The high expression of Long noncoding RNA TEX41 promotes the proliferation, migration, and invasion of hepatocellular carcinoma

Introduction

After lung cancer and colorectal cancer, liver cancer is the third leading cause of cancer death worldwide, with over 750,000 liver cancer-related deaths occurring in 2022 (1). Hepatocellular carcinoma (HCC) is the primary type of malignant liver disease. The 5-year survival rate for patients with primary localized HCC is 32.6%, while that for patients with regional or metastatic HCC is only 10.8% and 2.4%, respectively (2). Therefore, thoroughly examining the pathogenesis of HCC and finding new biomarkers have important guiding significance for determining treatment plans and improving prognosis. In recent studies, long noncoding RNA (lncRNA) has played an important role in the development of many cancers (3,4). Although lncRNA and microRNA (miRNA) cannot encode proteins, they also play an important role in regulating gene expression and protein function as noncoding transcripts (5). The mechanism of action of lncRNA includes gene imprinting, chromatin remodeling, cell cycle regulation, splicing regulation, messenger RNA degradation and translation regulation, and protein binding. Among them, lncRNA can adsorb miRNA through complementary base sequences, thereby acting as a competing endogenous RNA (ceRNA) to interact with miRNA and affect target gene expression. LncRNA TEX41 is a newly discovered lncRNA, and studies have confirmed its high expression and molecular sponge effect in various malignancies including cervical cancer, acute lymphoblastic leukemia, melanoma, lung adenocarcinoma, and colorectal cancer (6-8). Studies have shown that miR-200a-3p plays an important role in the emergence and development of HCC (9,10). BIRC5 is an antiapoptotic protein that participates in various physiological and pathological processes. Studies on this subject have shown that the high expression of BIRC5 increases the mortality rate of patients with HCC (11), and it may thus be valuable for diagnosing and treating this disease (12-14). We speculated that lncRNA TEX41 may target the miR-200a-3p-BIRC5 molecular axis and affect the development of HCC. In this study, we identified the binding sites between lncRNA TEX41 and miR-200a-3p, miR-200a-3p, and BIRC5 through the starBase database (https://rnasysu.com/encori/index.php). Subsequently, we sought to clarify the effects of lncRNA TEX41 on the biological behavior of HCC cells and the miR-200a-3p-BIRC5 molecular axis, providing a basis for the targeted therapy of HCC. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-812/rc).

Methods

Patients and samples

We retrospectively collected cancer tissues and adjacent tissues from patients with HCC who underwent surgery at The Second Affiliated Hospital of Nantong University from September 2022 to December 2024. The inclusion criteria were as follows: (I) meeting the diagnostic criteria for HCC and (II) successful acquisition and intact preservation of HCC and adjacent tissue specimens during operation. Meanwhile, the exclusion criteria were as follows: (I) a history of liver surgery; (II) malignant tumors in other parts of the body; (III) incomplete case data; and (IV) presence of autoimmune diseases. The basic clinical data of all patients with HCC, such as age, gender, tumor diameter, and tumor-node-metastasis (TNM) staging, were collected. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Nantong University (No. 2024KT186). Informed consent was taken from all individual participants.

Quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was separated with TRIzol reagent (15596026; Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was prepared using the Takara Primer RT kit (RR036A; Takara Bio, Kusatsu, Japan), and quantitative polymerase chain reaction (qPCR) was conducted using BeyoFast^TM SYBR Green qPCR Mix (D7262; Beyotime, Nantong, China) and a Bio-Rad CFX96 real-time fluorescence qPCR instrument (Bio-Rad Laboratories, California, USA). The qPCR conditions were as follows: 95 ℃ for 15 minutes, followed by 40 cycles, with each cycle consisting of 95 ℃ for 10 seconds and 66 ℃ for 32 seconds. Table 1 summarizes the primer sequences (Sangon Biotech, Shanghai, China) for the genes used in the study. Finally, the experimentally obtained computed tomography (CT) values of lncRNA TEX41, miR-200a-3p, BIRC5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by Graphpad Pism9.5 (GraphPad Software, Santiago, USA) for data analysis using the 2^−ΔΔCt normalization method.

Cell culture and transfection

Human liver cancer cells (SK-Hep-1, LM-3, HepG2, Hep3B, and MHCC-97H) and normal liver cells (QSG-7701) were acquired from Cellverse (Jodhpur, India). The QSG-7701 cells were cultivated in RPMI-1640 (PM150110; Pricella Biotechnology, Wuhan, China) with 10% fetal bovine serum (FBS; 164210; Pricella Biotechnology) and 1% penicillin/streptomycin (PB180120; Pricella Biotechnology). SK-Hep-1, LM-3, HepG2, Hep3B, and MHCC-97H cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; PM150210; Pricella Biotechnology) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were grown in a 37 ℃ incubator containing a 5% CO₂ atmosphere. A TEX41 interference (sh-TEX41) lentiviral vector and corresponding negative control (NC) vector (sh-NC) were provided by Jikai Gene Chemical Technology Co., Ltd. (Shanghai, China). Three small hairpin RNA (shRNA) fragments targeting TEX41 were inserted into hU6-MCS-Ubiquitin-EGFP-IRES-puromycin, and an NC group was used. Sh-TEX41-1 was used in a series of subsequent experiments due to its significant and stable knockdown effect.

Cell Counting Kit-8 (CCK-8) and colony formation detection

In the CCK-8 assay, liver cancer cells SK-HEP-1 and HCCLM3 were cultivated in a 96-well plate (3×10²/well; 5 replicates per well) and cultured at 37 ℃. To measure proliferation, a CCK-8 test reagent (C6005; NCM Biotech, Newport, RI, USA) was added to the cells at the same time of day for 3–4 days. A microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect the optical density at 450 nm.

For colony formation assay, liver cancer cell lines SK-HEP-1 and HCCLM3 (1×10³ cells/well) were seeded in six-well plates and grown for 14 days. When macroscopic colonies were visible at the bottom of the dish, the experiment was terminated. The cells were fixed with 4% paraformaldehyde for 15 min. Next, 1% crystal violet was added for 15 min of staining. Number of colonies in each was counted, performing these analyses in triplicate.

Wound healing test

HCC cell lines SK-HEP-1 and HCCLM3 (3×10⁵/cell) were seeded into a six-well plate and grown to a confluence of 80–90%. While the cells were being kept in serum-free culture medium, a pipette tip was used to scratch parallel lines in a monolayer. Wound healing was monitored with an inverted microscope on the same day for 3 days. The strength of cell migration ability was determined according to the degree of reduction in wound width and was analyzed via ImageJ software.

Migration and invasion in vitro

For the migration experiment, cells from each group were collected 48 hours after transfection. The cells were resuspended in DMEM without FBS, and the cell density was adjusted to 1×10⁵ cells/mL. A 200-µL cell suspension was inoculated into the upper chamber of a Transwell chamber, and 700 µL of DMEM containing 10% FBS was placed into the lower chamber. The cells were cultured in an incubator with a 5% CO₂ atmosphere at 37 ℃ for 48 hours. The cells invading the outer side of the upper ventricular membrane were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet, observed under an inverted microscope, and photographed. Five visual fields were randomly selected for cell counting. These procedures were repeated at least three times.

For the invasion experiment, matrix glue and culture medium were mixed at a ratio of 1:8, and diluted substrate gel was added into the upper chamber. After solidification, the cell suspension (1×10⁵ cells/mL; 200 µL) was inoculated in the upper chamber. Subsequently, 500 µL of culture medium containing 10% FBS was added to the lower chamber, and the cells were incubated for 48 hours. We fixed the invading cells by soaking in 4% paraformaldehyde for 15 min. Subsequently, we stained the cells by soaking them in 0.1% crystal violet for 15 min. A microscope was used to count five randomly selected areas to determine the number of invading cells. The experiment was repeated three times.

Western blotting

Proteins were separated using cell protein lysate (P0013B; Beyotime) and protease inhibitor mixture (P1005; Beyotime). After sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane. The membrane was sealed with 5% nonfat milk in Tris-buffered Saline Tween (TBST) for 1 hour and then incubated with antibodies against GAPDH (1:10,000, Proteintech, Rosemont, IL, USA), E-cadherin (1:20,000, Proteintech), N-cadherin (1:2,000, Proteintech) and Vimentin (1:20,000, Proteintech) at 4 ℃ for 2 hours. After four washes with TBST (10 min each time), the proteins were incubated with anti-rabbit (1:10,000, Proteintech) or anti-mouse immunoglobulin G (IgG; 1:10,000, Proteintech) at room temperature for 1.5 hours and subjected to detection staining. The GAPDH protein was used as an internal control.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 8.0 (Dotmatics, Boston, MA, USA). The measurement data of continuous variables are expressed as the mean ± standard deviation. The analysis of continuous variables was conducted via the t-test and repeated-measures analysis of variance. Categorical variables are expressed as numbers and were compared with Chi-squared tests. Survival curves were drawn according to the Kaplan-Meier method, and the log-rank test was used to determine significance, with a P value <0.05 indicating a statistically significant difference.

Results

The expression of lncRNA TEX41 in HCC tissues and cell lines

In order to investigate the differentially expressed genes in liver cancer tissues, we conducted correlation analysis on the dataset GSE41804 in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and found that LINC01093, lncRNA TEX41, LINC00238, LINC00844, and LINC01419 were differentially expressed in HCC. Through PubMed search, it was found that there is currently no research on the role and mechanism of lncRNA TEX41 in HCC. Therefore, this study targeted lncRNA TEX41 to explore its specific molecular regulatory mechanisms in the occurrence, development, and metastasis of liver cancer. We first discovered through the GEPIA2 database (http://gepia2.cancer-pku.cn/#index) that lncRNA TEX41 is significantly overexpressed in HCC tissues (Figure 1A). lncRNA TEX41 was highly expressed in the HCC cell lines, especially in SK-Hep-1 and HCCLM3 (Figure 1B).

Figure 1 The expression of lncRNA TEX41 in HCC tissues and cell lines. (A) LncRNA TEX41 had a significantly higher expression in HCC tissues. (B) The expression of lncRNA TEX41 was higher in SK-HEP-1 and HCCLM3 than in the other cell lines. ****, P<0.001. HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma.

The relationship between the relative expression level of lncRNA TEX41 and clinical pathological features in HCC tissues

We collected clinical and pathological information from 50 patients, such as sex, age, tumor size, lymph node metastasis and TNM stage. Through analysis, we found that the high relative expression level of lncRNA TEX41 is significantly correlated with lymph node metastasis and TNM stage in HCC patients. The clinical pathological features of the HCC patients are summarized in Table 2.

The success of silencing lncRNA TEX41 in SK-HEP-1 and HCCLM3 cells

We found that the virus was successfully transfected into SK-Hep-1 and HCCLM3 cell lines via fluorescence microscopy (Figure 2A,2B). The expression level of lncRNA TEX41 in the sh-TEX41 group was lower than that in the sh-NC group (Figure 2C,2D). This also showed that we successfully silenced lncRNA TEX41 by transfecting lentivirus.

Figure 2 The effect of silencing lncRNA TEX41 on the expression of lncRNA TEX41 in SK-HEP-1 and HCCLM3 cells. (A) Fluorescence indicated successful transfection of the SK-HEP-1 viruses. The scale in the figure is ×100. (B) Fluorescence indicated successful transfection of the HCCLM3 viruses. The scale in the figure is ×100. (C) Expression changes of TEX41 in SK-HEP-1 cells after silencing of TEX41 (D) Expression changes of TEX41 in HCCLM3 cells after silencing of TEX41. **, P<0.01. NC, negative control.

The effect of silencing lncRNA TEX41 on the proliferation, migration, and invasion of SK-HEP-1 and HCCLM3 cells

Through CCK8 experiments and cell colony formation experiments, we found that the cell proliferation ability of the sh-TEX41 group was weaker than that of the sh-NC group in both the SK-HEP-1 and HCCLM3 cell lines (Figure 3A,3B). This indicates that silencing lncRNA TEX41 could inhibit the proliferation ability of SK-HEP-1 and HCCLM3 cells. In addition, scratch experiments and Transwell experiments showed that silencing lncRNA TEX41 could inhibit the migration and invasion ability of the SK-HEP-1 and HCCLM3 cell line (Figure 3C-3E). We also examined the effect of TEX41 interference on key proteins involved in epithelial-mesenchymal transition (EMT). As a result, we found that TEX41 knockout increased E-cadherin levels and inhibited N-cadherin and vimentin levels (Figure 3F). This indicates that knockdown of TEX41 can reduce the metastasis of the HCC cell lines SK-HEP-1 and HCCLM3.

Figure 3 The effect of lncRNA TEX41 targeting of the miR-200a-3p-BIRC5 axis on the proliferation, migration, and invasion of SK-HEP-1 and HCCLM3 cells. (A) The CCK-8 assay was used to determine the influence of silencing TEX41 on the proliferation of SK-HEP-1 and HCCLM3 cells. (B) A cell colony formation experiment was used to determine the influence of silencing TEX41 on the proliferation of SK-HEP-1 and HCCLM3. Crystal violet staining method was used. (C) Wound healing experiments were used to determine the change of migration after the silencing of TEX41. (D) A Transwell assay was used to determine the influence of silencing TEX41 on migration. Crystal violet staining method was used. (E) A Transwell assay was used to determine the influence of silencing TEX41 on invasion. Crystal violet staining method was used. (F) Western blot analysis of vimentin, N-cadherin, and E-cadherin after silencing of TEX41. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. OD, optical density; NC, negative control; CCK-8, Cell Counting Kit-8.

Screening and validation of downstream target genes of lncRNA TEX41

We screened miRNAs that interact with lncRNA TEX41 through the starBase database (Figure 4A), predicted the interaction sites between lncRNA TEX41 and miR-200a-3p (Figure 4B), and found a linear correlation in their expression in multiple HCC samples. Through qRT-PCR experiments, the results showed that miR-200a-3p was significantly downregulated in SK-HEP-1 and HCCLM3 (Figure 4C). After knocking down lncRNA TEX41, miR-200a-3p expression was significantly increased (Figure 4D). We speculated that there may be an interaction between lncRNA TEX41 and miR-200a-3p. Possible downstream target gene BIRC5 was screened using GEPIA2 and starBase databases, it was associated with the overall survival of HCC patients (Figure 4E,4F). qRT-PCR experiments showed that the expression of BIRC5 was significantly reduced after knocking down lncRNA TEX41 (Figure 4G). We speculate that there may be a relationship between lncRNA TEX41 and miR-200a-3p-BIRC5.

Figure 4 Screening and validation of downstream target genes of lncRNA TEX41. (A) miRNAs that were predicted to interact with lncRNA TEX41. (B) The interaction site between lncRNA TEX41 and miR-200a-3p. (C) The expression of miR-200a-3p was lower in SK-HEP-1 and HCCLM3 than in the other cell lines. (D) Expression changes of miR-200a-3p in SK-HEP-1 and HCCLM3 cells after silencing of TEX41. (E) The target gene BIRC5 was identified via the intersection of the GEPIA2 and starBase databases. (F) The Kaplan-Meier curve downloaded from the GEPIA2 database indicating the correlation between BIRC5 expression and overall survival rate. (G) Expression changes of BIRC5 in SK-HEP-1 and HCCLM3 cells after silencing of TEX41. *, P<0.05; **, P<0.01. GEPIA, Gene Expression Profiling Interactive Analysis; HR, hazard ratio; NC, negative control.

Discussion

HCC is among the most common malignant tumors. Despite considerable advance in both basic and clinical in recent years, the incidence and mortality rates of liver cancer have not significantly decreased; in fact, they have risen year on year. Currently, the primary treatment methods for liver cancer include surgery, liver transplantation, radiotherapy, and chemotherapy (15). With the development of detection and treatment methods, the 5-year survival rate of patients with liver cancer is increasing year on year. There is a need to seek new diagnostic biomarkers to assist in early diagnosis and treatment. The human liver cancer cell lines HCCLM3 and SK-HEP-1 are often used to study the molecular mechanisms of HCC; for instance, they are used in studies focused on screening molecular markers for early diagnosis or the identification of targeted therapeutic targets. We aim to provide a new theoretical basis and clinically valuable targets for the diagnosis and treatment of HCC.

LncRNA figures prominently in the occurrence and development of tumors, not only participating in the regulation of epigenetics, cell cycle, and cell differentiation, but also regulating gene transcription and expression in chromatin modification, transcriptional activation, and interference (16). Therefore, lncRNA is considered a relatively novel diagnostic biomarker and therapeutic target for various cancers. With the rapid development of gene chips and next-generation sequencing technology, a growing number of lncRNAs are being identified and studied. In the past few years, a large number of studies have shown that lncRNA is essential to the proliferation, apoptosis, invasion, and metastasis of HCC, providing a new theoretical basis for the diagnosis and treatment of HCC (17-19). LncRNA TEX41 plays a cancer-promoting role in various malignant tumors, but its mechanism of action in HCC remains unclear.

This study analyzed the effect of lncRNA TEX41 on liver cancer cells through cell experiments. The results showed that the low expression of lncRNA TEX41 could inhibit the proliferation, migration, and invasion of the HCCLM3 and SK-HEP-1 HCC cell lines, further confirming the tumor-promoting effect of lncRNA TEX41 in HCC. One study proposed an ceRNA mechanism in which lncRNA and miRNA interact to form a competitive network (20), and this was further confirmed in various subsequent studies. For instance, Yang et al. found that lncRNA TEX41 can upregulate Atg5 through sponging miR-153-3p, thereby promoting the proliferation and migration of squamous cell carcinoma cells (21). In addition, a study has found that lncRNA TEX41 can promote the malignant behavior of melanoma cells by targeting the miR-103a-3p-C1QB axis (22). We examined the miRNAs that interact with lncRNA TEX41 in liver cancer through the use of the starBase database and found that miR-200a-3p plays a role in the occurrence and development of liver cancer. Other research (23) indicates that circBACH1 competes endogenously with miR-200a-3p to promote the replication of hepatitis B virus and the development of liver cancer. Meanwhile, other work (24) has found that miR-200a-3p can be used in the diagnosis and prognostic evaluation of liver cancer and that it is correlated with tumor stage. In our study, we found that silencing lncRNA TEX41 in SK-HEP-1 and HCCLM3 cells resulted in increased expression of miR-200a-3p and inhibited the proliferation and migration of liver cancer cells; this indicates a similar competitive effect between lncRNA TEX41 and miR-200a-3p as that reported previously. In addition, we predicted the target gene BIRC5 of miR-200a-3p using the starBase and GEPIA2 databases. One study reported that BIRC5, as an autophagy-related gene, plays an important role in predicting the prognosis of liver cancer (25). In addition, Li et al. indicated that BIRC5 is a target for the diagnosis and prognosis of hepatitis B virus-related HCC (26). In our study, we found that silencing lncRNA TEX41 reduced the expression of BIRC5 in the SK-HEP-1 and HCCLM3 HCC cell lines.

Conclusions

In summary, silencing lncRNA TEX41 may inhibit the biological functions of liver cancer cells, and its mechanism may involve the targeting of the miR-200a-3p-BIRC5 molecular axis. However, although we found that silencing lncRNA TEX41 may affect the biological behavior of the SK-HEP-1 and HCCLM3 cell lines through the miR-200a3p-BIRC5 axis, this was only examined at the cell level. Therefore, further validation will be conducted at the in vivo level.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-812/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-812/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-812/prf

Funding: This study was supported by the Nantong Health Bureau Foundation (No. MB2020007) and the Nantong Municipal Science and Technology Plan Project (No. JCZ21092).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-812/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Nantong University (No. 2024KT186). Informed consent was taken from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)