Inhibition of LINC01048 decreases cell proliferation and induces the apoptosis in gastric cancer

Abstract

With advancements in the study of long non-coding RNAs (lncRNAs) as significant biomarkers and therapeutic targets in various human diseases, including cancer, neurodegenerative conditions, and genetic disorders, emerging evidence highlights their regulatory roles in the development of gastric cancer (GC). However, the mechanisms underlying these functions remain largely unknown. In this study, we investigate the role of the LINC01048 in tumorigenesis and cell proliferation in GC cell lines, such as HGC27 and MKN45. Analysis of publicly available databases and experimental validation revealed that LINC01048 expression is elevated in GC tissues and cell lines and is associated with poor prognosis. Knockdown of LINC01048 significantly suppressed cell proliferation and induced apoptosis in GC cells. Additionally, depletion of LINC01048 reduced the nuclear localization of β-catenin while increasing its cytoplasmic retention. Furthermore, treatment with SKL2001, a Wnt/β-catenin pathway activator, reversed the effects of LINC01048 knockdown on cell proliferation and apoptosis. In vivo experiments confirmed that LINC01048 depletion decreased tumor growth in GC xenograft models. Overall, these findings suggest that LINC01048 plays an oncogenic role in GC by promoting cell proliferation and modulating the Wnt/β-catenin pathway. Therefore, LINC01048 may serve as a potential therapeutic target for GC treatment.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-02994-2.

Introduction

Gastric cancer (GC) is a significant global health concern, ranking as the fifth most common cancer and the third leading cause of cancer-related deaths worldwide [1, 2]. From traditional therapies and early targeted treatments to today’s precision medicine, the field of cancer treatment has achieved remarkable progress. Despite these advancements, drug resistance and high medical costs remain critical challenges that demand urgent resolution. With the rapid development of AI technology, the integration of genomics and proteomics data to optimize personalized treatment strategies may emerge as a central focus of future research. Therefore, it is urgent to explore novel molecular biomarkers for early diagnosis. GC is highly lethal, particularly in cases of metastatic disease, with a median survival of less than one year. Current treatment approaches primarily include surgical or endoscopic resection combined with chemotherapy. Although advancements in the clinical treatment of GC have been made, searching for novel and effective therapeutic targets for GC remains a pressing challenge, except vascular endothelial growth factor receptor 2 (VEGFR-2), receptor tyrosine protein kinase erbB-2 (HER2), and programmed cell death protein 1 (PD-1) and its ligands (PD-L1) for treating GC. Therefore, by exploring new avenues of research, we can uncover potential therapeutic targets and strategies that may improve patient outcomes and enhance the management of this devastating disease.

Increasing evidence recently has shown that epigenetic changes, such as aberrant DNA methylation, histone modifications, and noncoding RNAs (ncRNAs) expression, play substantial roles in the development and progression of malignancies. Among these changes, long non-coding RNAs (lncRNAs), a novel class of ncRNAs, which defined as transcripts exceeding 200 nucleotides, regulate gene expression at transcriptional, and post-transcriptional levels. Furthermore, Initial investigations found that lncRNAs have drawn attention as potential biological regulators involved in various cellular processes, including cell proliferation and apoptosis, migration, and invasion [3, 4].

Previous studies have shown that the aberrant expression of lncRNAs play a vital role in the tumorigenesis and progression of various types of human cancers [5–7]. For example, previously referred to as LINC01212 with a crucial role in melanoma survival has provided proof-of-principle that targeting a lncRNA in vivo is a potentially viable therapeutic option [8]; LINC00152 was also shown to bind to PRC2 protein, thereby inhibiting the expression of interleukin-24 and promoting Lung adenocarcinoma (LAD) cell proliferation [9]; Additionally, The long noncoding RNA HOTAIR activates the Hippo pathway by directly binding to SAV1 in renal cell carcinoma [10]. Therefore, these non-coding RNAs (ncRNAs) may impact normal gene expression and disease progression, making them a new class of targets for drug discovery [11]. Several studies have implicated lncRNAs in regulating GC progression through several mechanisms, including enhanced cell cycle progression and tumorigenesis via EGR1 mediated lnc01503 [12], increased cell proliferation and metastasis by LncRNA CASC11 [13], and inhibition of gastric cancer cell proliferation and metastasis by lncRNA MEG3 through activation of p53 [14]. The SP-1-induced overexpression of tissue differentiation-inducing non-protein coding RNA was previously shown to promote cell proliferation by influencing KLF2 mRNA stability in gastric cancer [15]. Moreever, targeting LINC00649 and LINC01296 could hinder cell proliferation and accelerate lung cancer cell apoptosis, suggesting that LINC00649 and LINC01296 may function as a potential biomarker for lung cancer and esophageal carcinoma treatment, respectively [16, 17]. Strikingly, Helicobacter pylori (H. pylori) infection is the greatest known risk factor for gastric cancer (GC). However, several studies provides insights into the involvement of lncRNAs and mRNAs in the pathogenesis of H. pylori infection and has identified several potential biomarkers that might help improve the strategy for the diagnosis and treatment of H. pylori-associated gastric carcinogenesis [18]. Therefore, exploring the functions and mechanisms of lncRNAs in GC is of great important.

Based on our current study, we have identified LINC01048 as a novel cancer-related long non-coding RNA (lncRNA) transcribed from an intergenic region of chromosome 13. Previous studies have implicated LINC01048 in various cancers, including its role as an oncogene in human cutaneous squamous cell carcinoma, where it activates YAP1 transcription through binding to TAF15 [19]. Moreover, LINC01048 has been associated with overall survival and disease-free survival in patients with esophageal squamous cell carcinoma. However, the functional significance of LINC01048 in gastric cancer (GC) is poorly characterized. Therefore, we aimed to elucidate the role of LINC01048 in GC by conducting in vitro and in vivo experiments. We focused on investigating the impact of LINC01048 knockdown on GC cell proliferation and apoptosis, aiming to shed light on the functional mechanisms underlying LINC01048 in the context of GC.

Our results provide valuable insights into the involvement of LINC01048 in GC progression, highlighting its potential as a target for therapeutic interventions. Understanding the role of LINC01048 in GC will contribute to the broader knowledge of molecular mechanisms underlying GC development and may pave the way for the development of novel diagnostic and therapeutic strategies.

Methods and reagent

Cell culture

The AGS, BGC823, HGC27, and MKN45 gastric cancer cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). A human gastric mucosal epithelial cell line called GES-1 was used as a control and was purchased from the Chinese Academy of Sciences Cell Bank. The cell lines were cultured in DMEM, RPMI 1640, or F-12 K medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) as recommended by the cell line provider. The cells were maintained in a humidified incubator with 5% CO₂ at 37 ℃. Cell were passaged upon reaching 80–90% confluence. Each cell line was used for a limited number of passages, typically 8–10 generations.

Cell transfection

Short hairpin RNAs (shRNAs) targeting LINC01048, namely sh-LINC01048#1, sh-LINC01048#2, and sh-LINC01048#3, along with a negative control shRNA (sh-NC), were synthesized by the Sangon Biotech company (Zhengzhou, China). The shRNAs were individually ligated into the pLKO.1 plasmid. To investigate the functional impact of LINC01048 knockdown in gastric cancer (GC) cells, HGC27 and MKN45 cells were cultured in six-well plates. Upon cell adherence, the plasmids containing the shRNAs were transfected into HGC27 and MKN45 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h after transfection.

RNA pulldown

RNA pulldown assay kit was purchased from the BersinBio CO., Ltd (BersinBio, Guangzhou, China) followed the instruction. Biotin-labelled LINC01048 probe were synthesised by BersinBio and followed by incubation with extracts from gastric cancer cells. Next, the proteins extract from the pulldown were separated using an SDS‐PAGE gel and subsequently detected by western blot analysis.

Reverse transcription‑quantitative polymerase chain reaction (RT‑PCR)

Following the manufacturer’s protocol, RNA was separated via Trizol reagent (Invitrogen, MA, USA). After reverse transcription through PrimeScript RT Reagent Kit (TAKARA, China) of RNA, complementary DNA (cDNA) was obtained. Next, PCR amplification was performed on 5 µL of cDNA using GAPDH as an internal parameter for LNC00853 and mRNA. The levels of genes were measured via applying the 2^{− ΔΔCT} method. All primers applied for RT-PCR were designed and constructed via Sangon Biotech (Shanghai, China) and the primer sequences used within this study are as follows: LINC01048: forward, 5’-CCCAGCCACATAGAATTGTGTC-3’ and reverse, 5’-AGCCGGGATTAGGAGTCTTAAC-3’; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward, 5’-AATCCCATCACCATCTTC-3’ and reverse, 5’-AGGCTGTTGTCATACTTC-3’.

RNA sequencing and TCGA data analysis

RNA-sequencing data and clinical information of GC cancer samples, as well as RNA-sequencing data of 50 adjacent non-cancerous samples were obtained from cBioPortal; we firstly to identify the differentially expressed genes (DEGs) between the control group and cancer group. EnrichR and clusterprofiler were used to conduct GO functional enrichment analyses, which includes biological process (BP), cellular components (CC), and molecular functions (MF). To categorize genes according to their functional significance, GSEA [20] was used. Gene set enrichment analysis (GSEA) was carried out using the GSEA preranked module on the GenePattern server, with log2 fold change values for all detected genes for the indicated comparisons as the ranking metric, and Hallmarks as the gene sets database to be tested for enrichment.

Cell proliferation assay

The impact of LINC01048 knockdown on cell proliferation was assessed using the CCK-8 assay. Transfected HGC-27 and MKN-45 cells (2 × 10³ cells) were seeded into 96-well plates and allowed to attach for 24 h at 37 °C with 5% CO₂. Afterward, 10 µl of Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Mashiki-machi, Kumamoto, Japan) was added to each well and the cells were incubated for an additional 2 h. Cell viability was subsequently measured at different time points (0, 24, 48, 72, and 96 h) with a FLx800 Fluorescence Microplate Reader (BioTek, Winooski, VT, USA) at an absorbance of 450 nm.

Crystal violate staining assay

The crystal violate staining assay was performed by seeding 300 cells/well in a 6-well plate and maintaining them in a CO₂ incubator for 2–3 weeks. The cells were then stained with 0.1% crystal violet regent (dissolve in methanol) for 10 min at room temperature and washed with DI water to remove the excessive staining solution. Finally, the plate was photographed.

Flow cytometry analysis

To investigate the impact of LINC01048 knockdown on apoptosis of HGC-27 and MKN-45 gastric cancer cells, we utilized the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Briefly, the transfected cells were seeded at a density of 2 × 10⁴ cells/mL and incubated at 37 ℃ with 5% CO₂ for 48 h. Next, cells were washed twice with PBS, centrifuged, and suspended in binding buffer. The cell suspension was then treated with Annexin V-FITC/PI and incubated in the dark for 15 min. Finally, flow cytometry (BD Biosciences) was used to measure the percentage of apoptotic cells.

Nuclear-cytoplasmic fractionation

Cells were harvested and washed twice with cold PBS 48 h after transfection of Linc01048. The NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific) was used to separate nuclear and cytoplasmic fractions from Gastric adenocarcinoma HGC27 and MKN45 cells according to the manufacturer’s instructions. The cells were lysed using cell lysis buffer containing phosphocreatine (ATP) on ice for 10 min after gentle shaking for 30 s. The lysates were centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was collected as the cytoplasmic fraction while the pellet was collected as the nuclear fraction. Total protein content in the nuclear and cytoplasmic fractions was measured using the BCA Protein Assay Kit (Beyotime Biotechnology). Subsequently, we used RT-qPCR to detect the expression levels of LINC01048 in the nuclear and cytoplasmic fractions. Specifically, we extracted total RNA from the nuclear and cytoplasmic fractions using TRIzol reagent (Invitrogen), followed by reverse transcription according to the manufacturer’s instructions. Expression levels of LINC01048 were subsequently detected using qPCR. GAPDH was chosen as the internal reference gene.

Western blotting

Cells were harvested and rinsed with cold PBS. Cells were then treated with RIPA lysis buffer on ice for 30 min and then centrifuge at 12,000 rpm for 15 min. The protein concentration was assessed by the BCA kit. Each lane was loaded with 30–35 µg of protein. Next, the protein samples were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidence difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h and incubated with primary antibodies (1:1000 dilution) at 4 ℃ for overnight. Finally, chemiluminescence (ECL) reagent was used for detection. Prior to incubation with antibodies, all membranes were cut horizontally or vertically based on the molecular weights of the target proteins. This cutting step, a standard part of our protocol, was performed before antibody incubation to reduce non-specific binding. Primary antibodies used within this study are as follows: anti-β-catenin (#8480, CST), anti-H3 (#4499, CST), and anti-GAPDH (#5174, CST). After washing three times, the membrane was incubated with correspondent secondary antibodies (1:3000-1:5000 dilution). The bands were detected with enhanced chemiluminescence and normalized to GAPDH.

In vivo analysis

Four to five-weeks-old BALB/C nude mice were commercially acquired from the Slaccas Laboratory Animal Center (shanghai, China). The mice were randomly divided into two groups (n = 3 per group). HGC-27 cells (1 × 10⁶) stably transfected with sh-LINC01048 or sh-NC were subcutaneously injected into the right flank of the athymic nude mice. The tumor size and body weight of the mice were monitored and measured every three days over a period of 6 weeks. Afterward, the nude mice were sacrificed and the tumor weight and volume were calculated according to the formula: volume = length x width x height x 0.52. All animal studies acquired the approval of the Committee Ethics of Animal Center of Henan Cancer Hospital of Zhengzhou University. The maximal tumor size/burden permitted by the Ethics Committee is no more than 2000 mm³ in volume and the diameters were less than 15 mm in any dimension. No mouse has violated this standard. The maximal tumor size/burden was not exceeded.

Statistical analysis

All experiments were repeated at least two times. All data are shown as mean ± SD. Student’s t-test (two groups) and one-way ANOVA (multiple groups) were utilized to analyze the groups’ comparisons. Kaplan–Meier method and log-rank analysis were used to analyze the survival curves. Data were considered to be statistically significant when p values were less than 0.05. All statistical analyses were conducted using GraphPad Prism 8.0 software.

Results

The expression of LINC01048 in GC and negatively correlated with patient survival

To investigate the biological role of LINC01048 in GC, we initially determined its expression level in various cancer types using the TNMplot database. The analysis revealed that LINC01048 was highly expressed in breast cancer, lung cancer, pancreas cancer, and stomach cancer (Fig. 1A). Furthermore, clinical association analysis revealed that LINC01048 expression was significantly elevated in patients of all stages (Fig. 1B), and was associated with large tumor size (T stage) (Fig. 1C). Based on these results, we also evaluated LINC01048 expression in gastric cancer cell lines and gastric epithelial cells using qPCR. As shown in Fig. 1D. (P < 0.01), and the result have been clarified that LINC01048 was significantly upregulated in gastric cancer cell lines. Moreover, Kaplan-Meier survival curves demonstrated that patients with higher LINC01048 level presented worse overall survival (OS) than those with lower LINC01048 level (Fig. 1E-F). These data indicated that the expression level of LINC01048 showed a significant negative correlation with the overall survival rate and cure rate of GC patients, indicating its potential involvement in GC development. Therefore, we speculated that early detection of lncRNA LINC01048 in patients has predictive value for prognosis, suggesting that this lncRNA could serve as a valuable potential diagnostic marker and therapeutic target for GC patients. Furthermore, to determine the role of LINC01048 plays in GC, we then to silence the LINC01048 in HGC27 and MKN45 cells by transfecting with LINC01048-specific short hairpin RNAs (sh-LINC01048#1, sh-LINC01048#2, and sh-LINC01048#3) or control shRNA (sh-NC), and qRT-PCR was performed to assess the interference efficiency. The results indicated that sh-LINC01048#1 and sh-LINC01048#2 presented the highest interference efficiency (Fig. 1G). Therefore, sh-LINC01048#1 and sh-LINC01048#2 were chosen for use in the subsequent experiments. Above all these data indicated that LINC01048 contributes to the GC proliferation.

Fig. 1.

Expression and functional analysis of LINC01048 in gastric cancer (GC) progression. A Expression levels of LINC01048 in various cancers based on TNMplot database analysis. LINC01048 is highly expressed in breast cancer, lung cancer, pancreatic cancer, and stomach cancer. B Clinical association analysis reveals elevated expression of LINC01048 in GC patients of all stages. C LINC01048 expression is significantly associated with large tumor size (T stage) in GC patients. D qPCR analysis demonstrates heightened expression of LINC01048 in GC cell lines compared to normal gastric epithelial cells. E Analysis of TCGA data reveals increased expression of LINC01048 in GC tumors. F Kaplan-Meier analysis shows that GC patients with high LINC01048 expression have shorter overall survival than those with low LINC01048 expression. G Efficiency of LINC01048 knockdown in HGC27 and MKN45 cells using LINC01048-specific short hairpin RNAs or control shRNA was assessed by qRT-PCR. sh-LINC01048#1 and sh-LINC01048#2 show the highest interference efficiency. Statistical significance was determined using one-way ANOVA. *p < 0.05, p < 0.01, p < 0.001 (n = 3, independent experiments)

Subcellular localization of LINC01048 in gastric cancer (GC)

To explore the underlying mechanism of LINC01048-mediated tumor progression and metastasis of Gastric Cancer. We analyzed RNA-sequencing data and clinical information of gastric cancer samples, along with RNA-sequencing data from adjacent non-cancerous samples, which were obtained from cBioPortal. We then found Gene Ontology (GO) analysis implied that LINC01048 could regulate a series of biological processes that are associated with tumor progression, such as cell proliferation, cell adhesion, and focal adhesion, the results as shown in Fig. 2A. Furthermore, we also conducted gene set enrichment analysis (GSEA) to investigate the potential mechanism underlying the contribution of LINC01048 to the GC through the gene sets enriched among the most up- or downregulated mRNAs. The results identified that the Wnt/β-catenin is hallmark of this gene sets. (Fig. 2B). These findings highlights the important of LINC01048 in the Wnt/β-catenin signaling pathway. Then, we checked the relationship between the LINC01048 and β-catenin using the GAPIA (Fig. 2C), and the result indicated that LINC01048 may regulated the cell proliferation through the Wnt/β-catenin signaling pathway. Next, to investigate the subcellular localization of LINC01048 in the gastric adenocarcinoma cell lines HGC27 and MKN45, a nuclear-cytoplasmic fractionation assay was performed. After fractionation, qPCR analysis was performed to quantify the expression levels of LINC01048 in the nuclear and cytoplasmic fractions of HGC27 and MKN45 cells. The results of the qPCR analysis revealed that the expression of LINC01048 was elevated in the nuclear fraction compared to the cytoplasmic fraction in both cell lines (Fig. 2D). This finding suggests that LINC01048 is predominantly localized in the nucleus in HGC27 and MKN45 cells. The higher expression of LINC01048 in the nuclear fraction suggests its potential involvement in nuclear processes and functions.

Fig. 2.

Subcellular Localization in Gastric Cancer (GC) and the functional of LINC01048 enrichment analyzed. A Functional enrichment of network proteins that interact with LINC01048. B GSEA plots for Wnt-β-catenin pathway. C The correlation between β-catenin and LINC01048 expression based on data derived from GEPIA. DThe expression of LINC01048 in the nuclear and cytoplasm fractions of GC cancer cell lines was detected by q-PCR. Statistical significance was determined using one-way ANOVA. *p < 0.05 (n = 3, independent experiments)

LINC01048 knockdown inhibits growth of GC cells in vitro

Next, we aimed to assess the functional relevance of LINC01048 in gastric cancer cell carcinoma (GC) by investigating the effects of LINC01048 depletion. Our results indicate that knockdown of LINC01048 significantly impairs the proliferative capacity of GC cells (Fig. 3A), which is particularly noteworthy given the correlation between high LINC01048 levels and unfavorable clinical outcomes in GC patients. These findings underscore the potential clinical relevance of targeting LINC01048 as a therapeutic strategy for GC. In addition, we investigated whether LINC01048 depletion could facilitate GC cancer cell apoptosis. Indeed, flow cytometry analysis also revealed that knockdown of LINC01048 significantly increased the rate of apoptosis in GC cells (Fig. 3B), indicating that LINC01048 plays a crucial role in regulating this process. Additionally, we examined the expression of β-catenin in the nuclear and cytoplasmic fractions upon LINC01048 knockdown in two GC cell lines. Our finding as depicted in Fig. 3C, demonstrated a significant increase in β-catenin expression in the cytoplasmic fraction, accompanied by a decrease in nuclear expression. Furthermore, we also checked the expression of p21 and c-Myc, which play an important role in apoptosis when they interact with β-catenin during transcriptional processes. This suggests that LINC01048 may play a role in modulating the subcellular localization of β-catenin in GC cells. Furthermore, to further investigate its potential oncogenic role, we overexpressed LINC01048 in GC cell lines (AGS and NCI-N87) that exhibit low endogenous LINC01048 expression at the RNA level. Results showed that LINC01048 overexpression significantly promoted cell proliferation (Fig. 3D).

Fig. 3.

LINC01048 knockdown inhibits growth of GC cells in vitro. Endogenous LINC01048 was efficiently decreased by shRNA in HGC27 and MKN45 cells. Cell proliferation was measured by MTT upon LINC01048 knockdown. B Apoptosis assay was detected in the indicated cell lines depleted of LINC01048. C The representative images demonstrating nuclear localization of β-catenin, p21, and c-Myc showed altered patterns following LINC01048 knockdown. Conversely, cytoplasmic localization of these proteins (β-catenin, p21, and c-Myc) was also significantly affected. To validate these observations, endogenous protein levels in cytoplasmic and nuclear fractions of LINC01048-depleted GC cells were quantified by Western blot analysis, with H3 and GAPDH serving as respective nuclear and cytoplasmic markers. The Western blot images shown are representative of at least three independent experiments. C Proliferation after overexpression LINC01048 was assessed by MTT, and plate crystal violet staining assays. Statistical significance was determined using one-way ANOVA. *p < 0.05 (n = 3, independent experiments)

The LINC01048 regulate the GC proliferation through Wnt/β-catenin pathway

Based on the above data obtained, it has been observed that LINC01048 promotes cell proliferation in GC. To further validate this finding, we conducted an experiment using SKL2001, an activator of the Wnt/β-catenin pathway. We treated GC cells with SKL2001 after depleting LINC01048 and observed an increase in cell proliferation, as shown in Fig. 4A. Furthermore, cell apoptosis upon treatment with SKL2001 was significantly inhibited (Fig. 4B). Consistently, analysis of β-catenin, c-Myc and p21 expression in different cell compartments revealed similar result, furthermore, recue experiments was shown the (Fig. 4C). Collectively, these findings provide further evidence that LINC01048-mediated promotion of cell proliferation in GC is associated with the activation of the Wnt/β-catenin pathway. Furthermore, Western blotting experiments demonstrated that the LINC01048 can adsorb protein of β-catenin and the control probe was not capable of adsorbing protein of β-catenin, RNA IP, western blotting, and RT-PCR experiments verified that anti-β-catenin antibody can adsorb β-catenin protein and then pull the LINC01048 mRNA, and IgG did not adsorb β-catenin protein and did not pull the LINC01048 mRNA (Fig. 4D), These experiments confirmed that LINC01048 can interact with β-catenin protein, which may regulate the function of LINC01048.

Fig. 4.

The LINC01048 regulate the GC proliferation through Wnt/β-catenin pathway. A Treatment of GC cells with SKL2001 after LINC01048 depletion enhanced cell proliferation, as determined by MTT assay. B Apoptosis assay was applied to examine cell apoptosis upon treatment with SKL2001 in knockdown the LINC01048. C Western blotting assay was performed to analyze the expression of β-catenin, p21, and c-Myc in different cellular compartments (nucleus and cytoplasm). The Western blot images shown are representative of at least three independent experiments. D RNA IP revealed that β-catenin protein can capture LINC01048. IgG and β-catenin antibodies were used to adsorb protein-RNA complex and then subjected to western blotting, detecting β-catenin protein and RT-PCR detected LINC01048 mRNA

LINC01048 stimulate tumor growth in vivo

To further validate the role of LINC01048 in gastric cancer (GC) progression, we conducted xenograft experiments using HGC27 cells stably transfected with either sh-LINC01048 or sh-NC (negative control). After a 28-day period post-inoculation, tumors from both groups were carefully harvested and subjected to a series of analyses. Tumor volume was measured using a caliper every other day and calculated using the formula: V (mm³) = 0.5 × length × width². We observed that tumors in the sh-LINC01048 group exhibited a marked reduction in size, volume, and weight compared to the sh-NC control group (Fig. 5A-C), suggesting that LINC01048 knockdown significantly suppresses tumor growth in vivo. This result aligns with our in vitro findings, further supporting the hypothesis that LINC01048 plays a crucial role in promoting GC cell proliferation. Additionally, the expression level of LINC01048 was significantly reduced in the sh-LINC01048 group, confirming the successful knockdown of the lncRNA (Fig. 5D). To assess the effect of LINC01048 knockdown on tumor cell proliferation, we performed immunofluorescence staining for Ki-67, a well-established marker of cell proliferation. Our results demonstrated a significant reduction in Ki-67 positive cells in the sh-LINC01048 group (Fig. 5E), further indicating that knockdown of LINC01048 inhibits GC cell proliferation in vivo. Taken together, these in vivo findings corroborate our in vitro data, providing strong evidence that LINC01048 plays a key role in regulating GC cell growth and proliferation. The consistent reduction in tumor growth and proliferation across both experimental platforms strengthens our conclusion that LINC01048 may serve as a potential therapeutic target for GC.

Fig. 5.

LINC01048 promotes GC cell growth in vivo. A A CDX model was generated for checking LINC01048 function. Whole body imaging was performed on mice prior to tumor extraction. B HGC27 cells stably infected with lentivirus scramble or shLINC01048 were subcutaneously injected into the right flank of each mouse. After 1 month, tumor size was measured every 3 days over a period of 4 weeks. C Tumors were excised from SCID mice at the end of the experiment. Photographs of tumors from each group are illustrated. Tumor weight was measured after the tumors were excised as shown. D LINC01048 expression in harvested xenograft tissues were assessed by immunohistochemistry. Representative photographs for antibody in different groups were shown. E Immunofluorescence analysis was performed to detect the expression of indicated cell proliferation markers in both groups. Indicated LINC01048 could promote the cancer cell proliferation

Discussion

LncRNAs have garnered significant interest as potential biomarkers and therapeutic targets in various human cancers, including colon cancer, prostate cancer, endometrial cancer, gastric cancer, keratinocyte carcinomas and lung cancer [6, 7, 21–25] and to serve as tumor promoters or suppressors [26]. We also mentioned that H. pylori infection can induce and activate the cancer-promoting signaling pathway and affect the occurrence and outcome of gastric cancer through controlling the regulatory functions of long non-coding RNAs (lncRNAs). However, there is still a lack of experimental researches about the molecular function of some lncRNAs including the LINC01048, and we will collect specimens from H. pylori (+) and H. pylori (−) GC patients and carry out for further experimental study. Moreover, lincRNAs play a pivotal role in mediating chemoresistance. A significant proportion of gastric cancer patients exhibit resistance to chemotherapy, leading to diminished treatment outcomes. Notably, targeting lincPRKD [27] is implicated not only in regulating cancer cell proliferation and migration but also potentially in driving chemoresistance. Thus, elucidating the mechanisms of lincRNAs represents a key direction for future exploration.

In the present study, we identified that lncRNA LINC01048 8 as a tumor promoter in the progression of GC and it expression was correlation with the prognosis of GC patients based on analysis of the TCGA database. Furthermore, we confirmed the upregulation of LINC01048 in GC tissues and cell lines. Kaplan-Meier analysis provided additional evidence that high expression of LINC01048 was significantly associated with unfavorable patient outcomes (Fig. 1), suggesting that LINC01048 could serve as an independent prognostic indicator for GC patients. These results highlight the potential of LINC01048 as a promising biomarker and therapeutic target in GC. Functionally, dysregulated lncRNAs can regulate various biological processes, such as cell proliferation and apoptosis [28–30]. Furthermore, some evidences have suggested that lncRNAs can interact with several components of the Wnt/β-catenin signalling pathway to regulate the expression of Wnt target genes in cancer, such as c-Myc [31]. Moreover, the expression of lncRNAs can also be influenced by the pathway [32–34]. These results demonstrate a potential regulatory network involving Wnt/β-catenin pathway-related lncRNAs, their interactions, and chemoresistance in gastric cancer. Meanwhile, this observation aligns with the functional enrichment results obtained from our analysis. Therefore, to investigate the functional role of LINC01048 in GC, we conducted loss-of-function assays in GC cells considering its high expression. The experimental results revealed that knockdown of LINC01048 significantly suppressed cell proliferation and induced cell apoptosis. Both the expression of p21 and c-Myc were increased demonstrated that LINC01048 promoted gastric tumor growth that is related to the activation of the Wnt/β-catenin signaling. All these findings strongly suggest that LINC01048 plays an oncogenes role in GC (Fig. 3). Moreover, emerging evidence indicates that long non-coding RNAs (lncRNAs) predominantly modulate epithelial-mesenchymal transition (EMT) progression and malignant invasion via Wnt/β-catenin signaling activation. Notably, these Wnt/β-catenin-regulated EMT-associated lncRNAs demonstrate dual clinical potential as both predictive biomarkers and precision therapeutic targets for suppressing metastatic dissemination in oncology patients.

Furthermore, in the rescue experiment, we utilized an activator of the Wnt/β-catenin pathway, SKL2001. The results revealed that the effects of LINC01048 knockdown on cell proliferation and apoptosis could be reversed by treatment with SKL2001. Specifically, cell proliferation was enhanced while apoptosis was inhibited upon SKL2001 treatment. These findings suggest that LINC01048 may stimulate cell proliferation in GC, potentially through the activation of the Wnt/β-catenin pathway. The data presented in Fig. 4 support this notion and provide further evidence for the involvement of the Wnt/β-catenin pathway in mediating the effects of LINC01048 on cell proliferation in GC.

Indeed, lncRNAs play a crucial role in the development and progression of cancers [35]. lncRNAs exert their effects through various mechanisms, which are influenced by factors such as subcellular localization, expression levels, and stability. One well-known signaling pathway in cancer is the canonical Wnt/β-catenin pathway which is characterized by the accumulation and translocation of β-catenin, an adherens junction-associated protein, to the nucleus. As we know that canonical Wnt signaling pathway plays a crucial role in the proliferation and differentiation of cancer cell. Therefore, in this study, we investigated the expression of β-catenin in both the nuclear and cytoplasmic compartments to examine the impact of Linc01048 on its subcellular localization. The results of our analysis demonstrated that Linc01048 abundance is correlated with nuclear translocation of β-catenin. Specifically, we observed a decrease in the nuclear expression of β-catenin and an increase in its cytoplasmic expression upon depletion of Linc01048 (Fig. 3C). This suggests that Linc01048 plays a role in facilitating the translocation of β-catenin from the cytoplasm to the nucleus. Furthermore, SKL2001 was utilized to investigate the role of the Wnt/β-catenin pathway in GC. Treatment with SKL2001 can activate the Wnt/β-catenin pathway and potentially modulate cell proliferation, apoptosis and subcellular localization, as illustrated in Fig. 4. Therefore, all these findings highlight the regulatory role of Linc01048 in modulating the subcellular localization of β-catenin and suggest that Linc01048 may contribute to the activation of the Wnt/β-catenin pathway by facilitating the nuclear accumulation of β-catenin.

Finally, to further validate the impact of LINC01048 on GC cell growth, we performed in vivo experiments. The results of these experiments demonstrated that LINC01048 depletion led to a significant reduction in GC tumor growth in vivo, as shown in Fig. 5. These findings provide additional evidence supporting the positive effect of LINC01048 on GC cell growth. Furthermore, PDX models demonstrated closer replication of patient clinical profiles, consistently maintaining tumor heterogeneity and drug response fidelity when compared to CDX models. Based on these findings, we therefore selected the PDX platform for subsequent investigations. Collectively, our current in vitro and in vivo experiments consistently highlight the functional significance of LINC01048 in promoting GC progression, emphasizing its potential as a therapeutic target for GC treatment. Meanwhile, it can also have the effect of blocking the Wnt/β-catenin signaling pathway, and is also a potential effective way to treat diseases related to this pathway, which deserves more attention.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the study. JinXi Huang, YuLong Chen, Hai Huang: Conceptualization, Data curation, Methodology, Validation and writ ten manuscript. Weiwei Yuan, Gaofeng Li, Beibei Chen: Conceptualization, Data curation, Methodology and Validation. Material preparation, data collection and analysis were also performed by Beibei Chen, Lihong Wang. JinXi Huang, Hai Huang: Conceptualization, Funding acquisition, Supervision. All authors commented on the manuscript. All authors have reviewed and approved the final manuscript.

Funding

This work was supported by grants from Medical Science and Technology Research Project of Henan Province (Key project jointly built by the province and the Ministry) (Grant No. SBGJ202102072). The Science and Technology Research Project of Henan Province (No.232102311039); Henan provincial Medical Science and Technology Research Project (No. LHGJ20210172); Science and Technique Foundation of Henan Province (No. 222102310424); Natural Science Foundation of Henan Province (No. 242300420091).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Affiliated Cancer Hospital of ZhengZhou University [ZZU-LAC20220729(07)]. As stipulated in the approval document: " No animal in this study exceeded the tumor size/volume thresholds and No ulceration or impaired mobility” The authors confirmed that the maximal tumors size/burden was not exceeded. The volume of tumors in mice did not exceed 1500 mm³ and the diameters were less than 12 mm in any dimension. All animal experiments complied with the ARRIVE guidelines [36] (https://arriveguidelines.org). All methods were carried out in accordance with relevant guidelines and regulations.

Patient consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.