosis, and immune evasion. Knockdown of TCONS-00026762 suppressed autophagy, promoted ferroptosis, and enhanced sensitivity to sorafenib in HCC cells, as evidenced by the decrease in levels of the autophagy marker LC3, as well as ferroptosis markers GPX4 and SLC7A11, and an increase in Huh7-SR cell viability. However, these changes were reversed by overexpression of AKR1C1. Moreover, ZnS treatment significantly downregulated the expression of TCONS-00026762 and AKR1C1, leading to inhibition of autophagy, induction of ferroptosis, and increased susceptibility of HCC cells to sorafenib. Notably, these effects were reversible upon the overexpression of TCONS-00026762. Conclusions Our findings suggest that ZnS inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells through the lncRNA TCONS-00026762/AKR1C1 pathway. autophagy ferroptosis Hepatocellular carcinoma lncRNA TCONS-00026762/AKR1C1 pathway Zingiberensis new saponin Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Liver cancer is a significant burden on global cancer incidence. Over the past few decades, the incidence of this disease has been steadily increasing in many countries ( 1 ). It is anticipated that globally, between 2020 and 2040, there will be a 55% increase in new cases of liver cancer, resulting in an estimated 1.4 million new cases by 2040 ( 2 ). Hepatocellular carcinoma (HCC) represents the dominant type of liver cancer, contributing to around 90% of all incidences. ( 3 ). Owing to the difficulty in detecting the symptoms and physical characteristics of HCC, more than 80% of patients are unable to undergo curative treatment upon diagnosis ( 4 ). Despite extensive efforts in the past decade to develop novel drugs and treatment strategies, there has been limited progress in the treatment of HCC ( 5 ). Consequently, the exploration of novel treatment approaches and drugs holds significant importance for the treatment of HCC. Increasing evidence is underscoring the critical role that long non-coding RNAs (lncRNAs) play in the onset, progression, and spread of various cancers, including HCC ( 6 – 8 ). For instance, the lncRNA RP11-620J15.3 is known to boost HCC cell multiplication and metastasis by targeting miR-326/GPI, thereby enhancing glycolysis ( 6 ). Furthermore, mounting evidence is showing a close correlation between the expression of lncRNAs and various forms of cell death, including autophagy and ferroptosis ( 9 – 11 ). For example, in breast cancer, metformin induces ferroptosis by suppressing autophagy through the lncRNA H19 ( 11 ). Our previous study found that Zingiberensis new saponin (ZnS) inhibits HCC growth by downregulating the expression of a novel LncRNA TCONS-00026762 ( 12 ); however, the exact mechanism by which LncRNA TCONS-00026762 inhibits HCC growth is unclear. LncRNA TCONS-00026762 is located on chromosome 10p15.1 and significantly overlaps with the gene for aldo-keto reductase family 1 member C1 (AKR1C1), a protein reported to be overexpressed in several cancers, including HCC ( 13 ). Moreover, AKR1CI is not only an autophagy-regulated joint protein but has also been implicated in the regulation of ferroptosis and drug sensitivity ( 14 – 17 ). Bioinformatics analysis suggests that AKR1C1 may serve as a prognostic biomarker for HCC. Moreover, it presents itself as a compelling target for the regulation of autophagy, ferroptosis, and drug sensitivity in HCC cells. Based on these insights, we hypothesize that ZnS could modulate these cellular processes in HCC through the lncRNA TCONS-00026762/AKR1C1 pathway, thereby inhibiting the malignant progression of the disease. Given the above background, this study aims to determine whether ZnS regulates autophagy, ferroptosis, and drug sensitivity in HCC cells through the lncRNA TCONS-00026762/AKR1C1 pathway. We aim to provide new mechanistic insights and identify potential therapeutic targets for the comprehensive treatment of HCC. Methods Bioinformatics analysis First, GEPIA ( http://gepia.cancer-pku.cn/ ) was utilized to analyze the survival rate of AKR1C1 in TCGA liver cancer data. Then, we downloaded the TCGA transcriptomic and clinical data from UCSC Xena to analyze the differential expression of AKR1C1 across different tumor stages. Next, in the liver cancer TCGA dataset, samples were categorized into high-expression and low-expression groups based on the median expression level of AKR1C1. Subsequently, we selected 26 autophagy-related and 5 ferroptosis-related gene sets from the MSigDB and then employed gene set variation analysis (GSVA) to compute the pathway activity scores for each sample in the HCC dataset from CGA. Following this, we conducted a t-test to analyze the relationship between these pathway activity scores and the expression levels of AKR1C. Additionally, we analyzed the correlation between AKR1C1 and the immune spot (PD1) using Pearson’s correlation analysis. Cell culture and treatment This study employed the human HCC cell lines Huh7 and SMMC-7721, along with the human normal liver cell line HL-7702. These cell lines, sourced from COBIOER BIOSCIENCES CO., LTD (Nanjing, China), were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, NY, USA), supplemented with 10% fetal bovine serum (FBS; Gibco). All cell culture practices were executed in a humidified incubator set at 37°C with 5% CO 2 . Sorafenib-resistant cells Huh7 (Huh7-SR) were established through long-term exposure to sorafenib, starting at an initial concentration of 0.5 µM and gradually escalating the dose up to 10 µM. Huh7 or Huh7-SR cells underwent treatment with 5 µM sorafenib for 24 h and/or 1 µM ZnS for 48 h. Huh7 cells treated with the equal amount of dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) served as the controls. Cell transfection Huh7 and Huh7-SR cells were seeded into 96-well or 6-well plates and allowed to reach 80% confluence. Following this, lentiviral vectors engineered for the knockdown of TCONS-00026762 (sh-TCONS-00026762-1/2), as well as for the overexpression of AKR1C1 (oe-AKR1C1) and TCONS-00026762 (oe-TCONS-00026762), were transfected into the cells, alongside their respective negative controls (sh-NC or oe- NC). Transfections were performed using HighGene transfection reagent (ABclonal, Wuhan, China) and allowed to proceed for 48 h, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine transfection efficiency. ELISA To evaluate the ferroptosis level in different groups, the evaluation of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron levels were detected in each group. The contents of MDA, 4-HNE, and iron in cell lysates were assessed using MDA Assay Kit (Solarbio; BC0020), 4-HNE Assay Kit (Elabscience, Wuhan, China; E-EL-0128c) and Iron Assay Kit (Solarbio; #BC5315) according to the manufacturer’s instructions, respectively. qRT-PCR qRT-PCR assay was conducted following a reported method ( 12 ). The specific primers used included TCONS-00026762 (forward: 5’-AAT GAG GAG CAG GTT GGA CT-3’, reverse: 5’-GAT CAC TTC CTC ACC TGG CT-3’), AKR1C1 (forward: 5’-TTT GGC ACC TAT GCG CCT G-3’, reverse: 5’-CAG AAT CAA TAT GGC GGA AGC CAG-3’), and the housekeeping gene GAPDH (forward: 5’-GGT GAA GGT CGG AGT CAA CG-3’, reverse: 5’-CAA AGT TGT CAT GGA TGA ACC-3’). Cell viability and cell apoptosis As per previously described methods ( 12 ), the viability and apoptosis levels of Huh7 or Huh-SR cells were assessed using the CCK-8 assay and flow cytometry, respectively. Cell migration Cells underwent trypsin digestion and were resuspended in serum-free culture medium, adjusting the cell density to 1×10 6 /mL. Then, 200 µL of cells were pipetted into the upper chamber of the Transwell insert, while the lower chamber was filled with 600 µL of FBS. After incubating for 24 h, cells were subjected to a 30 min formaldehyde fixation and subsequently stained with 0.1% crystal violet for 20 min. Any non-migratory cells on the upper surface were wiped off. Cell counting was performed by observing three random fields under a microscope (Olympus, Japan). Immunofluorescence staining Huh7 cells were plated onto sterilized coverslips situated in a 12-well plate. Post removal of the culture medium, cells were fixed with a 3% paraformaldehyde solution for 15 min and permeabilized with 1% Triton-X 100 for 10 min. After blocking non-specific binding sites with 3% bovine serum albumin for 30 min, the coverslips were incubated at 4°C overnight with a primary antibody against LC3 (Cell Signaling Technology, MA, USA; 4108), at a dilution of 1:100. Subsequently, a secondary antibody (ABclonal Technology; AS011) was introduced to the samples at a dilution of 1:500, along with a 4',6-diamidino-2-phenylindole staining solution for nuclear counterstaining. The immunofluorescence signals were captured using a laser scanning confocal microscope (Perkin Elmer, MA, USA). Western blot analysis The expression of LC3, GPX4, and SLC7A11 in cell samples was evaluated using western blot analysis as previously described ( 18 ). For primary antibody incubation, anti-LC3 antibody (Cell Signaling Technology; 4108; dilution of 1:1,000), anti-glutathione peroxidase 4 (GPX4) antibody (Affinity, OH, USA; DF6701; dilution of 1:1,000), anti-solute carrier family 7a member 11 (SLC7A11) antibody (Affinity; DF12509; dilution of 1:1,000), and anti-GAPDH antibody (Abcam, UK; ab9485; dilution of 1:2,500) were applied. Data analysis All data are displayed as mean ± standard deviation. Statistical analysis was performed using GraphPad 7.0 software (GraphPad Software, Inc., CA, USA), with one-way analysis of variance and Tukey's test employed to compare differences among groups. Statistical significance was set at a p -value of less than 0.05. Results AKR1C1 could be a prognostic marker for HCC To investigate the role of the LncRNA TCONS-00026762/AKR1C1 pathway in the treatment of HCC, we employed bioinformatics techniques to delineate the mechanistic involvement of AKR1C1 in HCC pathogenesis. Bioinformatic analysis revealed a significant upregulation of AKR1C1 in the HCC tumor cohort when compared to the normal group (t-test, P < 0.05; Fig. 1 A). Intriguingly, HCC patients characterized by lower AKR1C1 expression levels exhibited longer overall survival as opposed to those with elevated expression levels (Log-rank test, P < 0.05; Fig. 1 B). Further, differential expression analysis disclosed that AKR1C1 was overexpressed in early-stage tumors (stages 1–2) as compared to late-stage tumors (stages 3–4) (t-test, P < 0.05; Fig. 1 C). Collectively, these findings underscore the potential of AKR1C1 as a prognostic marker for HCC. Complementing these observations, existing literatures have implicated AKR1C1 in a myriad of cellular processes, including cancer autophagy, ferroptosis, and chemoresistance. In our study, we ascertained that heightened expression levels of AKR1C1 significantly influenced the activity scores across 16 distinct autophagy-related pathways and one ferroptosis-related pathway, as cataloged in Table 1 . Additionally, Pearson’s correlation analysis yielded a notable negative association between AKR1C1 and PD1 ( P = 0.00026; Fig. 1 D). Considering these empirical insights and bioinformatic interpretations, we hypothesize that targeted therapeutic interventions against AKR1C1 in HCC may be intricately associated with modulating autophagy, ferroptosis, and chemotherapy sensitivity. Table 1 Identification of the pathways significantly related to autophagy and ferroptosis between high and low AKR1C1 expression groups through gene set variation analysis Pathway Group1 Group 2 P value Method GOBP_LYSOSOMAL_MICROAUTOPHAGY AKR1C1_high AKR1C1_low 0.031 T-test GOBP_POSITIVE_REGULATION_OF_MACROAUTOPHAGY AKR1C1_high AKR1C1_low 0.03 T-test GOBP_CHAPERONE_MEDIATED_AUTOPHAGY AKR1C1_high AKR1C1_low 0.038 T-test GOBP_NEGATIVE_REGULATION_OF_AUTOPHAGY_OF_MITOCHONDRION AKR1C1_high AKR1C1_low 0.015 T-test REACTOME_LATE_ENDOSOMAL_MICROAUTOPHAGY AKR1C1_high AKR1C1_low 0.018 T-test WP_AUTOPHAGY AKR1C1_high AKR1C1_low 0.012 T-test KUMAR_AUTOPHAGY_NETWORK AKR1C1_high AKR1C1_low 0.0028 T-test GOBP_REGULATION_OF_AUTOPHAGY AKR1C1_high AKR1C1_low 0.0067 T-test GOBP_NEGATIVE_REGULATION_OF_AUTOPHAGY AKR1C1_high AKR1C1_low 0.0014 T-test GOBP_POSITIVE_REGULATION_OF_AUTOPHAGY AKR1C1_high AKR1C1_low 0.0054 T-test GOBP_NEGATIVE_REGULATION_OF_MACROAUTOPHAGY AKR1C1_high AKR1C1_low 0.0052 T-test GOBP_AUTOPHAGY_OF_PEROXISOME AKR1C1_high AKR1C1_low 0.0017 T-test GOBP_REGULATION_OF_AUTOPHAGY_OF_MITOCHONDRION_IN_RESPONSE_TO_MITOCHONDRIAL_DEPOLARIZATION AKR1C1_high AKR1C1_low 0.0015 T-test REACTOME_AUTOPHAGY AKR1C1_high AKR1C1_low 0.0016 T-test REACTOME_SELECTIVE_AUTOPHAGY AKR1C1_high AKR1C1_low 0.0053 T-test WP_CLOCKCONTROLLED_AUTOPHAGY_IN_BONE_METABOLISM AKR1C1_high AKR1C1_low 0.0028 T-test Ferroptosis AKR1C1_high AKR1C1_low 0.02 T-test TCONS-00026762 knockdown inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells by reducing AKR1C1 expression To corroborate the findings from our bioinformatics analysis, we assessed the impact of the LncRNA TCONS-00026762/AKR1C1 axis on autophagy, ferroptosis, and sensitivity to sorafenib in HCC cells. Initially, the expression of TCONS-00026762 and AKR1C1 were upregulated in HCC cell lines (Huh7 and SMMC-7721 cells) compared to normal live line (HL-7702 cells) ( P < 0.01; Fig. 2 A). Since the expression levels of TCONS-00026762 andAKR1C1 were slightly higher in Huh7 cells than in SMMC-7721 cells, Huh7 cells were selected for subsequent assays (Fig. 2 A). Then, transfection with sh-TCONS-00026762-1 and sh-TCONS-00026762-2 led to a marked decrease in the relative mRNA expression of both TCONS-00026762 and AKR1C1 in Huh7 cells ( P < 0.01; Fig. 2 B). Given slightly lower TCONS-00026762 and AKR1C1 expression levels in sh-TCONS-00026762-1-transfecetd Huh7 cells than TCONS-00026762-2-transfecetd cells (Fig. 2 B), the sh-TCONS-00026762-1 lentiviral plasmid was chosen for subsequent experiments. Notably, the viability of Huh7 cells was compromised following sh-TCONS-00026762 transfection, a decrement that was effectively reversed upon AKR1C1 overexpression ( P < 0.01; Fig. 2 C). Furthermore, immunofluorescence staining revealed that sh-TCONS-00026762 substantially attenuated the fluorescence signal corresponding to LC3, an autophagy marker ( P < 0.01; Fig. 2 D). This attenuation was counteracted by the introduction of oe-AKR1C1 ( P < 0.01; Fig. 2 D). Concordantly, western blot analysis of LC3 protein levels corroborated the results obtained through immunofluorescence staining ( P < 0.01; Fig. 2 E). Collectively, these data suggest that TCONS-00026762 knockdown inhibits autophagy in HCC cells by downregulating AKR1C1 expression. In terms of ferroptosis, sh-TCONS-00026762-mediated elevations in the contents of MDA, 4-HNE, and iron were significantly alleviated by AKR1C1 overexpression ( P < 0.01; Fig. 3 A). Similarly, knockdown of TCONS-00026762 led to a marked downregulation in the two key ferroptosis regulators, GPX4 and SLC7A11 ( P < 0.01; Fig. 3 B), and lowered levels of them are commonly deemed as ferroptosis indicators ( 19 , 20 ). However, the levels of GPX4 and SLC7A11 were restored by AKR1C1 overexpression ( P < 0.01; Fig. 3 B). Taken together, these findings indicate that knockdown of TCONS-00026762 promotes ferroptosis in HCC cells by decreasing AKR1C1 expression. Lastly, the Huh7-SR group exhibited elevated levels of TCONS-00026762 and AKR1C1 mRNA expression in comparison to the control group ( P < 0.05; Fig. 3 C). Concurrently, overexpression of AKR1C1 mitigated the decreased viability of Huh7-SR cells induced by TCONS-00026762 knockdown in the context of sorafenib treatment ( P < 0.01; Fig. 3 D). These cumulative data imply that knockdown of TCONS-00026762 enhances the sensitivity of sorafenib-resistant HCC cells to sorafenib treatment by reducing AKR1C1 expression. ZnS inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells by blocking the TCONS-00026762/AKR1C1 axis To investigate the role of ZnS in modulating autophagy, ferroptosis, and sorafenib sensitivity in HCC cells via the lncRNA TCONS-00026762/AKR1C1 pathway, we treated Huh7 cells with ZnS, either in the presence or absence of oe-TCONS-00026762. Initially, we assessed the impact of ZnS on the expression levels of TCONS-00026762 and AKR1C1. qRT-PCR analysis revealed that ZnS treatment led to a notable reduction in the expression of both TCONS-00026762 and AKR1C1 when compared to untreated controls ( P < 0.01; Fig. 4 A). This downregulation was effectively counteracted by the overexpression of TCONS-00026762 ( P < 0.01; Fig. 4 A). Following this, ZnS was observed to significantly diminish both cell viability ( P < 0.01; Fig. 4 B) and migratory capabilities of Huh7 cells ( P < 0.01; Fig. 4 D), while concurrently enhancing apoptosis ( P < 0.01; Fig. 4 C). Interestingly, these effects of ZnS were abolished upon oe-TCONS-00026762 transfection ( P < 0.01; Fig. 4 B-D). Importantly, overexpression of TCONS-00026762 effectively reversed the ZnS-mediated downregulation of LC3 ( P < 0.01; Fig. 4 E-F). In relation to ferroptosis, TCONS-00026762 overexpression significantly attenuated the ZnS-induced increases in levels of MDA, 4-HNE, and iron, while simultaneously reversing the decreased GPX4 and SLC7A11 expression ( P < 0.01; Fig. 4 G-H). Additionally, the ZnS-induced reduction in the viability of sorafenib-resistant Huh7-SR cells was also significantly nullified by TCONS-00026762 overexpression ( P < 0.01; Fig. 4 I). Discussion The burgeoning role of lncRNAs in carcinogenesis has been evident in recent years, with increasing literature supporting their potential as therapeutic targets ( 6 – 8 ). Our study uniquely advances this field by investigating the anticancer effects of ZnS through the modulation of autophagy and possibly ferroptosis and drug sensitivity in HCC cells, via the lncRNA TCONS-00026762/AKR1C1 pathway. Firstly, AKR1C1 was found to be notably upregulated in HCC cells, with higher expression associated with lower survival rates in patients. These findings echo earlier studies underscoring the potential of AKR1C1 as a predictive biomarker for cancer prognosis ( 21 , 22 ). Notably, the observation of higher AKR1C1 expression in early-stage tumors suggests that this marker might be implicated in the early onset of HCC, warranting further exploration in future research. Autophagy is a cellular survival mechanism that prevents cellular damage and promotes survival under conditions of energy or nutrient deprivation, as well as in response to various cytotoxic insults ( 23 ). Qu et al reported that PNO1 promotes autophagy and inhibits apoptosis in HCC cells via the MAPK signaling pathway ( 24 ). Ferroptosis is a form of regulated cell death that involves iron-dependent lipid peroxidation. Emerging evidence suggests that ferroptosis plays a critical role in cancer biology, including inhibiting cancer progression ( 16 ). In HCC, targeting ferroptosis has shown potential in controlling tumor growth and enhancing the efficacy of existing treatments ( 25 ). Chemoresistance is a significant barrier to effective cancer treatment. Sorafenib is the only targeted drug for the treatment of advanced HCC ( 26 ). Resistance to chemotherapeutic agents like sorafenib is a major clinical challenge, and understanding the underlying mechanisms is crucial for developing effective treatment strategies ( 27 ). Furthermore, our bioinformatics analysis confirmed the involvement of AKR1C1 in modulating autophagy, ferroptosis, and chemoresistance during the progression of HCC. Intriguingly, the lncRNA TCONS-00026762 significantly overlaps with the AKR1C1 gene, leading us to hypothesize a parallel function for lncRNA TCONS-00026762. Our subsequent experiments revealed that TCONS-00026762 and AKR1C1 were upregulated in HCC cell lines, further supporting the involvement of dysregulated lncRNAs in HCC pathogenesis ( 28 ). Moreover, knockdown of TCONS-00026762 led to reduced expression of both AKR1C1 and the autophagy marker LC3. Furthermore, the knockdown of TCONS-00026762 was associated with a significantly decline in cellular levels of MDA, 4-HNE, and iron, as well as decreased expression of ferroptosis markers GPX4 and SLC7A11. Importantly, we observed a notable reduction in cell viability in Huh7-SR cells upon TCONS-00026762 knockdown. Most significantly, overexpression of AKR1C1 counteracted these effects, aligning our findings with existing literature and thereby validating our initial hypothesis ( 14 , 18 , 29 , 30 ). In our previous report, we demonstrated that ZnS could inhibit the malignant progression of HCC while concurrently downregulating the expression levels of lncRNA TCONS-00026762 ( 12 ). Crucially, our findings demonstrated that the lncRNA TCONS-00026762/AKR1C1 pathway is involved in mediating the effects of ZnS on regulation of autophagy, ferroptosis, and drug sensitivity in HCC cells. Overexpression of TCONS-00026762 attenuated the ZnS-mediated downregulation of TCONS-00026762, AKR1C1, LC3, GPX4, and SLC7A11 as well as a decrease in the viability of Huh7-SR cells. These observations provide mechanistic insights into the role of this pathway in modulation of autophagy, ferroptosis, and chemoresistance. Conclusions In conclusion, our study enriches the existing body of literature by detailing the modulation of autophagy, and potentially ferroptosis and drug sensitivity, through the lncRNA TCONS-00026762/AKR1C1 pathway in HCC cells. It underscores the prognostic and therapeutic potential of targeting this pathway, opening new avenues for further research and drug development in HCC. Abbreviations HCC: Hepatocellular carcinoma; lncRNAs: long non-coding RNAs; ZnS: Zingiberensis new saponin; AKR1C1: aldo-keto reductase family 1 member C1; qRT-PCR: quantitative real-time polymerase chain reaction; MDA: malondialdehyde; 4-HNE: 4-hydroxynonenal Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Science and Technology Research Project of Jiangxi Provincial Department of Education [GJJ2201639], Doctoral Research Start-up Fund Project of Jinggangshan University [JZB2117] and the Science and Technology Project of Jiangxi Provincial Administration of Traditional Chinese Medicine [2021B695]. Authors' contributions LL: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Visualization and Writing-original draft. KH: Data curation, Investigation, Funding acquisition, Resources, Supervision, Validation. PZ: Data curation, Methodology, Software, Validation. XL: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing-review & editing. All authors reviewed the results and approved the final version of the manuscript. Acknowledgements Not applicable. References McGlynn KA, Petrick JL, El-Serag HB. Epidemiology of Hepatocellular Carcinoma. 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Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin. International journal of cancer. 2007;120:2019-27. Zhou Y, Lin Y, Li W, Liu Q, Gong H, Li Y, et al. Expression of AKRs superfamily and prognostic in human gastric cancer. Medicine (Baltimore). 2023;102:e33041. Wen X, Klionsky DJ. At a glance: A history of autophagy and cancer. Seminars in cancer biology. 2020;66:3-11. Han Z, Liu D, Chen L, He Y, Tian X, Qi L, et al. PNO1 regulates autophagy and apoptosis of hepatocellular carcinoma via the MAPK signaling pathway. Cell Death Dis. 2021;12:552. Zheng Y, Wang Y, Lu Z, Wan J, Jiang L, Song D, et al. PGAM1 Inhibition Promotes HCC Ferroptosis and Synergizes with Anti-PD-1 Immunotherapy. Advanced science (Weinheim, Baden-Wurttemberg, Germany). 2023;10:e2301928. Zhu YJ, Zheng B, Wang HY, Chen L. New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta pharmacologica Sinica. 2017;38:614-22. Guo L, Hu C, Yao M, Han G. Mechanism of sorafenib resistance associated with ferroptosis in HCC. Frontiers in pharmacology. 2023;14:1207496. Hashemi M, Mirzaei S, Zandieh MA, Rezaei S, Amirabbas K, Dehghanpour A, et al. Long non-coding RNAs (lncRNAs) in hepatocellular carcinoma progression: Biological functions and new therapeutic targets. Progress in biophysics and molecular biology. 2023;177:207-28. Wohlhieter CA, Richards AL, Uddin F, Hulton CH, Quintanal-Villalonga À, Martin A, et al. Concurrent Mutations in STK11 and KEAP1 Promote Ferroptosis Protection and SCD1 Dependence in Lung Cancer. Cell reports. 2020;33:108444. Shiiba M, Yamagami H, Yamamoto A, Minakawa Y, Okamoto A, Kasamatsu A, et al. Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity. Oncology reports. 2017;37:2025-32. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4315084","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304740520,"identity":"5792981e-f28f-491b-9728-5a3ac3dd60be","order_by":0,"name":"Liang Luo","email":"","orcid":"","institution":"Affiliated Hospital of Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Luo","suffix":""},{"id":304740521,"identity":"4764cc0f-491f-4466-b21e-50323a57c9aa","order_by":1,"name":"Keqing He","email":"","orcid":"","institution":"Affiliated Hospital of Jiangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Keqing","middleName":"","lastName":"He","suffix":""},{"id":304740523,"identity":"9c251202-db4f-43bf-af9c-e99f21636cf3","order_by":2,"name":"Pingsheng Zhou","email":"","orcid":"","institution":"Jiangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pingsheng","middleName":"","lastName":"Zhou","suffix":""},{"id":304740526,"identity":"b884a355-a111-464d-b55c-ccdf78ea3d1f","order_by":3,"name":"Xing Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACPmYgkcDAIMfYAOYzE9bCBtViTIIWKJ0I0UGUFnYeM4mHO2rTm/uXP5NgqLBObGA/e4CAw3iMDRLPHM9tnPHGTILhTHpiA09eAiEthg8S244BtZxhk2BsO5zYIMFjQEiLwQGglnTGGcefSTD+I04LyJaaBMb+BjMJxgaitLAVGyS2HTBsnMFjbJFwLN24jScHvxZ+/sPbJH+21ckb9h9/eONDjbVsP/sZ/Fqg4DCD4YwEcJzCY4oQqGOQ5z9ApNpRMApGwSgYcQAAIOlAE2uwQogAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Jinggangshan University","correspondingAuthor":true,"prefix":"","firstName":"Xing","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-04-24 02:56:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4315084/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4315084/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56958765,"identity":"fab4a938-c842-4576-9312-74f4bf8bc703","added_by":"auto","created_at":"2024-05-22 16:44:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":966051,"visible":true,"origin":"","legend":"\u003cp\u003eAKR1C1 expression and survival analysis in hepatocellular carcinoma (HCC) patients. A. AKR1C1 expression in the HCC tumor group compared to the normal group. B. Survival analysis based on AKR1C1 expression levels in HCC patients. C. in early-stage (stage 1-2) tumors versus late-stage (stage 3-4) tumors. D. Correlation analysis between PD1 and AKR1C1 expression levels. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4315084/v1/daa73edf35ba86da9eca749c.png"},{"id":56958764,"identity":"f1f7056e-0062-45ce-922d-0cae317ff564","added_by":"auto","created_at":"2024-05-22 16:44:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1904883,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TCONS-00026762 knockdown on autophagy in hepatocellular carcinoma (HCC) cells. A. Assessment of mRNA expression of TCONS-00026762 and AKR1C1 in HCC cell lines (Huh7 and SMMC-7721 cells) and normal live line (HL-7702 cells) using qRT-PCR. B. Assessment of mRNA expression of TCONS-00026762 and AKR1C1 in Huh7 cells transfected with sh-TCONS-00026762-1 and sh-TCONS-00026762-2 using qRT-PCR. C. Assessment of Huh7 cell viability following TCONS-00026762 knockdown and AKR1C1 overexpression using CCK-8 assay. D. Assessment of LC3 expression in Huh7 cells following TCONS-00026762 knockdown and AKR1C1 overexpression using immunofluorescence staining; scale bar: 25 μm. E. Assessment of LC3 expression in Huh7 cells following TCONS-00026762 knockdown and AKR1C1 overexpression using western blotting analysis. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4315084/v1/4e75b30cde376ef8002920a4.png"},{"id":56958768,"identity":"6a847a25-6b06-40c4-b2e2-dfecc7d39ae1","added_by":"auto","created_at":"2024-05-22 16:44:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1315308,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TCONS-00026762 knockdown on ferroptosis and sorafenib sensitivity in hepatocellular carcinoma (HCC) cells. A. Assessment of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron levels following TCONS-00026762 knockdown and AKR1C1 overexpression using ELISA. B. Assessment of expression levels of ferroptosis markers glutathione peroxidase 4 (GPX4) and solute carrier family 7a member 11 (SLC7A11) using western blotting analysis. C. Assessment of mRNA expression levels of TCONS-00026762 and AKR1C1 in sorafenib-resistant Huh7-SR cells sing qRT-PCR. D. Assessment of Huh7-SR cell viability following sh-TCONS-00026762 and oe-AKR1C1 transfection using CCK-8 assay. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4315084/v1/1a9f763a5fd4245a90e88923.png"},{"id":56959168,"identity":"e827ab21-0a30-4a8a-962c-4fdfdf386376","added_by":"auto","created_at":"2024-05-22 16:52:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4500944,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Zingiberensis new saponin (ZnS) treatment via the TCONS-00026762/AKR1C1 pathway in hepatocellular carcinoma (HCC) cells. A. Assessment of TCONS-00026762 and AKR1C1 expression in response to ZnS or oe-TCONS-00026762 transfection using qRT-PCR. B. Evaluation of the effect of ZnS and TCONS-00026762 on HCC cell viability using CCK-8 assay. C. Evaluation of the influence of ZnS and TCONS-00026762 on HCC cell apoptosis using flow cytometry assay. D. Evaluation of the impact of ZnS and TCONS-00026762 on HCC cell migration using Transwell assay; scale bar: 50 μm. E-F. Immunofluorescence staining and western blotting analysis of LC3 expression in response to ZnS and TCONS-00026762; scale bar: 25 μm. G. The contents of MDA, 4-HNE, and iron were detected using ELISA. H. Western blotting analysis of GPX4 and SLC7A11 expression. I. CCK-8 assay to evaluate the effect of ZnS, TCONS-00026762, and sorafenib on HCC cell viability. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4315084/v1/37649cf92bb14e4f1adce88c.png"},{"id":59506607,"identity":"91e6cc8c-e7c7-4e03-9219-ad76a2f6ed5e","added_by":"auto","created_at":"2024-07-02 15:16:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9033046,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4315084/v1/3b0f3a93-d1f8-44c3-8afb-e77a24ea7bca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zingiberensis New Saponin Inhibits LncRNA TCONS-00026762/AKR1C1 Pathway, Revealing Unique Insights into Cellular Processes and Drug Response in Hepatocellular Carcinoma","fulltext":[{"header":"Background","content":"\u003cp\u003eLiver cancer is a significant burden on global cancer incidence. Over the past few decades, the incidence of this disease has been steadily increasing in many countries (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is anticipated that globally, between 2020 and 2040, there will be a 55% increase in new cases of liver cancer, resulting in an estimated 1.4\u0026nbsp;million new cases by 2040 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Hepatocellular carcinoma (HCC) represents the dominant type of liver cancer, contributing to around 90% of all incidences. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Owing to the difficulty in detecting the symptoms and physical characteristics of HCC, more than 80% of patients are unable to undergo curative treatment upon diagnosis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Despite extensive efforts in the past decade to develop novel drugs and treatment strategies, there has been limited progress in the treatment of HCC (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Consequently, the exploration of novel treatment approaches and drugs holds significant importance for the treatment of HCC.\u003c/p\u003e \u003cp\u003eIncreasing evidence is underscoring the critical role that long non-coding RNAs (lncRNAs) play in the onset, progression, and spread of various cancers, including HCC (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). For instance, the lncRNA RP11-620J15.3 is known to boost HCC cell multiplication and metastasis by targeting miR-326/GPI, thereby enhancing glycolysis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Furthermore, mounting evidence is showing a close correlation between the expression of lncRNAs and various forms of cell death, including autophagy and ferroptosis (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For example, in breast cancer, metformin induces ferroptosis by suppressing autophagy through the lncRNA H19 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Our previous study found that Zingiberensis new saponin (ZnS) inhibits HCC growth by downregulating the expression of a novel LncRNA TCONS-00026762 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e); however, the exact mechanism by which LncRNA TCONS-00026762 inhibits HCC growth is unclear.\u003c/p\u003e \u003cp\u003eLncRNA TCONS-00026762 is located on chromosome 10p15.1 and significantly overlaps with the gene for aldo-keto reductase family 1 member C1 (AKR1C1), a protein reported to be overexpressed in several cancers, including HCC (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Moreover, AKR1CI is not only an autophagy-regulated joint protein but has also been implicated in the regulation of ferroptosis and drug sensitivity (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Bioinformatics analysis suggests that AKR1C1 may serve as a prognostic biomarker for HCC. Moreover, it presents itself as a compelling target for the regulation of autophagy, ferroptosis, and drug sensitivity in HCC cells. Based on these insights, we hypothesize that ZnS could modulate these cellular processes in HCC through the lncRNA TCONS-00026762/AKR1C1 pathway, thereby inhibiting the malignant progression of the disease.\u003c/p\u003e \u003cp\u003eGiven the above background, this study aims to determine whether ZnS regulates autophagy, ferroptosis, and drug sensitivity in HCC cells through the lncRNA TCONS-00026762/AKR1C1 pathway. We aim to provide new mechanistic insights and identify potential therapeutic targets for the comprehensive treatment of HCC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis\u003c/h2\u003e \u003cp\u003eFirst, GEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to analyze the survival rate of AKR1C1 in TCGA liver cancer data. Then, we downloaded the TCGA transcriptomic and clinical data from UCSC Xena to analyze the differential expression of AKR1C1 across different tumor stages. Next, in the liver cancer TCGA dataset, samples were categorized into high-expression and low-expression groups based on the median expression level of AKR1C1. Subsequently, we selected 26 autophagy-related and 5 ferroptosis-related gene sets from the MSigDB and then employed gene set variation analysis (GSVA) to compute the pathway activity scores for each sample in the HCC dataset from CGA. Following this, we conducted a t-test to analyze the relationship between these pathway activity scores and the expression levels of AKR1C. Additionally, we analyzed the correlation between AKR1C1 and the immune spot (PD1) using Pearson\u0026rsquo;s correlation analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eThis study employed the human HCC cell lines Huh7 and SMMC-7721, along with the human normal liver cell line HL-7702. These cell lines, sourced from COBIOER BIOSCIENCES CO., LTD (Nanjing, China), were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, NY, USA), supplemented with 10% fetal bovine serum (FBS; Gibco). All cell culture practices were executed in a humidified incubator set at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Sorafenib-resistant cells Huh7 (Huh7-SR) were established through long-term exposure to sorafenib, starting at an initial concentration of 0.5 \u0026micro;M and gradually escalating the dose up to 10 \u0026micro;M. Huh7 or Huh7-SR cells underwent treatment with 5 \u0026micro;M sorafenib for 24 h and/or 1 \u0026micro;M ZnS for 48 h. Huh7 cells treated with the equal amount of dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) served as the controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eHuh7 and Huh7-SR cells were seeded into 96-well or 6-well plates and allowed to reach 80% confluence. Following this, lentiviral vectors engineered for the knockdown of TCONS-00026762 (sh-TCONS-00026762-1/2), as well as for the overexpression of AKR1C1 (oe-AKR1C1) and TCONS-00026762 (oe-TCONS-00026762), were transfected into the cells, alongside their respective negative controls (sh-NC or oe- NC). Transfections were performed using HighGene transfection reagent (ABclonal, Wuhan, China) and allowed to proceed for 48 h, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine transfection efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eTo evaluate the ferroptosis level in different groups, the evaluation of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron levels were detected in each group. The contents of MDA, 4-HNE, and iron in cell lysates were assessed using MDA Assay Kit (Solarbio; BC0020), 4-HNE Assay Kit (Elabscience, Wuhan, China; E-EL-0128c) and Iron Assay Kit (Solarbio; #BC5315) according to the manufacturer\u0026rsquo;s instructions, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR\u003c/h2\u003e \u003cp\u003eqRT-PCR assay was conducted following a reported method (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The specific primers used included TCONS-00026762 (forward: 5\u0026rsquo;-AAT GAG GAG CAG GTT GGA CT-3\u0026rsquo;, reverse: 5\u0026rsquo;-GAT CAC TTC CTC ACC TGG CT-3\u0026rsquo;), AKR1C1 (forward: 5\u0026rsquo;-TTT GGC ACC TAT GCG CCT G-3\u0026rsquo;, reverse: 5\u0026rsquo;-CAG AAT CAA TAT GGC GGA AGC CAG-3\u0026rsquo;), and the housekeeping gene GAPDH (forward: 5\u0026rsquo;-GGT GAA GGT CGG AGT CAA CG-3\u0026rsquo;, reverse: 5\u0026rsquo;-CAA AGT TGT CAT GGA TGA ACC-3\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and cell apoptosis\u003c/h2\u003e \u003cp\u003eAs per previously described methods (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), the viability and apoptosis levels of Huh7 or Huh-SR cells were assessed using the CCK-8 assay and flow cytometry, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell migration\u003c/h2\u003e \u003cp\u003eCells underwent trypsin digestion and were resuspended in serum-free culture medium, adjusting the cell density to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL. Then, 200 \u0026micro;L of cells were pipetted into the upper chamber of the Transwell insert, while the lower chamber was filled with 600 \u0026micro;L of FBS. After incubating for 24 h, cells were subjected to a 30 min formaldehyde fixation and subsequently stained with 0.1% crystal violet for 20 min. Any non-migratory cells on the upper surface were wiped off. Cell counting was performed by observing three random fields under a microscope (Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eHuh7 cells were plated onto sterilized coverslips situated in a 12-well plate. Post removal of the culture medium, cells were fixed with a 3% paraformaldehyde solution for 15 min and permeabilized with 1% Triton-X 100 for 10 min. After blocking non-specific binding sites with 3% bovine serum albumin for 30 min, the coverslips were incubated at 4\u0026deg;C overnight with a primary antibody against LC3 (Cell Signaling Technology, MA, USA; 4108), at a dilution of 1:100. Subsequently, a secondary antibody (ABclonal Technology; AS011) was introduced to the samples at a dilution of 1:500, along with a 4',6-diamidino-2-phenylindole staining solution for nuclear counterstaining. The immunofluorescence signals were captured using a laser scanning confocal microscope (Perkin Elmer, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe expression of LC3, GPX4, and SLC7A11 in cell samples was evaluated using western blot analysis as previously described (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). For primary antibody incubation, anti-LC3 antibody (Cell Signaling Technology; 4108; dilution of 1:1,000), anti-glutathione peroxidase 4 (GPX4) antibody (Affinity, OH, USA; DF6701; dilution of 1:1,000), anti-solute carrier family 7a member 11 (SLC7A11) antibody (Affinity; DF12509; dilution of 1:1,000), and anti-GAPDH antibody (Abcam, UK; ab9485; dilution of 1:2,500) were applied.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll data are displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was performed using GraphPad 7.0 software (GraphPad Software, Inc., CA, USA), with one-way analysis of variance and Tukey's test employed to compare differences among groups. Statistical significance was set at a \u003cem\u003ep\u003c/em\u003e-value of less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eAKR1C1 could be a prognostic marker for HCC\u003c/h2\u003e\n\u003cp\u003eTo investigate the role of the LncRNA TCONS-00026762/AKR1C1 pathway in the treatment of HCC, we employed bioinformatics techniques to delineate the mechanistic involvement of AKR1C1 in HCC pathogenesis. Bioinformatic analysis revealed a significant upregulation of AKR1C1 in the HCC tumor cohort when compared to the normal group (t-test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Intriguingly, HCC patients characterized by lower AKR1C1 expression levels exhibited longer overall survival as opposed to those with elevated expression levels (Log-rank test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Further, differential expression analysis disclosed that AKR1C1 was overexpressed in early-stage tumors (stages 1\u0026ndash;2) as compared to late-stage tumors (stages 3\u0026ndash;4) (t-test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Collectively, these findings underscore the potential of AKR1C1 as a prognostic marker for HCC.\u003c/p\u003e\n\u003cp\u003eComplementing these observations, existing literatures have implicated AKR1C1 in a myriad of cellular processes, including cancer autophagy, ferroptosis, and chemoresistance. In our study, we ascertained that heightened expression levels of AKR1C1 significantly influenced the activity scores across 16 distinct autophagy-related pathways and one ferroptosis-related pathway, as cataloged in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Additionally, Pearson\u0026rsquo;s correlation analysis yielded a notable negative association between AKR1C1 and PD1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00026; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Considering these empirical insights and bioinformatic interpretations, we hypothesize that targeted therapeutic interventions against AKR1C1 in HCC may be intricately associated with modulating autophagy, ferroptosis, and chemotherapy sensitivity.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eIdentification of the pathways significantly related to autophagy and ferroptosis between high and low AKR1C1 expression groups through gene set variation analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePathway\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroup1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroup 2\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP\u003c/em\u003evalue\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMethod\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_LYSOSOMAL_MICROAUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.031\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_POSITIVE_REGULATION_OF_MACROAUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_CHAPERONE_MEDIATED_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.038\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_NEGATIVE_REGULATION_OF_AUTOPHAGY_OF_MITOCHONDRION\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eREACTOME_LATE_ENDOSOMAL_MICROAUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.018\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWP_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKUMAR_AUTOPHAGY_NETWORK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0028\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_REGULATION_OF_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0067\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_NEGATIVE_REGULATION_OF_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_POSITIVE_REGULATION_OF_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0054\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_NEGATIVE_REGULATION_OF_MACROAUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0052\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_AUTOPHAGY_OF_PEROXISOME\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGOBP_REGULATION_OF_AUTOPHAGY_OF_MITOCHONDRION_IN_RESPONSE_TO_MITOCHONDRIAL_DEPOLARIZATION\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eREACTOME_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eREACTOME_SELECTIVE_AUTOPHAGY\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0053\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWP_CLOCKCONTROLLED_AUTOPHAGY_IN_BONE_METABOLISM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0028\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFerroptosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_high\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAKR1C1_low\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT-test\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTCONS-00026762 knockdown inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells by reducing AKR1C1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo corroborate the findings from our bioinformatics analysis, we assessed the impact of the LncRNA TCONS-00026762/AKR1C1 axis on autophagy, ferroptosis, and sensitivity to sorafenib in HCC cells. Initially, the expression of TCONS-00026762 and AKR1C1 were upregulated in HCC cell lines (Huh7 and SMMC-7721 cells) compared to normal live line (HL-7702 cells) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Since the expression levels of TCONS-00026762 andAKR1C1 were slightly higher in Huh7 cells than in SMMC-7721 cells, Huh7 cells were selected for subsequent assays (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Then, transfection with sh-TCONS-00026762-1 and sh-TCONS-00026762-2 led to a marked decrease in the relative mRNA expression of both TCONS-00026762 and AKR1C1 in Huh7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Given slightly lower TCONS-00026762 and AKR1C1 expression levels in sh-TCONS-00026762-1-transfecetd Huh7 cells than TCONS-00026762-2-transfecetd cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), the sh-TCONS-00026762-1 lentiviral plasmid was chosen for subsequent experiments. Notably, the viability of Huh7 cells was compromised following sh-TCONS-00026762 transfection, a decrement that was effectively reversed upon AKR1C1 overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eFurthermore, immunofluorescence staining revealed that sh-TCONS-00026762 substantially attenuated the fluorescence signal corresponding to LC3, an autophagy marker (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). This attenuation was counteracted by the introduction of oe-AKR1C1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Concordantly, western blot analysis of LC3 protein levels corroborated the results obtained through immunofluorescence staining (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these data suggest that TCONS-00026762 knockdown inhibits autophagy in HCC cells by downregulating AKR1C1 expression.\u003c/p\u003e\n\u003cp\u003eIn terms of ferroptosis, sh-TCONS-00026762-mediated elevations in the contents of MDA, 4-HNE, and iron were significantly alleviated by AKR1C1 overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, knockdown of TCONS-00026762 led to a marked downregulation in the two key ferroptosis regulators, GPX4 and SLC7A11 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), and lowered levels of them are commonly deemed as ferroptosis indicators (\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e). However, the levels of GPX4 and SLC7A11 were restored by AKR1C1 overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Taken together, these findings indicate that knockdown of TCONS-00026762 promotes ferroptosis in HCC cells by decreasing AKR1C1 expression.\u003c/p\u003e\n\u003cp\u003eLastly, the Huh7-SR group exhibited elevated levels of TCONS-00026762 and AKR1C1 mRNA expression in comparison to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Concurrently, overexpression of AKR1C1 mitigated the decreased viability of Huh7-SR cells induced by TCONS-00026762 knockdown in the context of sorafenib treatment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). These cumulative data imply that knockdown of TCONS-00026762 enhances the sensitivity of sorafenib-resistant HCC cells to sorafenib treatment by reducing AKR1C1 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZnS inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells by blocking the TCONS-00026762/AKR1C1 axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of ZnS in modulating autophagy, ferroptosis, and sorafenib sensitivity in HCC cells via the lncRNA TCONS-00026762/AKR1C1 pathway, we treated Huh7 cells with ZnS, either in the presence or absence of oe-TCONS-00026762. Initially, we assessed the impact of ZnS on the expression levels of TCONS-00026762 and AKR1C1. qRT-PCR analysis revealed that ZnS treatment led to a notable reduction in the expression of both TCONS-00026762 and AKR1C1 when compared to untreated controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). This downregulation was effectively counteracted by the overexpression of TCONS-00026762 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eFollowing this, ZnS was observed to significantly diminish both cell viability (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) and migratory capabilities of Huh7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), while concurrently enhancing apoptosis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Interestingly, these effects of ZnS were abolished upon oe-TCONS-00026762 transfection (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Importantly, overexpression of TCONS-00026762 effectively reversed the ZnS-mediated downregulation of LC3 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE-F).\u003c/p\u003e\n\u003cp\u003eIn relation to ferroptosis, TCONS-00026762 overexpression significantly attenuated the ZnS-induced increases in levels of MDA, 4-HNE, and iron, while simultaneously reversing the decreased GPX4 and SLC7A11 expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). Additionally, the ZnS-induced reduction in the viability of sorafenib-resistant Huh7-SR cells was also significantly nullified by TCONS-00026762 overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe burgeoning role of lncRNAs in carcinogenesis has been evident in recent years, with increasing literature supporting their potential as therapeutic targets (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Our study uniquely advances this field by investigating the anticancer effects of ZnS through the modulation of autophagy and possibly ferroptosis and drug sensitivity in HCC cells, via the lncRNA TCONS-00026762/AKR1C1 pathway.\u003c/p\u003e \u003cp\u003eFirstly, AKR1C1 was found to be notably upregulated in HCC cells, with higher expression associated with lower survival rates in patients. These findings echo earlier studies underscoring the potential of AKR1C1 as a predictive biomarker for cancer prognosis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Notably, the observation of higher AKR1C1 expression in early-stage tumors suggests that this marker might be implicated in the early onset of HCC, warranting further exploration in future research.\u003c/p\u003e \u003cp\u003eAutophagy is a cellular survival mechanism that prevents cellular damage and promotes survival under conditions of energy or nutrient deprivation, as well as in response to various cytotoxic insults (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Qu et al reported that PNO1 promotes autophagy and inhibits apoptosis in HCC cells via the MAPK signaling pathway (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Ferroptosis is a form of regulated cell death that involves iron-dependent lipid peroxidation. Emerging evidence suggests that ferroptosis plays a critical role in cancer biology, including inhibiting cancer progression (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In HCC, targeting ferroptosis has shown potential in controlling tumor growth and enhancing the efficacy of existing treatments (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Chemoresistance is a significant barrier to effective cancer treatment. Sorafenib is the only targeted drug for the treatment of advanced HCC (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Resistance to chemotherapeutic agents like sorafenib is a major clinical challenge, and understanding the underlying mechanisms is crucial for developing effective treatment strategies (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Furthermore, our bioinformatics analysis confirmed the involvement of AKR1C1 in modulating autophagy, ferroptosis, and chemoresistance during the progression of HCC. Intriguingly, the lncRNA TCONS-00026762 significantly overlaps with the AKR1C1 gene, leading us to hypothesize a parallel function for lncRNA TCONS-00026762.\u003c/p\u003e \u003cp\u003eOur subsequent experiments revealed that TCONS-00026762 and AKR1C1 were upregulated in HCC cell lines, further supporting the involvement of dysregulated lncRNAs in HCC pathogenesis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Moreover, knockdown of TCONS-00026762 led to reduced expression of both AKR1C1 and the autophagy marker LC3. Furthermore, the knockdown of TCONS-00026762 was associated with a significantly decline in cellular levels of MDA, 4-HNE, and iron, as well as decreased expression of ferroptosis markers GPX4 and SLC7A11. Importantly, we observed a notable reduction in cell viability in Huh7-SR cells upon TCONS-00026762 knockdown. Most significantly, overexpression of AKR1C1 counteracted these effects, aligning our findings with existing literature and thereby validating our initial hypothesis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous report, we demonstrated that ZnS could inhibit the malignant progression of HCC while concurrently downregulating the expression levels of lncRNA TCONS-00026762 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Crucially, our findings demonstrated that the lncRNA TCONS-00026762/AKR1C1 pathway is involved in mediating the effects of ZnS on regulation of autophagy, ferroptosis, and drug sensitivity in HCC cells. Overexpression of TCONS-00026762 attenuated the ZnS-mediated downregulation of TCONS-00026762, AKR1C1, LC3, GPX4, and SLC7A11 as well as a decrease in the viability of Huh7-SR cells. These observations provide mechanistic insights into the role of this pathway in modulation of autophagy, ferroptosis, and chemoresistance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study enriches the existing body of literature by detailing the modulation of autophagy, and potentially ferroptosis and drug sensitivity, through the lncRNA TCONS-00026762/AKR1C1 pathway in HCC cells. It underscores the prognostic and therapeutic potential of targeting this pathway, opening new avenues for further research and drug development in HCC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHCC: Hepatocellular carcinoma; lncRNAs: long non-coding RNAs; ZnS: Zingiberensis new saponin; AKR1C1: aldo-keto reductase family 1 member C1; qRT-PCR: quantitative real-time polymerase chain reaction; MDA: malondialdehyde; 4-HNE: 4-hydroxynonenal\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Science and Technology Research Project of Jiangxi Provincial Department of Education [GJJ2201639], Doctoral Research Start-up Fund Project of Jinggangshan University [JZB2117] and the Science and Technology Project of Jiangxi Provincial Administration of Traditional Chinese Medicine [2021B695].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLL: Conceptualization, Data curation, Formal analysis, Methodology, Software, Validation, Visualization and Writing-original draft. KH: Data curation, Investigation, Funding acquisition, Resources, Supervision, Validation. PZ: Data curation, Methodology, Software, Validation. XL: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing-review \u0026amp; editing. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcGlynn KA, Petrick JL, El-Serag HB. Epidemiology of Hepatocellular Carcinoma. Hepatology (Baltimore, Md). 2021;73 Suppl 1:4-13.\u003c/li\u003e\n\u003cli\u003eRumgay H, Arnold M, Ferlay J, Lesi O, Cabasag CJ, Vignat J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040. Journal of hepatology. 2022;77:1598-606.\u003c/li\u003e\n\u003cli\u003eLlovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Nature reviews Disease primers. 2021;7:6.\u003c/li\u003e\n\u003cli\u003eZongyi Y, Xiaowu L. Immunotherapy for hepatocellular carcinoma. Cancer letters. 2020;470:8-17.\u003c/li\u003e\n\u003cli\u003eHuang A, Yang XR, Chung WY, Dennison AR, Zhou J. Targeted therapy for hepatocellular carcinoma. Signal transduction and targeted therapy. 2020;5:146.\u003c/li\u003e\n\u003cli\u003eLiu C, Xu K, Liu J, He C, Liu P, Fu Q, et al. LncRNA RP11-620J15.3 promotes HCC cell proliferation and metastasis by targeting miR-326/GPI to enhance glycolysis. Biology direct. 2023;18:15.\u003c/li\u003e\n\u003cli\u003eXia A, Yue Q, Zhu M, Xu J, Liu S, Wu Y, et al. The cancer-testis lncRNA LINC01977 promotes HCC progression by interacting with RBM39 to prevent Notch2 ubiquitination. 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Dioscorea Zingiberensis New Saponin Inhibits the Growth of Hepatocellular Carcinoma by Suppressing the Expression of Long Non-coding RNA TCONS-00026762. Frontiers in pharmacology. 2021;12:678620.\u003c/li\u003e\n\u003cli\u003eItoh S, Taketomi A, Harimoto N, Tsujita E, Rikimaru T, Shirabe K, et al. Antineoplastic effects of gamma linolenic Acid on hepatocellular carcinoma cell lines. Journal of clinical biochemistry and nutrition. 2010;47:81-90.\u003c/li\u003e\n\u003cli\u003ePhoo NLL, Dejkriengkraikul P, Khaw-On P, Yodkeeree S. Transcriptomic Profiling Reveals AKR1C1 and AKR1C3 Mediate Cisplatin Resistance in Signet Ring Cell Gastric Carcinoma via Autophagic Cell Death. International journal of molecular sciences. 2021;22.\u003c/li\u003e\n\u003cli\u003eChang LL, Li YK, Zhao CX, Zeng CM, Ge FJ, Du JM, et al. AKR1C1 connects autophagy and oxidative stress by interacting with SQSTM1 in a catalytic-independent manner. Acta pharmacologica Sinica. 2022;43:703-11.\u003c/li\u003e\n\u003cli\u003eGagliardi M, Cotella D, Santoro C, Cor\u0026agrave; D, Barlev NA, Piacentini M, et al. Aldo-keto reductases protect metastatic melanoma from ER stress-independent ferroptosis. Cell Death Dis. 2019;10:902.\u003c/li\u003e\n\u003cli\u003eChang WM, Chang YC, Yang YC, Lin SK, Chang PM, Hsiao M. AKR1C1 controls cisplatin-resistance in head and neck squamous cell carcinoma through cross-talk with the STAT1/3 signaling pathway. Journal of experimental \u0026amp; clinical cancer research : CR. 2019;38:245.\u003c/li\u003e\n\u003cli\u003eHe K, Liu X, Cheng S, Zhou P. Zingiberensis Newsaponin Inhibits the Malignant Progression of Hepatocellular Carcinoma via Suppressing Autophagy Moderated by the AKR1C1-Mediated JAK2/STAT3 Pathway. Evid Based Complement Alternat Med. 2021;2021:4055209.\u003c/li\u003e\n\u003cli\u003eLiu P, Feng Y, Li H, Chen X, Wang G, Xu S, et al. Ferrostatin-1 alleviates lipopolysaccharide-induced acute lung injury via inhibiting ferroptosis. Cell Mol Biol Lett. 2020;25:10.\u003c/li\u003e\n\u003cli\u003eChen P, Li X, Zhang R, Liu S, Xiang Y, Zhang M, et al. Combinative treatment of \u0026beta;-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation. Theranostics. 2020;10:5107-19.\u003c/li\u003e\n\u003cli\u003eWang HW, Lin CP, Chiu JH, Chow KC, Kuo KT, Lin CS, et al. Reversal of inflammation-associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin. International journal of cancer. 2007;120:2019-27.\u003c/li\u003e\n\u003cli\u003eZhou Y, Lin Y, Li W, Liu Q, Gong H, Li Y, et al. Expression of AKRs superfamily and prognostic in human gastric cancer. Medicine (Baltimore). 2023;102:e33041.\u003c/li\u003e\n\u003cli\u003eWen X, Klionsky DJ. At a glance: A history of autophagy and cancer. Seminars in cancer biology. 2020;66:3-11.\u003c/li\u003e\n\u003cli\u003eHan Z, Liu D, Chen L, He Y, Tian X, Qi L, et al. PNO1 regulates autophagy and apoptosis of hepatocellular carcinoma via the MAPK signaling pathway. Cell Death Dis. 2021;12:552.\u003c/li\u003e\n\u003cli\u003eZheng Y, Wang Y, Lu Z, Wan J, Jiang L, Song D, et al. PGAM1 Inhibition Promotes HCC Ferroptosis and Synergizes with Anti-PD-1 Immunotherapy. Advanced science (Weinheim, Baden-Wurttemberg, Germany). 2023;10:e2301928.\u003c/li\u003e\n\u003cli\u003eZhu YJ, Zheng B, Wang HY, Chen L. New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta pharmacologica Sinica. 2017;38:614-22.\u003c/li\u003e\n\u003cli\u003eGuo L, Hu C, Yao M, Han G. Mechanism of sorafenib resistance associated with ferroptosis in HCC. Frontiers in pharmacology. 2023;14:1207496.\u003c/li\u003e\n\u003cli\u003eHashemi M, Mirzaei S, Zandieh MA, Rezaei S, Amirabbas K, Dehghanpour A, et al. Long non-coding RNAs (lncRNAs) in hepatocellular carcinoma progression: Biological functions and new therapeutic targets. Progress in biophysics and molecular biology. 2023;177:207-28.\u003c/li\u003e\n\u003cli\u003eWohlhieter CA, Richards AL, Uddin F, Hulton CH, Quintanal-Villalonga \u0026Agrave;, Martin A, et al. Concurrent Mutations in STK11 and KEAP1 Promote Ferroptosis Protection and SCD1 Dependence in Lung Cancer. Cell reports. 2020;33:108444.\u003c/li\u003e\n\u003cli\u003eShiiba M, Yamagami H, Yamamoto A, Minakawa Y, Okamoto A, Kasamatsu A, et al. Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity. Oncology reports. 2017;37:2025-32.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"autophagy, ferroptosis, Hepatocellular carcinoma, lncRNA TCONS-00026762/AKR1C1 pathway, Zingiberensis new saponin","lastPublishedDoi":"10.21203/rs.3.rs-4315084/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4315084/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLong non-coding RNAs (lncRNAs) are often involved in regulating various cellular processes during cancer progression. This study aimed to investigate the role of Zingiberensis new saponin (ZnS) in hepatocellular carcinoma (HCC) cells through the lncRNA TCONS-00026762/AKR1C1 pathway.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBioinformatics analysis was initially used to assess the prognostic significance of AKR1C1 in TCGA liver cancer data. Huh7 and Huh7-SR cells were either transfected with sh-TCONS-0026762 and oe-AKR1C1 or treated with ZnS and oe-TCONS-00026762. The expression of TCONS-00026762 and AKR1C1 was quantified using quantitative real-time PCR. The effects of either TCONS-00026762 knockdown or ZnS treatment on autophagy, ferroptosis, and drug sensitivity were investigated using a combination of immunofluorescence staining, western blot, and CCK-8 assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBioinformatics analysis revealed that AKR1C1 is a prognostic marker for HCC and is association with autophagy, ferroptosis, and immune evasion. Knockdown of TCONS-00026762 suppressed autophagy, promoted ferroptosis, and enhanced sensitivity to sorafenib in HCC cells, as evidenced by the decrease in levels of the autophagy marker LC3, as well as ferroptosis markers GPX4 and SLC7A11, and an increase in Huh7-SR cell viability. However, these changes were reversed by overexpression of AKR1C1. Moreover, ZnS treatment significantly downregulated the expression of TCONS-00026762 and AKR1C1, leading to inhibition of autophagy, induction of ferroptosis, and increased susceptibility of HCC cells to sorafenib. Notably, these effects were reversible upon the overexpression of TCONS-00026762.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings suggest that ZnS inhibits autophagy, promotes ferroptosis, and enhances sensitivity to sorafenib in HCC cells through the lncRNA TCONS-00026762/AKR1C1 pathway.\u003c/p\u003e","manuscriptTitle":"Zingiberensis New Saponin Inhibits LncRNA TCONS-00026762/AKR1C1 Pathway, Revealing Unique Insights into Cellular Processes and Drug Response in Hepatocellular Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 16:44:40","doi":"10.21203/rs.3.rs-4315084/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5246105-1247-4e67-88f0-43d4878b5a46","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T15:08:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-22 16:44:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4315084","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4315084","identity":"rs-4315084","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}