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INTRODUCTION
Breast cancer (BC) is a prevalent malignancy among women worldwide, with its incidence continuing to rise1. According to recent data published by American Cancer Society, BC alone accounts for 32% of all new cancer diagnoses in women and it is a leading cause of cancer death for women younger than age 50 in the United States2. BC can be categorized into various types according to the expression of molecular markers such as human epidermal growth factor receptor 2 (HER2), progesterone, and estrogen3,4. Triple-negative BC (TNBC) is characterized by the absence of HER2, progesterone receptor, and estrogen receptor5. TNBC accounts for approximately 10-20% of total BC cases and is more prevalent in women < 50 years6. Treatments including surgery, chemotherapy, and radiotherapy have been widely applied and have made advances to some extent7. Nevertheless, many patients have missed the surgical window at the time of diagnosis7. Tumor recurrence poses another obstacle in the treatment of TNBC. For patients with TNBC, the median time to relapse ranges from approximately 19-40 months, and there is a 75% mortality rate within the first 3 months following relapse5,8. Moreover, therapeutic resistance of TNBC also deserve mentioning, including enhanced antioxidant ability, increased drug efflux and metabolism, and enhanced DNA repair during treatment6. Thus, it is imperative to find more effective therapeutical strategies.
Increasingly studies have identified long non-coding RNAs (lncRNA) as key players in various types of cancer, playing either tumor-suppressive or oncogenic roles, and holding promise for the development of effective targeted therapies in TNBC9. For example, lncRNA SEM13B-AS1 knockdown promoted the proliferation, migration, and invasion of TNBC cells (BT-549 and MDA-MB-231) by acting as a competing endogenous RNA against miR-3940 to regulate the downstream gene KLLN10. The transcription factor SP1-induced high expression of lncRNA AFAP1-AS1 facilitates tumorigenesis in TNBC by activating the mTOR signaling11. LINC01614 is a novel oncogene and was first demonstrated as a lung cancer-associated lncRNA in 201812. After that, Wang et al. further confirmed that LINC01614 is an epithelial-mesenchymal transition (EMT)-associated oncogene13. Based on these two studies, researchers have increasingly investigated its role in various types of cancer. For example, LINC01614 acts as an oncogene in gastric cancer and reduces the chemosensitivity of gastric cancer cells to 5-fluorouracil14. Cai et al. reported that LINC01614 promoted the proliferative and invasive ability of osteosarcoma cells15. Especially, Vishnubalaji et al. analyzed that LINC01614 was upregulated in patients with BC and was correlated with worse disease-free survival, having the potential to be an unfavorable prognostic marker16. Zhang et al. reported that the interaction of LINC01614 with COL3A1 can predict drug sensitivity in TNBC patients, although the accuracy still needs improvement17. Nevertheless, its specific role and underlying mechanisms in TNBC have not been discussed yet.
Transcriptional activation is critical for the upregulation of lncRNAs18. Transcription factor SP1 was reported to upregulate LINC01614 in glioma and thereby promotes cancer progression19. In addition, SP1 was reported to affect TNBC progression by activating the expression of other lncRNAs such as AFAP1-AS111 and LINC0123420. Hence, it was hypothesized that SP1 may also contribute to the transcriptional upregulation of LINC01614 in TNBC cells.
The current study aimed to explore the influence of LINC01614 on the viability, migration, invasion, and EMT process of TNBC cell lines (BT-549 and MDA-MB-231). Experiments were designed to confirm its oncogenic role and interaction with the transcription factor SP1 in TNBC cells. This study may provide a novel theoretical basis for the targeted treatment for TNBC.
METHODS
Patient samples
TNBC tissues (n = 78) and matching non-cancerous tissues (n = 7 8) were obtained from the patients diagnosed with TNBC (Stage I–IIA) from Yichang Central People´s Hospital. All the patients gave written informed consent, and the protocols for the use of human tissues were approved by the Ethics Committee of Yichang Central People´s Hospital (2022-164-01). Before sampling, none of the patients underwent radiotherapy, chemotherapy, or hormone therapy. The tissue samples were confirmed by histopathological examination. All the samples were then immediately frozen in liquid nitrogen and stored at −80ºC for the subsequent experiments.
Cell culture and transfection
TNBC cell lines (CAL-51, BT-549, MDA-MB-231, and MDA-MB-468) and normal human breast epithelial cells (MCF-10A) were purchased from ATCC and cultured in DMEM medium containing fetal bovine serum (10%), penicillin, and streptomycin were used for cell culture. The culture conditions were 5% CO2 at 37°C in a humidified incubator.
BT-549 and MDA-MB-231 cells were seeded in 6-well plates for 24 h. Short hairpin RNA targeting LINC01614 (sh-LINC01614#1 and sh-LINC01614#2) was used for the knockdown of LINC01614. The full sequence of SP1 was transfected into pcDNA3.1 vectors to amplify SP1 expression. These plasmids together with their negative controls (sh-NC and empty pcDNA3.1 vectors) were all obtained from GenePharma (Shanghai, China) and were transfected into TNBC cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). After 48 h of transfection, subsequent in vitro experiments were conducted.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted and then reverse-transcribed into complementary DNA. Quantitative PCR experiments were performed on the ABI 7500 Fast Real-Time PCR System with SYBR Fast qPCR Mix (Invitrogen). Relative expression of LINC01614 and SP1 was quantified using the 2−ΔΔCt method and normalized to GAPDH. Sequences of primers used in the study were listed as follows:
LINC01614: (forward) 5´- CAACCAAGAGCGAAGCCAAG-3´ and (reverse) 5´- CGCCCCAAAACAACTGAGTC-3´.
SP: (Forward) 5´- GGGTACTTCAGGAATCCAGG-3´ and (reverse) 5´-TGAGCTCCATGATCACCTG-3´.
GAPDH: (Forward) 5´-GATCATCAGCAATGCCTCC -3´ and (reverse) 5´-TCCACGATACCAAAGTTGTC -3´.
Subcellular fraction assay
To determine the location of LINC01614 in TNBC cells, the PARIS Kit (Life Technologies, Carlsbad, CA, USA) was used. Cells were harvested, lysed on ice for 10 min, and subjected to centrifugation at 12000 g for 3 min. The precipitation part was mainly the nucleus, and the supernatant was the cytoplasm. The distribution of LINC01614 in the cytoplasmic or nuclear part was measured by RT-qPCR. U6 and GAPDH were regarded as the nuclear and cytoplasmic controls, respectively.
Cell counting kit-8 (CCK-8) assay
After the indicated transfection, TNBC cells were plated into 96-well plates (2000 cells/well) and incubated overnight. Then, cell viability was evaluated by adding 15 µL of CCK-8 solution (Glpbio, Montclair, USA) to each well at the time points of 24 h, 48 h, and 72 h, and the absorbance at 450 nm was examined by a microplate reader (BioTek, Vermont, USA).
Wound healing assay
The assay was performed to assess cell migration by measuring the widths of scratches at 0 h and 24 h. TNBC cells (3 × 105 cells) were seeded into 6-well plates and subjected to cell transfection. After that, cells were incubated with a serum-free medium for 24 h. Scratches were made on cell layers using a 200 µL pipette tip. Next, cells were cultured in a complete medium for 24 h. Cell fragments were removed by washing with phosphate-buffered saline, and the images of wound closures at 0 and 24 h after scratch were taken using an inverted microscope (Olympus, Tokyo, Japan). The experiment was repeated in triplicate.
Transwell assay
Transwell assays were conducted to evaluate cell migration and invasion using 8 µm pore Transwell chambers (Corning Incorporated, Corning, USA). For cell invasion assays, TNBC cells with indicated transfection (2 × 104 cells) were suspended in 300 µL of serum-free medium and added to the upper chamber pre-coated with Matrigel (Sigma Aldrich, St. Louis, USA) 2 h before cell inoculation. The lower chamber was added with 500 µL complete medium containing 10% fetal bovine serum. After 24 h, cells invading the lower chamber were fixed with 4% paraformaldehyde (Zeye Biotechnology, Shanghai, China), stained with crystal violet (0.2%, Beyotime, Shanghai, China), and then imaged using a light microscopy (Olympus). For cell migration experiments, similar procedures were performed except for the precoat of Matrigel. Each assay was performed in triplicate.
Western blot analysis
Proteins were isolated from the TNBC cells using cell lysis buffer (Abcam, UK), and the protein concentrations were confirmed by Bicinchoninic acid assay kits (Thermo Fisher Scientific, Waltham, USA). Then, equal quantities of proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (10%) and transferred onto polyvinylidene difluoride membranes. Primary antibodies against E-cadherin (ab40772; 1:1000), vimentin (ab92547: 1:1000), Wnt (ab219412; 1:1000), β-catenin (ab32572; 1:5000) and GAPDH (ab8245; 1:500) were purchased from Abcam and prepared as recommended. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with these primary antibodies overnight at 4°C. The next day, secondary antibodies were incubated with the membranes for another 1 h. Finally, the blots were illuminated using enhanced chemiluminescence kit (Absin, Shanghai, China), and the band intensity was calculated with the ImageJ software (National Institutes of Health, Bethesda, USA).
Chromatin immunoprecipitation (ChIP) assay
The assay was performed using ChIP assay kit (Beyotime). TNBC cells were first cross-linked with 1% formaldehyde for 10 min and then fragmented through ultrasonication to 200-500 base pairs. Cell lysates were incubated with protein A/G beads coated with immunoglobulin G antibody (anti-IgG; ab6757, Abcam) and anti-SP1 (ab231778, Abcam). The IgG group acted as the negative control. Finally, DNA fragments were purified from the protein-DNA complex using a DNA extraction kit (Beckman Coulter, Miami, USA), and the precipitated DNA was analyzed by qPCR.
Luciferase reporter assay
The wild type (Wt) or mutant (Mut) promoter region of LINC01614 containing SP1 binding area was cloned into the pGL3-basic vector (Promega, Madison, USA) to construct Wt-pGL3-LINC01614 and Mut-pGL3-LINC01614. TNBC cells were cotransfected with the above Wt/Mut vectors and pcDNA3.1-SP1/empty pcDNA3.1 vectors for 48 h. After that, the luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity/renilla activity was calculated in the Wt or Mut group.
Statistical analysis
All the experiments were repeated in triplicate and data were processed by GraphPad Prism (La Jolla, USA). All data are presented as mean ± SD. Student’s t-test was performed to compare differences between two groups. One-way analysis of variance followed by Tukey’s post hoc analysis was conducted to evaluate the significance among multiple groups. A value of p < 0.05 was regarded as statistically significant.
RESULTS
LINC01614 was highly expressed in TNBC tissues and cell lines
The bioinformatics tool GEPIA (http://gepia2.cancer-pku.cn/#analysis) was used to analyze the expression of LINC01614 (ENSG00000230838) in 1085 breast invasive carcinoma (BRCA) samples and 291 corresponding normal samples. The box plot showed that LINC01614 was highly expressed in BRCA tissues in contrast to its expression in normal tissues (Fig. 1A, p < 0.05). Since TNBC accounts for 15-20% of all BRCA21, this study aimed to explore whether LINC01614 is also abnormally expressed in TNBC cell lines. RT-qPCR revealed that the LINC01614 expression was elevated significantly in TNBC cell lines compared with the normal cells (more than 6 folds) (Fig. 1B). Consistently, LINC01614 expression was increased significantly in the TNBC tissues (n = 78) relative to that in the non-cancerous tissues (n = 78) (Fig. 1C). Therefore, two TNBC cell lines (BT-549 and MDA-MB-231) with the highest LINC01614 expression were selected for subsequent experiments. The localization of LINC01614 in TNBC cells was determined using subcellular fraction assay. As displayed in Fig. 1D, LINC01614 was distributed in both nucleus and cytoplasm of TNBC cells.
Figure 1 LINC01614 was highly expressed in TNBC tissues and cell lines. A: LINC01614 (ENSG00000230838) expression in breast invasive carcinoma samples (n = 1085) and corresponding normal samples (n = 291) was analyzed using bioinformatics tool GEPIA. B: RT-qPCR was performed to measure LINC01614 expression in four TNBC cell lines with normalization to its expression in normal human breast epithelial cells (MCF-10A). C: LINC01614 expression levels in the collected TNBC tissues (n = 78) and matched noncancerous tissues (n = 78) were measured by RT-qPCR. D: subcellular fractionation assays were conducted to determine the localization of LINC01614 in cytoplasmic and nuclear parts of TNBC cells. *p < 0.05, ***p < 0.001. TNBC: triple-negative breast cancer.
Knockdown of LINC01614 suppressed the migration, invasion, and EMT process of TNBC cells
In this part, loss-of-function experiments were performed to explore the effect of LINC01614 depletion in the TNBC cell process. After shRNAs were transfected into TNBC cells, LINC01614 expression was markedly diminished in the sh-LINC01614 groups compared to that in sh-NC group (p < 0.001, Fig. 2A). In response to LINC01614 deficiency, there was a significant decrease in the viability of both BT-549 and MDA-MB-231 cells (Fig. 2B, p < 0.001). Wound healing and transwell migration assays revealed that wound closure rate and the number of migrated cells were noticeably reduced in the context of LINC01614 deficiency compared with those in the sh-NC group (Fig. 2C and D). The findings indicate the repressive effect of sh-LINC01614 on cell migration. In addition, cell invasive ability was also inhibited by the silencing of LINC01614, as evidenced by less invaded cells in the sh-LINC01614 groups in comparison to cell number in the sh-NC group (Fig. 2E). Meanwhile, LINC01614 knockdown led to the upregulation of E-cadherin protein levels and the downregulation of vimentin levels compared with their levels in the sh-NC group (Fig. 2F-H). The EMT process is characterized by the loss of epithelial surface markers (notably E-cadherin) and the acquisition of mesenchymal markers such as N-cadherin and vimentin22. Hence, the data suggest the inhibitory effect of LINC01614 knockdown on the EMT process of TNBC cells.
Figure 2 Knockdown of LINC01614 suppressed TNBC cell migration, invasion, and EMT process. A: RT-qPCR was performed to assess LINC01614 expression in TNBC cells transfected with sh-LINC01614#1 and sh-LINC01614#2. B: CCK-8 assays were conducted for detection of cell viability in response to LINC01614 knockdown. C and D: wound healing assays and Transwell migration assays were carried out to evaluate the effect of sh- LINC01614 on the migratory ability of TNBC cells. E: Transwell invasion assays were performed to examine cell invasive ability in the context of LINC01614 depletion. ***p < 0.001. TNBC: triple-negative breast cancer; CCK-8: cell counting kit-8; EMT: epithelial-mesenchymal transition; RT-qPCR: reverse transcription quantitative polymerase chain reaction Knockdown of LINC01614 suppressed TNBC cell migration, invasion, and EMT process. F-H: Western blotting was used to measure the effect of LINC01614 knockdown on protein levels of EMT markers in TNBC cells. GAPDH acts as a loading control. ***p < 0.001. TNBC: triple-negative breast cancer; CCK-8: cell counting kit-8; EMT: epithelial-mesenchymal transition; RT-qPCR: reverse transcription quantitative polymerase chain reaction.
Transcription factor SP1 directly bound to the promoter of LINC01614
The specific SP1 binding sites in the promoter region of LINC01614 were analyzed using JASPAR tool (Fig. 3A). According to the results of RT-qPCR, SP1 expression was significantly downregulated in TNBC cells transfected with sh-SP1 (p < 0.01) and was prominently upregulated in the cells transfected with pcDNA3.1-SP1 vectors (Fig. 3B, p < 0.001). In addition, LINC01614 expression was diminished in TNBC cells silencing SP1 whereas being upregulated in TNBC cells overexpressing SP1 (Fig. 3C). The results indicate the positive expression correlation between SP1 and LINC01614. Data from ChIP assays showed that SP1 directly bound to the chromatin fragments of predicted regions of LINC01614 in BT-549 and MDA-MB-231 cells (Fig. 3D). In addition, luciferase reporter assay was conducted to further validate the activation of SP1 on LINC01614 transcription. The results showed an increased luciferase activity of Wt-pGL3-LINC01614 in the TNBC cells overexpressing SP1, while the luciferase activity of Mut-pGL3-LINC01614 showed no significant difference between pcDNA3.1-SP1 group and corresponding control group (Fig. 3E). The above findings suggest that LINC01614 acts as a direct transcription target for SP1.
Figure 3 Transcription factor SP1 directly bound to the promoter of LINC01614. A: the specific SP1 binding sites in the promoter region of LINC01614 were analyzed using JASPAR tool. B: RT-qPCR was performed to verify the knockdown or overexpression efficacy of SP1 in TNBC cells. C: The effects of SP1 knockdown (or SP1 overexpression) on LINC01614 expression in TNBC cells were measured by RT-qPCR. D and E: chromatin immunoprecipitation assays and luciferase reporter assays were conducted to explore the interaction between SP1 and LINC01614. **p < 0.01, ***p < 0.001. TNBC: triple-negative breast cancer; RT-qPCR: reverse transcription quantitative polymerase chain reaction.
SP1 reversed the inhibitory effect of LINC01614 knockdown on TNBC cell migration, invasion, and EMT process
Subsequently, rescue experiments were performed to investigate whether SP1 is responsible for the functions of LINC01614 in TNBC cells. RT-qPCR showed that LINC01614 expression was noticeably reduced in TNBC cells post-transfection of sh-LINC01614, and the alteration was mostly rescued by SP1 overexpression (Fig. 4A). In addition, the reduction of TNBC cell viability induced by LINC01614 knockdown was partially reversed by SP1 overexpression (Fig. 4B, p < 0.001). According to the results of wound closure assays and transwell invasion assays, SP1 upregulation rescued the decrease in wound closure rate and the number of invaded cells caused by LINC01614 deficiency (Fig. 4C, p < 0.001). The data suggests that SP1 counteracted the inhibitory impact of LINC01614 knockdown on cell migration and invasion. Data from Western blotting showed that the gain of E-cadherin and the loss of vimentin induced by sh-LINC01614 were counteracted in the context of SP1 overexpression (Fig. 4E-G). Overall, SP1 reversed the inhibitory impact of LINC01614 depletion on TNBC cell viability, migration, invasion, and EMT process. The above data indicate that SP1-induced upregulation of LINC01614 promotes the malignant behavior of TNBC cells.
Figure 4 SP1 reversed the inhibitory effect of LINC01614 knockdown on the migration, invasion, and EMT process of TNBC cells. A: RT-qPCR was performed to measure LINC01614 expression in TNBC cells silencing LINC01614 and overexpressing SP1. B: CCK-8 assays, C: wound healing assays, and. D: Transwell invasion assays were carried out to evaluate the effects of LINC01614 knockdown and SP1 overexpression on cell viability, cell migration, and cell invasion, respectively. **p < 0.01, ***p < 0.001. TNBC: triple-negative breast cancer; RT-qPCR: reverse transcription quantitative polymerase chain reaction, EMT: epithelial-mesenchymal transition SP1 reversed the inhibitory effect of LINC01614 knockdown on the migration, invasion, and EMT process of TNBC cells. E-G: Western blotting was performed to quantify protein levels of EMT markers in TNBC cells of sh-NC, sh-LINC01614, sh-LINC01614 + pcDNA3.1, and sh-LINC01614 + pcDNA3.1-SP1 groups. **p < 0.01, ***p < 0.001. TNBC: triple-negative breast cancer; RT-qPCR: reverse transcription quantitative polymerase chain reaction, EMT: epithelial-mesenchymal transition.
SP1-induced LINC01614 activated the Wnt/β-catenin signaling pathway
The aberrant Wnt/β-catenin signaling can promote cancer cell differentiation, proliferation, and stem cell renewal, and targeting the pathway is a promising strategy for personalized cancer treatment23. Previously, LINC01614 was reported to activate the Wnt signaling in pancreatic cancer24. Therefore, protein levels of factors involved in the Wnt/β-catenin pathway were measured by Western blotting. As displayed in Fig. 5A-C, LINC01614 knockdown prominently reduced protein levels of Wnt and β-catenin in BT-549 and MDA-MB-231 cells, and the trend was partially upregulated by SP1 overexpression. The finding indicates that SP1-induced upregulation of LINC01614 may promote TNBC cell processes by activating the Wnt/β-catenin signaling pathway.
Figure 5 SP1-induced LINC01614 activated the Wnt/β-catenin signaling pathway. A: Western blotting was performed to measure protein levels of Wnt and β-catenin in triple-negative breast cancer cells of four groups: sh-NC, sh-LINC01614, sh-LINC01614 + pcDNA3.1, and sh-LINC01614 + pcDNA3.1-SP1. B and C: relative protein levels of Wnt and β-catenin in each group were quantified with normalization to GAPDH. **p < 0.01, ***p < 0.001.
DISCUSSION
TNBC, a subtype of BC, is related to high morbidity and mortality worldwide25. Recently, several lncRNAs, such as DDIT4-AS126, MALAT127, and SEMA3B-AS128, have been reported to modulate TNBC progression by mediating various mechanisms. Nevertheless, the functions and underlying mechanisms of lncRNAs in TNBC have not been clearly illustrated. The present study validated that nuclear transcription factor SP1 induced upregulation of LINC01614 to activate Wnt/β-catenin signaling in TNBC cells, thereby promoting cell viability, migration, invasion, and EMT process.
According to previous studies, LINC01614 can act as an oncogene and exerts regulatory control over diverse cellular processes across various types of cancer. For example, LINC01614 promotes cell proliferation and represses cell apoptosis in lung adenocarcinoma12. LINC01614 knockdown suppressed pancreatic cancer cell process and inhibit tumor proliferation24. Consistent with these articles, the present study also verified the oncogenic role of LINC01614 in TNBC cells. More specifically, this article showed the upregulation of LINC01614 in TNBC cells and tissues, and that the silencing of LINC01614 repressed cell migration, invasion, and EMT process.
As an aggressive type of cancer, TNBC is linked to a high probability of metastasis29. EMT process has been reported to associated with tumor metastasis30, and E-cadherin and Vimentin are regarded as important epithelial and mesenchymal markers, respectively31. The process is characterized by the absence of epithelial surface markers and the acquisition of mesenchymal markers22. Interestingly, LINC01614 was analyzed to be an EMT-associated oncogene by a previous study13. Consistent with that, the current work revealed that LINC01614 knockdown led to increased E-cadherin expression and decreased vimentin level, indicating the inhibitory effect of LINC01614 knockdown on the EMT process of TNBC cells. In another words, LINC01614 facilitates the EMT process of TNBC cells.
Particularly, subcellular fraction assays confirmed the location of LINC01614 in both the cytoplasm and nucleus of TNBC cells, indicating that LINC01614 may exert its role in TNBC cells both transcriptionally and post-transcriptionally. At the post-transcriptional level, the ceRNA role of LINC01614 has been demonstrated in other types of cancer such as osteosarcoma and lung cancer12,15. The study concentrated on the role of LINC01614 at the transcriptional level.
Recently, an increasing number of transcription and epigenetic regulatory factors have been revealed to influence lncRNA expression levels32. SP1 is a transcription factor frequently reported in cancer-related studies33. For example, SP1 could interact with the promoter of lncRNA LUCAT1 and induce its expression in cervical cancer cells, thereby facilitating cell growth, migration, and invasion34. In addition, overexpression of LINC00511 is significantly motivated by SP1 and contributes to glioma progression35. SP1 could bind to the lncRNA TINCR promoter and activate its transcription in colorectal cancer36. In line with the regulatory trend mentioned in previous articles, SP1 in this study was shown to directly bind to the promoter of LINC01614 and significantly activate LINC01614 expression in TNBC cells. Moreover, the current rescue experiments demonstrated that SP1 overexpression partially reversed the inhibitory effect of LINC01614 knockdown on the malignant behavior of TNBC cells, suggesting the involvement of SP1 in the oncogenic mechanisms of LINC01614 in TNBC. The regulatory relationship between SP1 and LINC01614 is consistent with the article focused on the two players in glioma19.
Wnt/β-catenin signaling participates in tumor growth and metastasis37-39. In a very recent study, Wang et al. revealed that Wnt/β-catenin signaling was a downstream pathway that can be regulated by LINC01614 in bladder cancer40. Consistently, the study discovered that LINC01614 depletion led to reduced protein expression of Wnt and β-catenin, and the trend was partly rescued by SP1 overexpression. The findings indicate that SP1-induced upregulation of Wnt/β-catenin signaling. It is worth mentioning that SP1 overexpression can partly but not completely recover the effect of LINC01614 depletion on cell viability and protein levels of EMT markers, Wnt, and β-catenin. That may be due to the involvement of other molecules in the regulatory networks related to SP1 and LINC01614. For example, there could be additional potential lncRNAs that are activated by SP1 and positively regulates the Wnt/β-catenin signaling in TNBC cells.
Since TNBC is perceived as a complex clinical entity, the study lacks further exploration of the SP1/LINC01614/Wnt/β-catenin axis in TNBC, which is a limitation of the study. Another limitation is the absence of animal experiments for further verification of in vitro findings.
In conclusion, experimental results in this study showed that LINC01614 upregulation induced by SP1 promoted TNBC cell migration, invasion, and EMT process by activating the Wnt/β-catenin signaling. The study might provide a novel therapeutical strategy for the advancement of targeted therapy in TNBC treatment. In the future, more experiments shall be performed to further explore other molecules involved in the regulatory axis of SP1/LINC01614/Wnt/β-catenin, and the post-transcriptional role of LINC01614 in TNBC can also be investigated.
