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

doi:10.21203/rs.3.rs-4315084/v1

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

https://doi.org/10.21203/rs.3.rs-4315084/v1

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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

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.

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% CO₂. 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 1×10⁶/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.

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. 1A). 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. 1B). 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. 1C). 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. 1D). 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	Pvalue	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. 2A). 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. 2A). 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. 2B). Given slightly lower TCONS-00026762 and AKR1C1 expression levels in sh-TCONS-00026762-1-transfecetd Huh7 cells than TCONS-00026762-2-transfecetd cells (Fig. 2B), 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. 2C).

Furthermore, immunofluorescence staining revealed that sh-TCONS-00026762 substantially attenuated the fluorescence signal corresponding to LC3, an autophagy marker (P < 0.01; Fig. 2D). This attenuation was counteracted by the introduction of oe-AKR1C1 (P < 0.01; Fig. 2D). Concordantly, western blot analysis of LC3 protein levels corroborated the results obtained through immunofluorescence staining (P < 0.01; Fig. 2E). 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. 3A). Similarly, knockdown of TCONS-00026762 led to a marked downregulation in the two key ferroptosis regulators, GPX4 and SLC7A11 (P < 0.01; Fig. 3B), 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. 3B). 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. 3C). 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. 3D). 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. 4A). This downregulation was effectively counteracted by the overexpression of TCONS-00026762 (P < 0.01; Fig. 4A).

Following this, ZnS was observed to significantly diminish both cell viability (P < 0.01; Fig. 4B) and migratory capabilities of Huh7 cells (P < 0.01; Fig. 4D), while concurrently enhancing apoptosis (P < 0.01; Fig. 4C). Interestingly, these effects of ZnS were abolished upon oe-TCONS-00026762 transfection (P < 0.01; Fig. 4B-D). Importantly, overexpression of TCONS-00026762 effectively reversed the ZnS-mediated downregulation of LC3 (P < 0.01; Fig. 4E-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. 4G-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. 4I).

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.

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.

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

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.

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No competing interests reported.

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

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Bioinformatics analysis

Cell culture and treatment

Cell transfection

ELISA

qRT-PCR

Cell viability and cell apoptosis

Cell migration

Immunofluorescence staining

Western blot analysis

Data analysis

Results

AKR1C1 could be a prognostic marker for HCC

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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