Mechanistic study of LINC01296 mediating TRIP13 regulation of neuroblastoma progression

Introduction

Neuroblastoma (NB) is an embryonal cancer arising from neural crest cells, most commonly occurring in the adrenal medulla or parasympathetic ganglia of the spinal cord, and is the second most common extracranial malignant tumor in childhood and the most common solid tumor in infancy (1-4). Most children with stage 1 and 2 disease can be cured by surgery alone (5,6). In addition, most infants with disseminated disease have good outcomes after chemotherapy and surgery (7,8). In contrast, most children over 1 year of age with advanced NB die of progressive disease despite intensive multimodal therapy (9). There are approximately 600 new cases of NB in the United States each year, with an incidence rate of approximately 1 in 7,000 births (10). Although NB accounts for disproportionately high morbidity and mortality among childhood cancers, it has one of the highest rates of spontaneous and complete degeneration. Due to the rapid progression and high malignancy of advanced NB, the prognosis is consistently unsatisfactory, with a 5-year overall survival rate of 30–40% (11,12). This underscores the critical need to identify novel molecular drivers and therapeutic targets to improve outcomes. Therefore, it is necessary to understand the molecular mechanisms of NB and develop new therapeutic targets for NB.

Long non-coding RNA (lncRNA) is a nucleotide greater than 200 nucleotides in length. The human genome is extensively transcribed, generating thousands of lncRNA. Many of the lncRNAs are aberrantly expressed in a variety of tumors, and the aberrant expression of lncRNA has been shown to play an important role in regulating tumor biological behaviours, including tumor angiogenesis, drug resistance, apoptosis and metastasis (13-16). As a lncRNA that has attracted much attention in recent years, LINC01296 has been reported to be significantly upregulated in lung cell carcinoma, ovarian cancer, colorectal cancer, hepatocellular carcinoma, and others (17-19). In the GEPIA database, we found that LINC01296 was highly expressed in a variety of tumors. LINC01296 has been also identified as a key prognostic marker in NB, where its upregulation is significantly associated with high-risk disease and poor patient survival (20). However, the mechanism of LINC01296 in NB have not been systematically elaborated.

Thyroid hormone receptor interacting protein 13 (TRIP13) plays a key role in regulating mitotic processes, including spindle assembly checkpoints and DNA repair pathways, which may be responsible for chromosomal instability (CIN). Since CIN is a major hallmark of cancer, TRIP13 may act as a tumor susceptibility locus (21,22). The oncogenic role of TRIP13 has been of great interest, involving several aspects of malignant transformation such as cancer cell proliferation, drug resistance and tumor progression (22). A growing number of studies have shown that TRIP13 is overexpressed in several forms of cancer and is usually associated with low survival (23). It is worth noting that currently, there are no studies that systematically explore whether LINC01296 and TRIP13 have functional overlap or synergy in NB.

Based on the fact that high expression of LINC01296 in NB cells promotes malignant cell proliferation and cycle alterations, and that TRIP13 has the ability to regulate cell migration, in this study we focused on the roles of LINC01296 and TRIP13 in regulating NB in vivo and in vitro, and clarified that LINC01296 can mediate the role of TRIP13 in promoting the tumor cell proliferation, providing potential biomarkers and therapeutic targets for the diagnosis and treatment of tumor metastasis. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1255/rc).

Methods

Cell culture

The NB cell line SK-N-SH was obtained from Zolgene Biotechnology Co., Ltd. (Fuzhou, China). It was removed from liquid nitrogen and quickly placed in a 37 ℃ water bath. After lysis, the cells were transferred to a centrifuge tube containing 5 ml of medium. Cells were collected by centrifugation at 1,000 rpm for 5 min at room temperature, and the supernatant was discarded; the cells were suspended in minimum essential medium (MEM) complete medium containing 10% fetal bovine serum (FBS) +1% non-essential amino acids (NEAAs) +1% penicillin-streptomycin (P/S) double antibody (Boster, Wuhan, China), inoculated in medium, gently blown to mix, and incubated at 37 ℃, 5% CO₂ and 70–80% humidity.

Lentivirus infection experiment

pLVZG-U6-ZsGreen1-Puro (Zolgene Biotechnology Co., Ltd., #ZVE1006) BamHI-EcoRI, pLVZG-CMV-3xflag-copGFP-Puro (Zolgene Biotechnology Co., Ltd., #ZVE1009) XbaI-NotI was provided by Zolgene Biotechnology (Fuzhou, China). Human cDNA was used as template to amplify TRIP13 and LINC01296 fragments, and the TRIP13 and LINC01296 target fragments were ligated to pLVZG-U6-ZsGreen1-Puro by homologous recombination, ligated at 50 ℃ for 30 min, ligated product was added to T1 E. coli receptor cells, mixed in ice bath for 30 min, heat- excited at 42 ℃ for 90 s, immediately to the ice for 2 min, add 500 µL of non-antibiotic LB liquid medium, 37 ℃ shaking bed shaking 1 h, take 150 µL coated in LB solid medium containing 100 µg/mL ampicillin antibiotic, blow-drying, and put into the 37 ℃ incubator culture overnight. A single clone was picked in LB liquid medium containing 100 µg/mL ampicillin antibiotic for polymerase chain reaction (PCR) verification and positive clones were sent for testing. The cell culture medium was changed and virus-infected cells were added. After mixing, the cells continued to be cultured. cells were observed after 8–12 hours and replaced with fresh medium. Fluorescence expression was observed after 3–4 days of infection, and the medium was changed halfway to maintain cell activity. By observing the effect of cell infection, the infection conditions and parameters of target cells were determined to obtain the SK-N-SH cell lines of sh-LINC01296, OE-LINC01296, sh-TRIP13, OE-TRIP13, sh-LINC01296 + OE-TRIP13 and sh-TRIP13 + OE-LINC01296.

Reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted using NucleoZol RNA Extraction Reagent (Gene CO., Ltd., Shanghai, China, 740404.200). cDNA was synthesised using SweScript RT I First Strand cDNA Synthesis Kit (Servicebio, Wuhan, China, G3330-100). RT-qPCR system was: 2×SYBR The RT-qPCR system was: 2× SYBR Master Mix (Servicebio, Fuzhou, China, CW0957) 12.5 µL, forward primer (10 µM) 1 µL, reverse primer (10 µM) 1 µL, cDNA sample 2 µL and ddH₂O 8.5 µL, total volume 25 µL. The RT-qPCR reaction program was: 95 ℃ for 5 min (95 ℃ 10 s, 60 ℃ 30 s, 72 ℃ 30 s, 40 cycles). Relative gene expression was calculated automatically using 2^−ΔΔCt. In this study, β-actin served as the internal reference gene, all primer sequences are listed in Table S1.

Cell proliferation

The cells of each experimental group in logarithmic growth phase were trypsin digested and resuspended into cell suspension with complete medium, and the cell concentration was adjusted to 1×10⁶ cells/mL. The diluted single cell suspension was added to 96-well plates with three replicate wells for each concentration. The plates were incubated at 37 ℃ in an incubator for 0, 24, 48 and 72 h; 10 µL 10% Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China), was added to continue the incubation, and the optical density (OD) values were measured at 450 nm after 1 h by microplate reader (Molecular Devices, Shanghai, China), respectively.

Cell migration

After trypsin digestion of the cells of each experimental group in the logarithmic growth phase, the complete medium was resuspended into a cell suspension, and the concentration of the suspension was adjusted to 3×10⁵ cells/mL, added to the upper chamber of Transwell (Corning Inc., Corning, NY, USA) and continue to culture for 24 h. Add 70 µL of cell suspension to each well of the cell insert. When all the cells were adherent and just full-grown, the wound healing inserts were lifted out with forceps and serum-free medium was added for 0 h. The wound healing was observed for 24 h. The cells were then removed from the culture dish and the wound healing was monitored.

Transwell assay

The Martrigel matrix gel (Corning Inc., Corning, NY, USA) was placed in the refrigerator at 4 ℃ until it was completely melted, and the Martrigel standard gel was diluted with serum-free medium at 1:8 on ice; 50 µL of diluted Martrigel gel was taken to cover the upper chamber of the Transwell, air-dried at 4 ℃, and allowed to solidify completely at 37 ℃. Each group of 1×10⁵ cells/mL was inoculated with 100 µL of Transwell, and then the Transwell was cultured in a well plate with 10% FBS medium; after culturing the cells for 24 h, the Transwell was removed, and the cells inside the chamber and the residual Matrigel gel were wiped with a cotton swab, and the cells at the bottom back of the chamber were washed with phosphate-buffered saline (PBS) for three times. The cells were fixed by paraformaldehyde (Servicebio, Wuhan, China, G1101-500) at the bottom and back of the chamber, stained with crystal violet, counted by microscopy and analysed for graphs.

Flow cytometry

Cells were collected by digestion with trypsin after incubation at 37 ℃ in a 5% CO₂ incubator for an appropriate period of time. The cells were washed twice with 1× PBS at 4 ℃, and then centrifuged at 1,000 rpm for 3 min at room temperature, and then the precipitate was collected. Then 300 µL of 1× Binding Buffer was added to suspend the cells. Annexin V-ALEXA 647 labelling: add 5 µL of Annexin V-ALEXA 647 and mix well, then incubate for about 15 min under the condition of avoiding light and at room temperature. For propidium iodide (PI) labelling, add 5 µL of PI staining 5 min before mounting. Before loading, 200 µL of 1× Binding Buffer was added. The apoptosis of cells in each group was calculated by flow cytometry (Agilent, New York, USA, NovoCyte 2060R).

Animal study

Non-Obese Diabetic/severe combined immunodeficiency (NOD/scid) mice, aged 6–8 weeks, were purchased from SBF (Beijing) Biotechnology Co., Ltd. (Beijing, China). Feeding conditions: light and darkness alternated for 12 h, relative humidity 50%±10%, temperature 23±2 ℃, given free access to food and water. A total of 30 mice were randomly divided into 5 cages according to the random number method, with 1 cage for every 6 mice, and kept for 1 week to adapt to the environment before receiving the tumor cells. Cells were pre-inoculated in a 10 cm dish and cultured in an incubator until the cell density was about 80–90%, trypsin digestion was performed for cell counting, and the cell number was adjusted to 1×10⁷ cells/mL in each group, 900 rpm, centrifuged for 4 min, and the precipitate was resuspended with 3,000 µL of matrix gel, and stored on ice. Groups: mice from the same group were housed in the same cage (one cage per group), and there were a total of 5 groups [negative control (NC) group, sh-LINC01296 group, sh-TRIP13 group, sh-LINC01296 + OE-TRIP13 group and sh-TRIP13 + OE-LINC01296 group]. Subcutaneous inoculation: the inoculation site was the axilla of the right forelimb of the mice, after local skin disinfection, the needle was inserted about 1–1.5 cm from the inoculation point and then submerged under the skin, the direction of the needle was changed and then the cell suspension was injected, after a semicircular mound was formed in the subcutaneous area, the needle was withdrawn, the needle eye was pressed with a wet swab, and after confirming that there was no leakage of the cell suspension, the mice were put back to the mouse cage and kept waiting for the formation of a tumor. Observation of the growth status of mice after inoculation of tumors: observe whether the inoculated part of mice is infected every day, observe the growth of tumors, and measure the long diameter (L) and short diameter (W) of the tumors of mice with vernier calipers every 2 days. Once the diameter of the tumor exceeded 20 mm, the mice were anesthetized and then sacrificed. After monitoring the mice for 23 days, after anesthesia with intraperitoneal injection of 62.5 mg/kg pentobarbital sodium (Fujian Children’s Hospital, Fuzhou, China), the animals were sacrificed by dislocation, and the subcutaneous tumor tissue was peeled off and photographed. Two of each group were taken and fixed in paraformaldehyde fixative, and the other 20 copies of tumors were stored at −80 ℃ for spare use. All animal experiments were performed under a project license (No. 2020KY074) granted by the Ethics Committee of Fujian Maternal and Child Health Hospital, in compliance with the Chinese Regulations for the Administration of Laboratory Animals for the care and use of animals. A protocol was prepared before the study without registration.

Immunohistochemistry (IHC)

Paraffin sections were dewaxed, hydrated and rinsed with running water. Sections were treated with 10% sodium citrate at 105 ℃ for 10 min to extract antigen. Endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol for 15 min at room temperature. After washes with PBS, tissues were closed with 5% normal donkey serum and incubated with the TRIP13 antibody (1:1,000) (Proteintech, Wuhan, China, 19602-1-AP) at 4 ℃ overnight. Sections were further incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:500) (Boster, Wuhan, China, SV0004) at room temperature for 2 h. Immunoreactivity was visualized using 3,3’-diaminobenzidine (DAB) substrate and then sections were counterstained with hematoxylin for 30 s, dehydrated, cleared and mounted. Images were observed under a microscope.

Statistical analysis

All experiments were subject to three technical replicates. Data were processed using SPSS 26.0 software and the corresponding histograms were plotted using GraphPadPrism 8.0 software, and all statistical results were dose information, expressed as mean ± standard deviation. Two-tailed Student’s t-test and one-way analysis of variance (ANOVA) was used, with P<0.05 for significant differences.

Results

LINC01296 promotes proliferation, migration and invasion of NB

To investigate the function of LINC01296 in NB cell lines, we constructed LINC01296 overexpression recombinant plasmid (pLvx-myc-LINC01296-puro) and knockdown plasmid (pLKO-sh-LINC01296), respectively. Lentiviral transfection of the LINC01296 recombinant plasmid was used to promote the expression of LINC01296 in NB cells, and the expression of LINC01296 in NB cells was knocked down by small hairpin RNA (shRNA). The constructed plasmids were transfected into SK-N-SH cells and the transfection efficiency was verified by the RT-qPCR. The results showed that the relative mRNA level of LINC01296 was significantly reduced after transfection of LINC01296-shRNA, while it was increased after transfection of LINC01296 (Figure 1A). This indicated that SK-N-SH cells were successfully transfected in this study. Next, we determined the role of LINC01296 in the growth of NB. CCK-8 kit was used to detect cell proliferation, and the results showed that overexpression of LINC01296 promoted the proliferation of SK-N-SH cells, whereas knockdown of LINC01296 significantly inhibited cell viability (Figure 1B). Similarly, the effects of LINC01296 on SK-N-SH cell migration and invasion were investigated using scratch and Transwell assays, respectively, and the results showed that overexpression of LINC01296 promoted SK-N-SH cell migration and invasion, whereas knockdown of LINC01296 markedly inhibited cell migration (Figure 1C) and invasion (Figure 1D). These results suggest that LINC01296 may promote NB cell proliferation, migration and invasion.

Figure 1 LINC01296 promoted the proliferation, migration and invasion of SK-N-SH cells, and inhibited apoptosis (mean ± SD, n=3). (A) RT-qPCR verified the transfection effect of LINC01296 gene after overexpression and knockdown in SK-N-SH cells. (B) The proliferation of LINC01296 gene after overexpression and knockdown in SK-N-SH cells was detected by CCK-8 method. (C) Transwell detected the migration of LINC01296 gene after overexpression and knockdown in SK-N-SH cells. The images were representative at ×10 magnification. (D) Transwell detected the invasion of LINC01296 gene after overexpression and knockdown in SK-N-SH cells. Invaded cells were stained with crystal violet, and the images were representative at ×10 magnification. All data were repeated three times and analyzed using two-tailed Student’s t-test or one-way ANOVA, with P<0.05 for significant differences, the same below. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation.

TRIP13 promotes NB proliferation, migration and invasion

Since TRIP13 is overexpressed in NB and expression is positively correlated with LINC01296, TRIP13 may also influence NB progression. To investigate the role of TRIP13 in NB cells. We constructed a TRIP13 gene overexpression recombinant plasmid (pLVX-myc-TRIP13-puro), designed TRIP13 specific shRNA (pLKO-shTRIP13), prepared OE-TRIP13 and shRNA-TRIP13 lentiviruses, and established TRIP13-stabilised OE-TRIP13 and sh-TRIP13 SK-N-SH cell lines. The relative expression of TRIP13 was significantly increased in cells transfected with OE-TRIP13, while that of shRNA-TRIP13 was significantly decreased in cells transfected with shRNA-TRIP13, indicating that the transfection was successful (Figure 2A). CCK-8 assay showed that TRIP13 overexpression promoted the proliferation of SK-N-SH cells, whereas TRIP13 knockdown significantly inhibited the cell proliferation (Figure 2B). To explore the effects of TRIP13 on cell migration and invasion, we performed scratch and Transwell assays. TRIP13 overexpressed cells showed significantly enhanced migration and invasion ability, while TRIP13 knockdown cells showed significantly weakened migration and decreased cell invasion ability compared with the NC group (Figure 2C,2D). These results suggest that TRIP13 may promote NB proliferation, migration and invasion.

Figure 2 TRIP13 promoted the proliferation, migration and invasion of SK-N-SH cells, and inhibited apoptosis (mean ± SD, n=3). (A) RT-qPCR verified the transfection effect of LINC01296 gene after overexpression and knockdown in SK-N-SH cells. (B) The proliferation of TRIP13 gene after overexpression and knockdown in SK-N-SH cells was detected by CCK-8 method. (C) Transwell detected the migration of TRIP13 gene after overexpression and knock-down in SK-N-SH cells. The images were representative at ×10 magnification. (D) Transwell detected the invasion of TRIP13 gene after overexpression and knockdown in SK-N-SH cells. Invaded cells were stained with crystal violet, and the images were representative at ×10 magnification. CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TRIP13, thyroid hormone receptor interactor 13.

In vitro studies show that LINC01296 mediates the role of TRIP13 in promoting tumor cell proliferation

To further demonstrate that LINC01296 mediates TRIP13 to play a role in promoting tumor cell proliferation. In vitro study of LINC01296 mediated TRIP13 play a role in promoting tumor cell proliferation. In SK-N-SH cell line with LINC01296 stable knockdown, the lentiviral vector overexpressing TRIP13 was introduced, and cell growth curves were used to observe the changes in the proliferation ability of the cells after LINC01296 stabilised knockdown and TRIP13 was overexpressed, and the proliferation ability of the sh-LINC01296 + OE-TRIP13 was significantly lower than that of the OE-TIRP13 group (Figure 3A). Changes in cell motility were observed by cell scratch assay and Transwell assay, and the migration and invasion abilities of sh-LINC01296 + OE-TRIP13 were significantly reduced compared with the OE-TIRP13 group (Figure 3B,3C). Flow cytometry analysis showed that apoptosis was significantly increased in sh-LINC01296 + OE-TRIP13 compared to OE-TIRP13 group (Figure 3D). Similarly, in the SK-N-SH cell line with stable knockdown of TRIP13, lentiviral vector overexpressing LINC01296 was introduced, and cell growth curves were used to observe the changes in the proliferative capacity of the cells after TRIP13 silencing and LINC01296 overexpression, and the proliferative capacity of the OE-LINC01296 + sh-TRIP13 was significantly higher than that of the OE-TIRP13 group (Figure 3D). LINC01296 group was significantly reduced (Figure 4A). Changes in cell motility were observed by cell scratch assay and Transwell assay, and the migration and invasion abilities of OE-LINC01296 + sh-TRIP13 were significantly reduced compared with the OE-LINC01296 group (Figure 4B,4C). Flow cytometry analysis showed that apoptosis was significantly higher in the OE-LINC01296 + sh-TRIP13 compared to the OE-LINC01296 group (Figure 4D). In summary, LINC01296 mediated TRIP13 to play a role in promoting tumor cell proliferation.

Figure 3 In vitro analysis LINC01296 mediated TRIP13 to promote SK-N-SH cell development (mean ± SD, n=3). (A) The proliferation of LINC01296 stabilized knockdown with TRIP13 overexpression were detected by CCK-8 method. (B) Transwell detected LINC01296 stabilized knockdown and TRIP13 overexpression of cells migration. The images were representative at ×10 magnification. (C) Transwell detected LINC01296 stabilized knockdown and TRIP13 overexpression of cell invasion. Invaded cells were stained with crystal violet, and the images were representative at ×10 magnification. (D) Flow cytometry was used to detect LINC01296 stabilized knockdown and TRIP13 overexpression of cells apoptosis. CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density; SD, standard deviation; TRIP13, thyroid hormone receptor interactor 13.

Figure 4 In vitro analysis LINC01296 mediated TRIP13 to promote SK-N-SH cell development (mean ± SD, n=3). (A) The proliferation of TRIP13 stabilized knockdown with LINC01296 overexpression were detected by CCK-8 method. (B) Transwell detected TRIP13 stabilized knockdown and LINC01296 overexpression of migration cells. The images were representative at ×10 magnification. (C) Transwell detected TRIP13 stabilized knockdown and LINC01296 overexpression of cell invasion. Invaded cells were stained with crystal violet, and the images were representative at ×10 magnification. (D) Flow cytometry was used to detect TRIP13 stabilized knockdown and LINC01296 overexpression of cells apoptosis. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density; SD, standard deviation; TRIP13, thyroid hormone receptor interactor 13.

In vivo study of LINC01296 mediates TRIP13 to play a role in promoting tumor cell proliferation

To further investigate LINC01296 mediated TRIP13 play a role in promoting tumor cell proliferation. We established a subcutaneous transplantation NB tumor model in NOD/scid mice, and statistically analysed the growth curves by observing the tumor growth in the NC, sh-LINC01296, sh-TRIP13, sh-LINC01296 + OE-TRIP13, and sh-TRIP13 + OE-LINC01296 groups, and statistically analysing the effects of LINC01296 and TRIP13 on the tumor-forming ability of NB in vivo. The results showed that the tumor growth in the sh-LINC01296 and sh-TRIP13 groups was significantly smaller compared with that of the NC group, the tumor growth in the sh-LINC01296 + OE-TRIP13 group was almost unchanged compared with that of the NC group, and the tumor growth in the sh-TRIP13 + OE-LINC01296 group was between the same as that of the NC group, sh-LINC01296 group and sh-TRIP13 group (Figure 5A,5B). The above results suggest that LINC01296 mediates TRIP13 to play a role in promoting tumor cell proliferation.

Figure 5 In vivo analysis LINC01296 mediates TRIP13 to promote SK-N-SH cell development. (A) Representative anatomical diagrams of the xenograft tumors harvested from NOD/scid mice in each group on day 23 (mean ± SD, n=6). (B) Growth curve of xenograft tumors in NOD/scid mice in each group from day 12 to 23. (C,D) The expression of LINC01296 and TRIP13 in tumor tissues of NOD/scid mice was detected by RT-qPCR (mean ± SD, n=3). (E) The expression of LINC01296 and TRIP13 in tumor tissues of NOD/scid mice was analyzed by IHC. The staining was performed using a TRIP13-specific antibody with DAB (brown) development and hematoxylin counterstaining (blue). The images were shown at ×20 magnification. IHC, immunohistochemistry; NC, negative control; NOD/scid, non-obese diabetic/severe combined immunodeficiency; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; TRIP13, thyroid hormone receptor interactor 13.

The tumor tissues obtained from NOD/scid mice were further executed, and the expression of LINC01296 and TRIP13 was detected using the RT-qPCR. The results showed that the mRNA level of LINC01296 was significantly down-regulated in the sh-LINC01296 group, the sh-TRIP13 group, and the sh-LINC01296 + OE-TRIP13 group and was significantly up-regulated in the sh-TRIP13 + OE-LINC01296 group (Figure 5C). While the mRNA level of TRIP13 was significantly down-regulated in the sh-LINC01296 group, sh-TRIP13 group, sh-TRIP13 + OE-LINC01296 group, but in the sh-LINC01296 + OE-TRIP13 group, the mRNA level of TRIP13 was significantly upregulated (Figure 5D). IHC staining was also used to observe LINC01296 and TRIP13 using laser confocal microscopy, and the results showed that the expression of TRIP13 was significantly down-regulated in the sh-LINC01296 group, the sh-TRIP13 group, and, the sh-TRIP13 + OE-LINC01296 group compared with the NC, and the expression of TRIP13 was significantly down-regulated in the sh-LINC01296 + TRIP13 expression was significantly up-regulated in the OE-TRIP13 group (Figure 5E).

Discussion

NB is an embryonic malignant tumor originating from neural crest cells and is also one of the more common extracranial malignant tumors in children (1). Although the treatment methods for NB have been continuously improving in recent years, the prognosis of advanced and high-risk patients remains a cause for concern (8). Therefore, an in-depth exploration of the molecular mechanism of NB and the search for new diagnostic markers and therapeutic targets are of crucial significance for improving the prognosis of patients.

lncRNA is a type of non-coding RNA with a length of more than 200 nucleotides, which plays a key role in processes such as gene expression regulation, transcription and post-transcriptional processing (24,25). In recent years, a large number of studies have revealed the close connection between abnormal expression of lncRNA and the occurrence and development of various tumors. LINC01296, a lncRNA that shows high expression in various tumors, has been confirmed to be carcinogenic (18,19,26). Nevertheless, the specific mechanism of action of LINC01296 in NB has not been fully studied. In this study, by establishing a SK-N-SH cell model with overexpression and knockdown of LINC01296, it was revealed that LINC01296 significantly promoted the proliferation, migration and invasion abilities of NB cells, while its knockdown inhibited these biological behaviors. This discovery is consistent with the carcinogenic effect of LINC01296 in other tumors, suggesting that LINC01296 may play a key carcinogenic role in NB. TRIP13 is a protein integral to the process of cell mitosis and has been confirmed to exhibit high expression in various tumors, correlating with their malignant progression and poor prognosis (27). This study also further confirmed the carcinogenic effect of TRIP13 in NB by constructing a SK-N-SH cell model with overexpression and knockdown of TRIP13. Furthermore, we also found that the expression level of TRIP13 was positively correlated with that of LINC01296, suggesting that there might be a synergistic effect between the two. Given that LINC01296 regulates gene expression during DNA repair and chromosome remodeling, and considering TRIP13’s pivotal role in the DNA repair pathway, we hypothesize that LINC01296 might facilitate tumor progression by modulating the expression of TRIP13.

To deeply investigate the interaction mechanism between LINC01296 and TRIP13, this study introduced lentiviral vectors overexpressing TRIP13 into cells with stable LINC01296 knockdown. The effects on cell biological behavior were observed through experiments on cell proliferation, migration, and invasion. The experimental results indicated that in cells with LINC01296 knockdown, the overexpression of TRIP13 did not significantly restore the cells’ proliferation, migration, and invasion abilities. This outcome suggests that LINC01296 plays a crucial mediating role in the behavior of tumor cells influenced by TRIP13. Conversely, in cells with TRIP13 knockdown, the overexpression of LINC01296 also did not significantly restore the cells’ proliferation ability, which further confirmed the synergistic effect between the two. This interaction may be achieved through multiple mechanisms. On one hand, LINC01296 might affect its intracellular localization and function via direct interaction with TRIP13. On the other hand, LINC01296 could influence the activity of TRIP13 by regulating its expression level or post-translational modifications. Additionally, LINC01296 and TRIP13 might jointly promote the proliferation, migration, and invasion of tumor cells by regulating downstream target genes or signaling pathways, thereby affecting processes such as the cell cycle, apoptosis, and cytoskeleton remodeling. Meanwhile, we established a subcutaneous transplanted tumor model in NOD/scid mice and evaluated the effects of LINC01296 and TRIP13 on tumor formation by observing the growth of the tumors. The results indicated that the knockdown of LINC01296 and TRIP13 significantly inhibited tumor growth, while their overexpression promoted tumor formation. This is consistent with the results of in vitro experiments, further confirming the carcinogenic effects of LINC01296 and TRIP13 in NB.

The results above indicate that the expression levels of LINC01296 and TRIP13 can be utilized for the diagnosis and prognosis evaluation of NB. Targeted treatment strategies for these biomarkers, such as small molecule inhibitors or RNA interference techniques, may provide new insights for treatment. Despite the clear mechanistic insights provided by our study, it is important to acknowledge its limitations. Our findings are primarily derived from experiments conducted in the SK-N-SH cell, the generalizability of the LINC01296/TRIP13 axis to all NB subtypes requires further validation. Future investigations should aim to corroborate these results in a broader spectrum of NB models to confirm the universal role of this pathway in the disease. In addition, the precise molecular mechanism by which LINC01296 regulates TRIP13, such as through direct binding or transcriptional control, remains to be fully elucidated and is the focus of our ongoing research.

Conclusions

This study delved deeply into the mechanism of LINC01296 in NB, particularly its impact on the proliferation, migration, and invasion of tumor cells through the regulation of TRIP13. Through in vitro cell experiments and in vivo animal model studies, we uncovered the pivotal roles of LINC01296 and TRIP13 in the progression of NB and suggested their potential as biomarkers and therapeutic targets.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1255/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1255/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1255/prf

Funding: This work was supported by the Health Science and Technology Project of Fujian Province (No. 2020CXA018), the Fujian Provincial Natural Science Foundation of China (No. 2021J01418), and the Startup Fund for Scientific Research, Fujian Medical University (No. 2023QH1222).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1255/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. All animal experiments were performed under a project license (No. 2020KY074) granted by the Ethics Committee of Fujian Maternal and Child Health Hospital, in compliance with the Chinese Regulations for the Administration of Laboratory Animals for the care and use of animals.

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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