LINC01082 inhibits malignant phenotypes of bladder cancer through sponging miR-1269a

Abstract

Objective

This work aims to delineate the involvement of LINC01082 in bladder cancer (BC) pathogenesis.

Methods

Significant downregulation of LINC01082 in BC tissues was identified through GEO database analysis (GSE89006). Subsequent validation in GEPIA, TCGA, and clinical cohorts supported these findings. The diagnostic utility was evaluated via ROC curve analysis. Interaction between LINC01082 and miR-1269a, predicted by lncRNASNP, was confirmed by dual luciferase reporter assays. RT-qPCR measured the expression of LINC01082, miR-1269a, and EMT-related genes, while Transwell assays characterized cell migration and invasion.

Results

LINC01082 was found to be significantly underexpressed in BC tissues and cell lines, with excellent diagnostic efficacy. LINC01082 can specifically bind to miR-1269a. When LINC01082 expression is silenced, BC cells exhibit increased migration and invasion capacities, accompanied by a decline in the epithelial marker E-cadherin and an upregulation of mesenchymal markers, including N-cadherin and vimentin. However, these effects can be effectively reversed by adding the miR-1269a inhibitor.

Conclusion

LINC01082 can downregulate the expression of miR-1269a, thereby inhibiting the migration, invasion, and EMT of BC cells, ultimately hindering the malignant progression of tumors. This study provides new insights into its anticancer effects and molecular mechanisms.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-025-04026-3.

Introduction

Bladder cancer (BC) is a prevalent urinary system malignancy [1] with rising incidence and mortality [3]. Due to its high recurrence/metastasis rates and stagnant five-year survival rates [4–7]，there is an urgent need for better biomarkers and treatment methods.

Long noncoding RNAs (lncRNAs) are RNA transcripts exceeding 200 nucleotides in length that cannot code for proteins [8]. They regulate key biological processes, such as the cell cycle and gene expression [9] and play a central role in cancer progression [10]. Taking BC as an example, previous literature has reported that CALML3-AS1 promotes BC development via the miR-4316/ZBTB2 pathway [11].It has been demonstrated that the lncRNA LINC01082 is not only associated with chromatin structure but is also involved in the regulation of gene expression [2]. Its location near FOXF1 may regulate FOXF1 and adjacent genes by influencing transcription factor binding and chromatin loops, and LINC01082 deletion is linked to decreased FOXF1 expression [14–16].Moreover, it is involved in diverse malignancies. In colon cancer, overexpression of this LINC01082 suppresses proliferation, migration, and invasion of cancer cells [18]. It also exerts its anticancer effect on non-small cell lung cancer by regulating the miR-543/TNRC6A signaling axis [19]. In addition, the abnormal expression of LINC01082 has been reported in BC [20]. However, although the carcinogenic role of LINC01082 is well documented in various tumors, in-depth research on its specific biological role and molecular mechanisms in BC is still lacking.

This study systematically analyzed differences in LINC01082 expression in BC tissues, adjacent non-cancerous tissues, and BC cell lines. Using in vitro cell models, this study also evaluated the impact of LINC01082 on migration, invasion, and epithelial-mesenchymal transition (EMT) of BC cells. By combining bioinformatic analysis and experimental validation, this study aims to clarify the mechanism of action of LINC01082 and provide a new theoretical basis and therapeutic strategy for the development of targeted treatment for BC.

Materials and methods

Participants and ethical guidelines

A total of 55 cases of primary BC diagnosed at Xinchang Hospital of Traditional Chinese Medicine and undergoing tumor resection were enrolled, along with matched adjacent non-cancerous tissue. None of the patients had received chemotherapy, radiation therapy, or any other form of anticancer treatment before surgery. Senior pathologists conducted professional examinations of all specimens, and all tissue specimens were immediately cryopreserved in liquid nitrogen. The Ethics Committee of Xinchang Hospital of Traditional Chinese Medicine evaluated and sanctioned the study protocol, and written informed consent was secured from every participant. The entire research process was conducted in full compliance with the ethical principles of the Declaration of Helsinki.

The sample size for this study was determined based on the available data. Prior to the study, GPower 3.1 was used to perform a power analysis (effect size 0.05, power 80%, significance level 5%) and estimated the minimum sample size for each group to be 46. To ensure reliability and account for potential sample loss, 55 participants were included, which met the predefined statistical power requirements.

Cell culture and transfection

Human BC cells (T24, UM-UC3) and urinary tract epithelial cells (SV-HUC-1) were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). These cells were maintained in DMEM medium, which was Supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) and 1% antibiotics. Cultivation was carried out at 37 °C in a Humidified incubator with 5% carbon dioxide.

Under the manufacturer’s standard procedures, the Lipofectamine 3000 transfection system (Invitrogen, USA) was used to introduce the following transfection material into the target cells: lentivirus particles carrying LINC01082 (LV-LINC01082) and empty vector (LV-NC, 1 × 10⁸ TU/mL), siRNA and si-LINC01082 (20 nM), negative control inhibitor, and miR-1269a inhibitor (50 nM). This procedure was performed when the cell density reached 70–80%. Two days after transfection, the culture medium was refreshed. Subsequently, the cells were cultured for an additional 24 h in a CO₂ incubator at 37 °C before the following experiments were performed. All cells were kept in the incubator for at least 24 h before temporary transfection. What’s more, puromycin (5 µM) was used for selecting the culture of LV-transfected cells for 5 days. Evaluate transfection efficiency through real-time quantitative PCR (RT-qPCR) analysis.

Transwell migration/invasion assay

Migration and invasion abilities of BC cells (T24, UM-UC3) were tested using Transwell chambers with or without Matrigel coating. In the migration assay, 5× 10⁴ BC cells were inoculated into a 24-well Transwell chamber. Medium without serum occupied the upper chamber, and the lower chamber contained a complete medium with 10% FBS. During the invasion experiment, the upper chamber was first coated with Matrigel (Corning Incorporated, Corning, New York, USA) and incubated at 37℃ for 24 h. After incubation, the chambers were removed, fixed with pre-cooled methanol, and stained with 0.1% crystal violet solution for 10 min. Finally, we counted the number of migrated or invaded cells using an optical microscope.

Subcellular fractionation

Using a cell nucleus and cytoplasmic protein extraction kit (Beyotime, Beijing), nuclear and cytoplasmic protein fractions were separated from T24 and UM-UC3 cells according to the manufacturer’s instructions. GAPDH was used as a reference RNA in the cytoplasm, and U6 was used as a reference RNA in the nucleus for calibration. The expression level of LINC01082 in the separated protein fractions was determined using RT-qPCR technology.

Dual luciferase-based gene reporter assay

Using the lncRNASNP database, we identified potential binding sites for miR-1269a in LINC01082. We designed two PCR amplification products: a wild-type (WT) sequence containing the entire binding site and a mutant (MUT) sequence in which a specific region was deleted. These amplification products were successfully cloned into the pmirGLO vector (Promega, Shanghai). As specified in the production manual, miR-1269a mimetic or its negative control (NC) was co-transfected with the corresponding recombinant plasmid for 48 h, and luciferase activity was quantitatively analyzed utilizing the Dual-Luciferase Reporter Gene Assay Kit (Promega).

RT-qPCR

A pre-cooled TRIzol LS extraction reagent (Invitrogen, USA) was employed to extract total RNA. Reverse transcription was performed according to the TaKaRa PrimeScript RT kit protocol (Japan) to synthesize cDNA. Quantitative real-time PCR detection was performed using the SYBR Premix Ex Taq system (Qiagen, USA). GAPDH was used as the housekeeping gene to normalize the expression levels of LINC01082 and markers related to EMT (E-cadherin, N-cadherin, and vimentin), while U6 was chosen as the reference gene for miRNA correction. Relative quantitative analysis was performed using the 2^−ΔΔCt method. The forward and reverse primer sequences are presented below:

LINC01082, F 5’-GAGATAGGACCAACCGTCAGGA-3’, R 5’-TGTGCTTCTCACTTGCAGGGT-3’; miR-1269a, F 5’-CGCTGGACTGAGCCGTGC-3’, R 5’-AGTGCAGGGTCCGAGGTATT-3’; GAPDH, F 5’-GTCTCCTCTGACTTCAACAGCG-3’, R 5’-ACCACCCTGTTGCTGTAGCCAA-3’; U6, F 5’-GCTCGCTTCGGCAGCACATATAC-3’, R 5’-AGTGCAGGGTCCGAGGTATT-3’.

Bioinformatics analysis

Abnormally expressed lncRNAs in BC tissues were screened using the GEO dataset (GSE89006). To further validate these findings, we collected clinical data from 404 tumor samples and 28 non-cancerous tissue samples from the GEPIA database, as well as 414 tumor samples and 19 non-cancerous tissue samples from the TCGA data to systematically evaluate the difference in LINC01082 expression between BC tissue and non-cancerous tissue. Using bioinformatic predictions based on the lncRNASNP database, we identified potential binding sites between LINC01082 and miR-1269a.

Statistical analysis

All experiments were performed with at least three independent replicates. Graphical representation was performed using GraphPad Prism 9.0 software. The data were analyzed using SPSS 22.0, with results expressed as mean ± standard deviation, and normalized via mean normalization to address systematic variations. Statistical analysis of differences between groups was performed using Student’s t-test or one- or two-factor analysis of variance (ANOVA). In addition, the interdependence between LINC01082 and miR-1269a expression patterns was analyzed through Pearson’s correlation. The threshold for statistical significance was set at P < 0.05.

Results

LINC01082 expression is diminished in BC tissues

This study first identified lncRNA molecules with abnormal expression in BC tissues. By analyzing the GSE89006 dataset, it was found that the expression level of LINC01082 in BC tissue samples was significantly lower than that in non-cancerous tissues (Fig. 1A). Further validation using the GEPIA and UALCAN databases confirmed that LINC01082 illustrated a substantial downward trend in BC tissues. (Fig. 1B-C). We also used RT-qPCR to detect LINC01082 expression in 55 paired BC and adjacent non-cancerous tissue samples. The experimental results confirmed that the expression of LINC01082 was markedly reduced in BC tissues (P < 0.001, Fig. 1D-E). To elucidate the clinical relevance of LINC01082, we analyzed the relationship between its expression and clinical pathological factors. As shown in Table 1, abnormal expression of LINC01082 was significantly associated with histological grade (P = 0.029) and lymph node metastasis (P = 0.022). ROC curve analysis results indicated that LINC01082 has good diagnostic value for BC (AUC = 0.83, sensitivity of 72.73%, specificity of 87.27%) (Fig. 1F).

Fig. 1.

The expression of LINC01082 is markedly reduced in bladder tumor samples. A. A volcano plot based on the GEO dataset (GSE89006) revealed that LINC01082, as a differentially expressed lncRNA molecule, exhibited significantly reduced expression in BC tissue. B. The GEPIA database showed that the expression of LINC01082 was markedly reduced in BC tissue. C. Analysis of TCGA samples showed that LINC01082 expressions were markedly downregulated in BC tissues in comparison with non-cancerous tissues. D-E. RT-qPCR analysis of paired tissues (tumor and adjacent non-cancerous tissues) from 55 BC patients showed that LINC01082 expressions were significantly reduced in BC tissues compared to adjacent non-cancerous tissues. F. ROC curve analysis indicated that the expression level of LINC01082 has a potential diagnostic value for BC. *** P < 0.001

Table 1.

Correlation between LINC01082 expression and clinicopathological factors

Characteristics	LINC01082 expression		P
	High (n = 26)	Low (n = 29)
Gender			0.588
Male	16	15
Female	10	14
Age			1.000
< 60 years	11	12
≥ 60 years	15	17
TNM stage			0.130
I-II	22	19
III–IV	4	10
Grade			0.029
Low stage	14	7
High stage	12	22
Lymph node metastasis			0.022
Absent	24	19
Present	2	10
Multiplicity			0.279
single	14	20
multiple	12	9

LINC01082 is downregulated in BC cell lines

Studies have shown that compared with bladder epithelial cells SV-HUC-1, the expression of LINC01082 is significantly downregulated in BC cell lines T24 and UM-UC3 (P < 0.01, Fig. 2A). To clarify the intracellular distribution characteristics of LINC01082, we performed subcellular fractionation experiments. The experimental results showed that in T24 cells, LINC01082 was primarily localized in the cytoplasm (Fig. 2B); similarly, in UM-UC3 cells, this molecule was also significantly enriched in the cytoplasm (Fig. 2C). These data collectively indicate that LINC01082 is primarily distributed in the cytoplasmic region of BC cells.

Fig. 2.

LINC01082 is downregulated and localized in the cytoplasm of BC cells. A. Detection of LINC01082 expression differences in SV-HUC-1 bladder epithelial cells and T24, UM-UC3 BC cells using RT-qPCR technology. B-C. Determination of LINC01082 distribution in T24 and UM-UC3 cells via nucleoplasm isolation experiment. ** P < 0.01, *** P < 0.001

Overexpression of LINC01082 effectively suppresses migration, invasion, and EMT in BC cells

To elucidate the biological functions of LINC01082, we successfully established a LINC01082 overexpression model in T24 and UM-UC3 cells using lentivirus-mediated stable transfection technology. RT-qPCR analysis confirmed that LINC01082 expression levels were significantly elevated in the LV-LINC01082 transfection group (P < 0.0001, Fig. 3A). Transwell analysis revealed that, compared with the empty vector control group, overexpression of LINC01082 significantly inhibited the migratory and invasive behavior of both BC cells (P < 0.05, Fig. 3B-C). Further RT-qPCR analysis of EMT marker expression changes revealed that in the LV-LINC01082-transfected group, the expression of the epithelial marker E-cadherin was significantly increased (P < 0.0001, Fig. 3D), while the expression of the mesenchymal markers N-cadherin and vimentin was markedly reduced (P < 0.001, Fig. 3E-F).

Fig. 3.

Increased expression of LINC01082 markedly hinders the capacity of BC cells to migrate, invade, and undergo EMT. A. Quantify LINC01082 expressions following lentivirus-mediated plasmid transfection using RT-qPCR. B-C. The effect of LINC01082 overexpression on the migration and invasion abilities of BC cells was evaluated using the Transwell assay. RT-qPCR to detect the expression levels of EMT markers (epithelial marker E-cadherin) (D), mesenchymal marker N-cadherin (E), and Vimentin (F) under LINC01082 overexpression conditions. * P < 0.05, ** P < 0.01, *** P < 0.001. **** P < 0.0001

LINC01082 exerts its influence by targeting miR-1269a downstream

Using the lncRNASNP database for bioinformatics prediction, we identified a potential binding interaction between LINC01082 and miR-1269a (Fig. 4A). Based on the predicted binding sites, we designed wild-type (LINC01082-WT) and mutant-type (LINC01082-MUT) reporter gene constructs for LINC01082. Dual luciferase reporter gene assay results revealed that under conditions of miR-1269a mimic transfection, the fluorescent activity of the wild-type vector was significantly reduced, while the mutant-type vector did not exhibit significant changes (P < 0.0001, Fig. 4B), confirming the targeted binding relationship between LINC01082 and miR-1269a. RT-qPCR-based comparative expression analysis of BC tissue and adjacent non-cancerous tissue revealed that miR-1269a was markedly overexpressed in BC samples (P < 0.0001, Fig. 4C-D). Moreover, correlation studies revealed that miR-1269a was negatively correlated to LINC01082 expression levels in BC tissues (r = −0.571, P < 0.0001, Fig. 4E). And miR-1269a levels were abnormally elevated in BC cells (T24 and UM-UC3) (P < 0.0001, Fig. 4F). Notably, when LINC01082 expression was increased, miR-1269a levels significantly decreased (P < 0.05, Fig. 4G). These results indicate that LINC01082 negatively regulates miR-1269a expression through direct targeting.

Fig. 4.

LINC01082 influences gene expression by targeting miR-1269a downstream. A. The potential binding sites between LINC01082 and miR-1269a were predicted using the lncRNASNP database. B. Confirmation of the binding interaction between LINC01082 and miR-1269a through dual luciferase reporter assays. C-D. Measurement of miR-1269a expression in bladder tumor samples via RT-qPCR. E. Assessment of the association between LINC01082 and miR-1269a levels in BC tissues using Pearson correlation analysis. F. Quantification of miR-1269a expression in BC cell lines by RT-qPCR. G. Examination of miR-1269a expression changes upon LINC01082 overexpression in BC cells using RT-qPCR. * P < 0.05, *** P < 0.001, **** P < 0.0001

By targeting miR-1269a, LINC01082 effectively restricts the migration, invasiveness, and EMT of BC cells

In the T24 and UM-UC3 cell lines, after silencing LINC01082 and miR-1269a, the expression of miR-1269a was measured using RT-qPCR. Experimental data showed that the miR-1269a inhibitor effectively blocked the upregulation of miR-1269a by si-LINC01082 (P < 0.0001, Fig. 5A). To further investigate the biological functions of LINC01082 and miR-1269a, we conducted Transwell experiments. The results showed that in T24 and UM-UC3 cells, where LINC01082 expression was inhibited, both migration and invasion activities exhibited a significant upward trend, and this effect could be reversed by the miR-1269a inhibitor (P < 0.05, Fig. 5B-C). Subsequently, the expression levels of key markers of EMT were detected by RT-qPCR. The results indicated that LINC01082 knockdown led to the downregulation of E-cadherin expression in BC cells while increasing the expression levels of N-cadherin and vimentin. However, these changes in EMT-related molecule expression could be corrected by the miR-1269a inhibitor (P < 0.05, Fig. 5D-F).

Fig. 5.

LINC01082 inhibits BC cell migration, invasion, and EMT by targeting miR-1269a. A. RT-qPCR confirmed miR-1269a upregulation after LINC01082 knockdown in T24 and UM-UC-3 cells. B-C. Transwell assays showed enhanced migration and invasion upon LINC01082 depletion, reversed by miR-1269a knockdown. D-F. The expression levels of EMT markers E-cadherin, N-cadherin, and vimentin were modulated by LINC01082/miR-1269a axis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Discussion

BC is a highly prevalent malignant tumor of the urinary system worldwide, severely impairing patients’ health and quality of life [21, 22]. Given its high recurrence rate and the obvious limitations of current diagnostic and therapeutic approaches [23–25], exploring effective biomarkers and innovative therapeutic targets has become a key focus in BC research.

Previous studies have shown that LINC01082 exhibits abnormal expression patterns in various malignant tumors [17–19]. This study, through database analysis, clinical tissue sample testing, and in vitro experiments, has for the first time discovered that the expression level of LINC01082 is significantly reduced in BC tissues and cell models. This abnormally expressed lncRNA demonstrates its value as a potential diagnostic biomarker. ROC curve analysis showed that LINC01082 May have diagnostic efficacy for BC patients, with a specificity and sensitivity of 87.27% and 72.73%, respectively, opening up new possibilities for clinical diagnosis.

Recent studies have revealed that lncRNAs can act as competitive endogenous RNAs (ceRNAs) to regulate miRNA activity through a molecular sponge mechanism, thereby influencing the miRNA-miRNA interaction network and participating in processes such as the cell cycle, metastasis, and tumorigenesis [28–30]. For example, LINC00472 inhibits epithelial-mesenchymal transition by specifically binding to miR-196b-5p [31].LINC01082 is primarily localized in the cytoplasm of BC cells, where bioinformatics analysis combined with dual luciferase assays confirmed its direct binding to miR-1269a. These findings not only substantiate the functional sequestration of miR-1269a by LINC01082 through canonical miRNA response elements, but also provide new experimental evidence for lncRNA regulating miRNA activity via the “molecular sponge” mechanism [32].As a key factor in gene expression regulation, microRNAs (miRNAs) have widespread functions in various biological processes [33]. Among them, miR-1269a, a non-coding RNA approximately 22 nucleotides in length, regulates gene expression at the post-transcriptional level by specifically binding to target mRNAs. Studies have shown that miR-1269a enhancing tumor cell invasion and metastasis by inhibiting PCDHGA9 and inducing epithelial-mesenchymal transition in colorectal cancer [34]. in Inlung cancer, it promotes carcinogenesis by targeting FOXO1 [35]. Its high expression and regulatory functions in various malignant tumors collectively confirm the carcinogenic properties of this molecule. Additionally, in the metastasis mechanism of BC, EMT, as a core process, is regulated by lncRNA/miRNA networks or drug effects. Our research indicates that the LINC01082/miR-1269a regulatory axis participates in the metastasis process by influencing the expression of EMT markers, and this mechanism may synergize with EMT processes mediated by signaling pathways such as TGF-β [36, 37]. Further experiments confirmed that inhibiting miR-1269a significantly alleviates the oncogenic effects caused by LINC01082 deficiency. Given the regulatory roles of LINC01082 and miR-1269a in BC progression, future targeted therapeutic strategies developed based on the ceRNA network may effectively intervene in BC progression by regulating the expression levels of these two molecules, thereby opening new avenues for personalized cancer treatment in clinical settings.

However, our study has some limitations. First, the sample size of this study is still insufficient. We will expand the number of participants in future studies to confirm the reliability of the current findings. Second, due to the lack of long-term follow-up of patients, the association between LINC01082 expression differences and treatment response and prognosis remains unclear. Therefore, we plan to conduct prospective cohort studies and long-term follow-up to further explore the potential clinical application value of LINC01082 in the diagnosis and treatment of BC. Additionally, the signaling pathways through which LINC01082 targets miR-1269a to regulate BC cell progression require further investigation. Although the clinical translation of LINC01082 is currently limited by the absence of prospective validation and insufficient molecular mechanism research, future strategies such as multi-center collaborative validation, combined biomarker assessment, and translational medicine studies may enable its development as a novel auxiliary biomarker for BC diagnosis.

In summary, the expression of LINC01082 in BC exhibits a significant downward trend, while the expression of miR-1269a exhibits a significant upward trend. This suggests that LINC01082, by negatively regulating the expression of miR-1269a, can effectively inhibit the migration and invasion capabilities of BC cells while triggering the EMT process, thereby accelerating the malignant progression of tumors. This important discovery provides a solid theoretical basis for the future development of novel therapeutic approaches based on RNA interference.

Abbreviations

BC

Bladder cancer

LncRNAs

Long noncoding RNAs

EMT

Epithelial-mesenchymal transition

WT

Wild-type

MUT

Mutant

NC

Negative control

MiRNAs

MicroRNAs

Authors’ contributions

XK S and LN H contributed to the study conception and design. Material preparation, data collection and analysis were performed by XK S, LN H, Y Y and JB Z. The first draft of the manuscript was written by XK S and LN H, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funding was received for conducting this study.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The authors state that they have obtained Xinchang Hospital of Traditional Chinese Medicine review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.