A novel lncRNA AC112721.1 promotes the progress of triple-negative breast cancer by directly binding to THBS1 and regulating the miR-491-5p/C2CD2L axis

doi:10.21203/rs.3.rs-4799911/v1

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A novel lncRNA AC112721.1 promotes the progress of triple-negative breast cancer by directly binding to THBS1 and regulating the miR-491-5p/C2CD2L axis

https://doi.org/10.21203/rs.3.rs-4799911/v1

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Triple-negative breast cancer (TNBC) seriously threatens women's health, long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and play fundamental roles in TNBC. This study aims to explore lncRNAs that can provide effective targets for the early diagnosis or treatment of TNBC. Here, we utilized the TCGA database to analyze differentially expressed genes, survival analysis and ROC curve analysis were also carried out. Notably, we identified a novel lncRNA, AC112721.1, which was significantly overexpressed in TNBC and associated with poor overall survival of TNBC patients. The loss and gain-of functional experiments revealed that AC112721.1 significantly promoted cells proliferation, migration, and suppressed cell apoptosis in vitro and inhibited tumorigenesis in vivo. Further study of underlying mechanisms demonstrated that AC112721.1 regulated the Ras pathway by directly binding to THBS1 protein and also functioned as ceRNA by sponging miR-491-5p to increase the expression of C2CD2L, thereby influencing the progression of TNBC. Taken together, our study indicated that AC112721.1 might be a new biomarker for the evaluation of TNBC prognosis and the treatment of TNBC.

Triple-negative breast cancer

AC112721.1

THBS1

miR-491-5p

C2CD2L

progression

The latest global cancer statistics for 2020 showed that there were as many as 2.26 million new cases of breast cancer, surpassing the 2.2 million cases of lung cancer, making it the leading cancer worldwide[1]. Like many other countries, breast cancer has also become the most common cancer among Chinese women, with a high incidence rate that ranks it as the number one killer of women's health[2].

Triple-negative breast cancer (TNBC) accounts for approximately 10-20% of all breast cancer cases. It is a type of breast cancer characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) expression[3]. TNBC tends to occur at a younger age, has poor differentiation, high invasiveness, and is prone to metastasize to internal organs[4]. It has a high tumor mutation frequency, making it challenging to study its disease mechanisms. TNBC also exhibits poor treatment effectiveness and is more likely to develop drug resistance, with a five-year survival rate of less than 80%. Additionally, conventional hormone therapy and targeted therapy methods are ineffective for TNBC. Currently, comprehensive treatment is typically used, including surgical resection, radiotherapy, and chemotherapy[5]. Chemotherapy is the main treatment modality for TNBC, but it has significant side effects[6]. Thus far, the precise pathogenesis and mechanism of TNBC had not been fully clarified yet. Therefore, elucidating the complex molecular mechanism underlying TNBC progression and identifying the key molecules regulating cancer progression is crucial for the treatment of TNBC.

Long non-coding RNA (lncRNA) is a class of RNA molecules that is longer than 200 nucleotides and plays various functions within organisms[7]. Firstly, they can serve as messenger molecules, transmitting information between the nucleus and cytoplasm, and regulating gene expression[8]. Secondly, lncRNA can bind to DNA sequences as transcriptional regulators, influencing chromatin structure and modifications, thereby affecting gene transcription levels[9]. In addition, lncRNA can also regulate post-transcriptional processes of RNA. Lastly, lncRNA can act as competitive endogenous RNA, influencing the regulatory effects of miRNA[10]. In addition to these functions, accumulating evidence has demonstrated that LncRNA can regulate cellular differentiation, proliferation, apoptosis, etc., and LncRNA abnormal expression and overactivation are usually involved in the cancer initiation and progression.

Recently, it has been proven that LncRNA dysregulation may paly an exclusively essential role in the development and recurrence of TNBC[11]. For example, Liu et al reported that JAG1 enhanced angiogenesis in TNBC through promoting the secretion of exosomal lncRNA MALAT1[12]. Our previous researches also showed that downregulation of HAGLROS could inhibit the proliferation and migration of MDA-MB-231 cell[13]. LINC01234 promoted TNBC progression through regulating the miR-429/SYNJ1 axis[14]. Therefore, LncRNA may be a novel promising biomarker for the treatment of TNBC, finding more specific LncRNAs and elucidated their detailed mechanisms in the pathogenesis are still of great importance.

In this project, we utilized the TCGA database to predict molecules that are differentially expressed in TNBC and specifically strongly associated with TNBC prognosis. Through preliminary screening, we have identified a novel LncRNA, AC112721.1, for the first time, which was overexpressed in TNBC. By conducting in vivo and in vitro experiments, we investigated the role of AC112721.1 in TNBC progression. Furthermore, through transcriptome sequencing, RNA pull-down-MS, and database analysis, we elucidated the potential target genes and signaling molecules regulated by AC112721.1. In summary, our data suggested that AC112721.1 was a potential biomarker and therapeutic target for TNBC.

2.1 Identification of novel lncRNA, AC112721.1, which was highly expressed in TNBC and associated with poor prognosis

To identify the novel lncRNAs in TNBCs, we used the TCGA database to analyze the differential gene expression in four subtypes of breast cancer (Figure 1A). Further, we analyzed the correlation between all differentially expressed genes and overall survival, and found that 159 genes were significantly associated with the survival time in TNBC patients (Figure 1B), including 14 novel LncRNAs (Supplementary Table 3). Among them, AC112721.1 expression was remarkably higher in TNBC tumors than in normal tissues and high-level expression of AC112721.1 was correlated with poor survival (Figure 1C and 1D). Then we undertook the receiver operating characteristic (ROC) curves of AC112721.1 expression to obtain the area under the ROC curve (AUC) values (0.889) (Figure 1E). Finally, we also evaluated the expression of AC112721.1 in clinical samples, showing that AC112721.1 was highly expressed in TNBC tissues (Figure 1F). Altogether, these data suggested that AC112721.1 was highly expressed in breast tumors and specifically correlated with poor survival in TNBC, it can also be used as a biomarker to diagnose TNBC with high sensitivity and specificity.

2.2 AC112721.1 promoted TNBC cell growth and migration.

To investigate the possible role of AC112721.1 in TNBC pathogenesis, we first measured the levels of AC112721.1 in four breast cancer cell lines and an immortalized human mammary cell line (MCF-10A), and we found that AC112721.1 exhibited significantly higher expressed in TNBC cells (MDA-MD-468, MDA-MB-231) (Figure 2A).

We next used shRNA to knockdown AC112721.1 expression in MDA-MB-231, the efficiency of knockdown was confirmed using qRT-PCR (Figure 2B). CCK-8, flow cytometry assay and migration assay were performed on the cell lines. The results showed that knockdown of AC112721.1 significantly decreased cell proliferation, promoted cell apoptosis, and inhibited cell migration (Figure 2C-2E). In contract, overexpression of AC112721.1 increased cell proliferation and migration (Figure 2F-2H). While, the same results were obtained in MDA-MB-468 cells (Supplementary Figure 1A-1D).

To further confirm the function of AC112721.1 on tumorigenicity of TNBC, sh- AC112721.1 or sh-negative control MDA-MB-231 stable cell lines were in situ injected into nude immunodeficient mice, the results showed that AC112721.1 knockdown significantly inhibited tumor growth as shown by decreased tumor volumes and weights (Figure 2I-2K). These finding indicated that AC112721.1 expression may contribute to the development and progression of TNBC.

2.3 AC112721.1 directly interacted with THBS1 and promoted the progression of TNBC by upregulating THBS1

To clarify how AC112721.1 exerts its function in TNBC, we conducted transcriptome sequencing to compare gene expression profiles between AC112721.1 knockdown MDA-MB-231 cells and scrambled cells. The results showed that, compared to the control group, there were a total of 290 differentially expressed genes in the si-AC112721.1 group, among which 242 had gene names (Figure 3A). Next, we performed an RNA pulldown assay to determine the potential binding proteins in MDA-MB-231 using biotinylated AC112721.1 probes, following mass spectrometry analysis (Figure 3B), a total of 77 proteins were detected to interact with AC112721.1 (Supplementary Table 4), and the intersection with the 242 differentially expressed genes identified in the previous data yielded THBS1 (Figure 3C and Supplementary Figure 2A). The interaction between AC112721.1 and THBS1 was further confirmed by RIP assay (Figure 3D). Additionally, we found that the expression level of THBS1 was significantly decreased or increased when AC112721.1 was knocked down or overexpressed, respectively (Figure 3E-3F and Supplementary Figure 2C), which was consistent with the sequencing results (Supplementary Figure 2B). Therefore, we focused on THBS1.

We then investigated whether THBS1 can promote the progression of TNBC. Firstly, we examined the expression of THBS1 in MDA-MB-231 cells and found that THBS1 was significantly upregulated compared to MCF-10A cells (Figure 3G). Moreover, knockdown of THBS1 significantly inhibited cell proliferation, migration, and promoted cell apoptosis (Figure 3H-3K and Supplementary Figure 2D), and we also obtained the same results in MDA-MB-468 cells (Supplementary Figure 2E-2G).

Subsequently, we performed KEGG analysis based on the sequencing results, and the results showed MAPK signaling pathway was the most downregulated pathway in AC112721.1-silenced cells (Figure 3L). Further analysis revealed the protein levels of three molecules involved in MAPK signaling pathway, including RAS, MEK phosphorylation and ERK phosphorylation, were obviously increased upon AC112721.1 overexpression and were decreased upon AC112721.1 knockdown (Figure 3M and Supplementary Figure 2H). While, these three protein levels were down-regulated when knocking down THBS1 (Figure 3N). The above results indicated that AC112721.1 may regulate the MAPK signaling pathway by targeting THBS1 protein, thus affecting the progression of TNBC.

2.4 AC112721.1 functioned as a miR-491-5p sponge in MDA-MB-231 cells

Existing studies have shown that lncRNAs can also act as ceRNA to bind miRNAs, playing an important regulatory role in the occurrence and development of tumors. To explore the miRNAs regulated by AC112721.1 in TNBC, 6 miRNAs with the highest correlation with AC112721.1 were predicted by correlation analysis and cis-trans regulation method, including hsa-miR-24-1-5p, hsa-miR-3926-1, hsa-miR-483-5p, hsa-miR-489-5p, hsa-miR-491-5p, hsa-miR-6788-5p (Supplementary Table 5). Further RT-PCR showed that only hsa-miR-491-5p expression was negatively correlated with AC112721.1 (Supplementary Figure 3A and 3B). And compared to MCF-10A cells, miR-491-5p was lowly expressed in MDA-MB-231 cells (Supplementary Figure 3C). Dual-luciferase reporter assay demonstrated that miR-491-5p mimic could significantly reduce the luciferase activity of wild-type AC112721.1 but not mutant group (Figure 4A), suggesting that miR-491-5p could bind directly to AC112721.1.

To investigate the biological functions of miR-491-5p in TNBC cells, we overexpressed or knocked down miR-491-5p in MDA-MB-231 cells respectively. The results showed that cell proliferation and migration were decreased, cell apoptosis was increased after the overexpression of miR-491-5p (Figure 4B-4E), the same results were also observed in MDA-MB-468 cells (Supplementary Figure 3D-3F). In contrast, the opposite results were observed when miR-491-5p was knocked down (Figure 4F-4I). Then, we co-transfected MDA-MB-231 cells with miR-491-5p mimic and AC112721.1, upregulating miR-491-5p could weaken the promoting effects of AC112721.1 overexpressing on cells proliferation and migration (Figure 4J and 4K).

2.5 AC112721.1 regulated the progression of TNBC through the miR-491-5p/C2CD2L axis

In order to further clarify the downstream target genes of miR-491-5p, TargetScan, RNA22, PolymiRTs, and MiRBase were used for prediction (Supplementary Table 6). As a result, 45 potential target genes were obtained (Figure 5A). Combining differential expression of target genes and literature mining, C2CD2L was preliminarily screened out. Then, qRT-PCR and western blot experiments showed a positive correlation between the expression levels of C2CD2L and AC112721.1 in MDA-MB-231 cells (Figure 5B and Supplementary Figure 4A), as well as a negative correlation between the expression levels of C2CD2L and miR-491-5p (Figure 5C), meanwhile, the increase in the mRNA levels of C2CD2L induced by AC112721.1 overexpression was reduced by miR-491-5p upregulation (Figure 5D). Luciferase assay further showed that miR-491-5p mimic reduced the luciferase activity of C2CD2L 3’UTR luciferase construct but not the mutant construct (Figure 5E), indicating that C2CD2L was the target gene of miR-491-5p, and AC112721.1 could antagonize the inhibitory effect of miR-491-5p on C2CD2L expression.

Given the above results, we investigated whether C2CD2L regulated the progression of TNBC. We found that C2CD2L was significantly upregulated in MDA-MB-231 cells (Supplementary Figure 4B) and TNBC tumors (Supplementary Figure 4C), and patients with high expression of C2CD2L exhibited poorer overall survival than those with low expression of C2CD2L (Supplementary Figure 4D). This suggested that C2CD2L may play an important role in the progression of TNBC. Then we knocked down C2CD2L in MDA-MB-231 cells (Figure 5F), and the results showed that knocking down C2CD2L significantly reduced cell proliferation and migration, increased cell apoptosis (Figure 5G-5I), the same results were also observed in MDA-MB-468 cells (Supplementary Figure 4E-4G). Collectively, these finding demonstrated that AC112721.1 could regulate the progression of TNBC through the miR-491-5p/C2CD2L axis.

TNBC is the fastest deteriorating malignant tumor in breast cancer and the most difficult subtype to diagnose and treat in clinical practice. Compared to other subtypes of breast cancer, TNBC patients have characteristics such as short overall survival, high malignancy, strong invasive potential, and high recurrence rate[6]. It is often diagnosed at an advanced stage, which poses a great challenge for clinical treatment. Because TNBC does not express ER, PR, and HER2, it often does not benefit from endocrine and targeted therapies[15]. Conventional chemotherapy remains the main treatment option for early and advanced TNBC patients. It has been reported that in recent years, neoadjuvant chemotherapy using platinum and ruthenium-containing drugs has shown positive therapeutic effects in some TNBC patients who are sensitive to chemotherapy[15]. However, over 50% of TNBC patients (stage I-III) still experience recurrence, and over 37% of patients die within 5 years[5]. Therefore, identifying new tumor markers and elucidating their pathogenic mechanisms are of great significance for promoting TNBC diagnosis and improving treatment efficacy.

LncRNAs are defined as transcripts of more than 200 nucleotides. They often interact with DNA, RNA, and proteins through complex three-dimensional structures, participating in a wide range of biological activities, including gene expression regulation, chromatin inactivation, pluripotency and differentiation, as well as regulation of alternative splicing[9, 16]. Recent studies have shown that LncRNAs were important regulatory factors in cancer-related cellular pathways[17, 18]. Dysregulation of LncRNAs was associated with cell proliferation, invasion, migration, chemotherapy resistance, and prognosis in TNBC[19, 20]. Due to these unique characteristics, lncRNAs have the potential to serve as novel diagnostic and prognostic biomarkers for TNBC treatment.

Therefore, the aim of this study is to explore LncRNAs that that can provide effective targets for the early diagnosis or treatment of TNBC. Based on this, we first obtained transcriptome sequencing data of TNBC and clinical information data of patients from the TCGA database website. Differential expression analysis of TNBC and normal samples was performed using three analysis methods: limma, edgeR, and DEGseq2. The differentially expressed genes were then subjected to survival analysis and ROC curve analysis. Among them, AC112721.1 expression was remarkably higher in TNBC tumors than in normal tissues and high-level expression of AC112721.1 was correlated with poor survival, moreover, the ROC curve analysis results also demonstrated that this molecule has high sensitivity and specificity in predicting TNBC. Therefore, we selected AC112721.1 as the focus of our study.

AC112721.1 is a novel LncRNA, and currently, there is no research available on the role and mechanism of AC112721.1 in TNBC. Therefore, we conducted the first investigation into the role of AC112721.1 in TNBC. The results showed that AC112721.1 was significantly upregulated in TNBC cells (MDA-MB-231) compared to normal breast cells (MCF-10A) and other types of breast cancer cells. Knockdown of AC112721.1 suppressed the proliferation and migration of MDA-MB-231 cells while promoting apoptosis, whereas overexpression of AC112721.1 yielded opposite results. In vivo experiments also demonstrated that AC112721.1 knockdown inhibited tumor growth. These findings suggested that AC112721.1 can promote the development of TNBC.

To further elucidate the mechanism of action of AC112721.1 in TNBC, we performed transcriptome sequencing and proteomic analysis, which led to the identification of thrombospondin 1 (THBS1) as a specific binding protein to AC112721.1. THBS1 is a major endogenous anti-angiogenic protein secreted by various cell types. Existing research has shown that THBS1 was overexpressed in late-stage tumors such as prostate cancer, pancreatic cancer, glioblastoma, breast cancer, gallbladder cancer, and melanoma[21-23]. Furthermore, THBS1 has been found to regulate immune response and metastasis of tumor cells[24]. In breast cancer, high expression of THBS1 was associated with poor prognosis[25]. However, the mechanism of action of THBS1 in TNBC remains unclear. In this study, we found that the expression of THBS1 was positively correlated with the expression of AC112721.1. Additionally, knocking down THBS1 significantly inhibited the proliferation and migration of MDA-MB-231 cells, promoted apoptosis, and suppressed the Ras/MAPK pathway. These data indicated that AC112721.1 may regulate the proliferation, migration, and apoptosis of TNBC cells by binding to THBS1.

Studies have proven that lncRNAs can also act as ceRNAs, regulating the expression of other mRNAs by competitively binding to miRNAs. They participate in the regulation of gene expression, form complex regulatory networks, and play important roles in cellular functions and disease processes. Here, through bioinformatics analysis and relevant experimental validation, we revealed that AC112721.1 can also upregulate the expression of C2CD2L by sponging miR-491-5p, promoting proliferation and migration of TNBC cells, while inhibiting apoptosis. C2CD2L is a membrane protein that is anchored to the endoplasmic reticulum[26]. It mediates ER-plasma membrane contacts, activates the AKT-mTOR pathway, and regulates cell growth and protein synthesis. C2CD2L was highly expressed in the brain and pancreatic islets[27]. Loss of C2CD2L in insulin-secreting cells led to defects in insulin release[28]. However, the mechanism of action of C2CD2L in TNBC remains unclear. Our study has preliminarily confirmed the promoting role of C2CD2L in TNBC progression.

Although our study has uncovered some new facts, there remain some limitations that should be mentioned. First, further investigation is needed to supplement the biological functions of THBS1 overexpression. Second, rescue experiments and in vivo experiments need to be added to clarify the regulatory role of the AC112721.1/miR-491-5p/C2CD2L axis in TNBC. Third, this study also needs to test the expression levels of AC112721.1 and its downstream target genes in clinical samples, and their correlation with various pathological indicators and prognosis of patients with TNBC.

In conclusion, our study identified a novel LncRNA, AC112721.1, as a regulator of TNBC. We have demonstrated for the first time at both in vitro and in vivo levels that AC112721.1 can promote cellular proliferation and migration, inhibit cellular apoptosis. Mechanistically, AC112721.1 regulated the Ras pathway by directly binding to THBS1 protein and also functioned as ceRNA by sponging miR-491-5p to increase the expression of C2CD2L, thereby influencing the progression of TNBC (Figure 5J). Our findings provided a new insight into the TNBC pathogenesis and will be useful for exploring candidates for therapeutic targets in TNBC in future.

4.1 Clinical specimens

20 triple-negative breast cancer samples and 20 paracancerous tissues were collected from 30 patients who underwent surgery at the First People's Hospital of Yunnan Province. All samples were histologically confirmed by a pathologist. The obtained tissue samples were immediately frozen in liquid nitrogen after the surgery. The protocol was approved by the Ethical Committee of First People's Hospital of Yunnan Province (No. KMUST-MEC-016). The informed consent was obtained from all human subjects. Research involving human research participants must have been performed in accordance with the Declaration of Helsinki

4.2 TCGA Data collection

The RNA-Seq data sets of 1,083 breast cancer and 104 normal samples were collected from TCGA (http://cancergenome.nih.gov/). Clinical information of the samples in TCGA was matched with the data of RNAs. The differentially expressed genes (DEG) in TNBC compared to normal tissues were analyzed using three R language packages, DEGseq2、edgeR and limma. In this study, absolute log2 fold change> 1 and p < 0.05 were set as the cut-off criteria to define DEG. For survival analysis, we first extracted relevant variables such as patient survival status and survival time from clinical data. We then grouped patients based on gene expression levels and finally used the "survival" package to plot Kaplan-Meier survival curves. We used the pROC package in R software to assess the diagnostic value of AC112721.1. The larger the area under the curve (AUC), the higher the sensitivity and specificity of the test.

4.3 Cell culture

Four human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, T47D) and

an immortalized mammary epithelial-like cell line, MCF-10A were purchased from the Peking Union Medical College Cell Culture Center (Beijing, China) and cultured at 37°C in a humidified incubator with 5% CO₂. MDA-MB-468, MDA-MB-231, and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; C11995500BT, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS, 10099141, Thermo Fisher Scientific), while T47D cells were maintained in Roswell Park Memorial Institute-1640 medium (C11875500BT, Thermo Fisher Scientific) supplemented with 10% FBS. MCF-10A cells were cultured in DMEM/F12 (12500-062, Thermo Fisher Scientific) medium supplemented with 10% horse serum (HyClone, Utah, USA), 1 mg/mL epidermal growth factor (Merck KGaA, Darmstadt, Germany), 1 mg/mL cholera toxin (Merck KGaA), 20 mg/mL insulin (Merck KGaA), and 1 mg/mL hydrocortisone (Merck KGaA). All cell lines were free from mycoplasma contamination.

4.4 Cell transfection

The lentivial-based small hairpin RNA (shRNA) target AC112721.1 was constructed by General Biosystems (Anhui, China) and cloned into pGreenPuro vector. The lentiviral vector was co-transfected with packing vectors psPAX2 and pMD2.G into 293T cells for lentivirus production. To establish stable cell lines, MDA-MB-231 cells were transduced by above lentivirus particles (2mL) with polybrene (10μg). After infection for 12h, the culture medium containing lentivirus particles was removed, and 4mL fresh medium containing puromycin was added to continue culture. After 2 weeks of section, qRT-PCR was performed to detect AC112721.1 expression and confirm the stable cell lines.

For transient transfection, MDA-MB-231 cells were transfected with small interfering RNAs (siRNAs), pcDNA3.1- AC112721.1 plasimd, miRNA mimics or miRNA inhibitor using TransIntro EL transfection reagent (TransGen Biotech, Beijing, China) according to the manufactures’s instructions. The AC112721.1 siRNA, THBS1 siRNA, C2CD2L siRNA, miR-491-5p mimic, miR-491-5p inhibitor and matched negative controls were purchased from RiboBio (Guangzhou, China). All the sequences were listed in supplementary Table 1.

4.5 RNA extraction and quantitative RT-PCR assays

Total RNA from tissues and cell lines was extracted using TRIzol Reagent (T9424, Merck KGaA) according to the manufacturer’s instructions. cDNA was synthesized from RNA using GoScript™ Reverse Transcription System (Promega, Wisconsin, USA). For qPCR, a specific forward primer was designed for each miRNA and reverse primers were targeted to the stem-loop sequence. Expression levels of miRNAs/mRNAs were analyzed by qPCR using SYBR Green Master Mix (Roche, Basel, Switzerland), and U6/GAPDH was used as an internal control. The expression level of each gene was calculated and normalized using the 2^−ΔΔct method. ABI 7300 instrument (Life technology, USA) was used to conduct the qRT-PCR and data collection. The primers and the sequences were shown in supplementary Table 1.

4.6 Cell proliferation assays

The cell proliferation assay was measured using Cell Counting Kit-8 (CCK-8) (Ribo Bio, Guangzhou, China). Cells were seeded in 24-well plates and transfected at different time points, 50μL of CCK-8 solution was added to each well, and they were incubated for 20-40 min at 37°C under 5% CO₂. Finally, the absorbance was measured using a microplate reader (Bio Tek) at 450nm.

4.7 Apoptosis analysis

Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (11988549001, Roche) was applied to detect cell apoptosis according to the manufacturer's instructions. Cells were digested with trypsin and washed by PBS twice, then, the harvested cells were incubated in a buffer supplemented with 1 μL Annexin V and 2 μL propidium iodide for 30 minutes at 4°C and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, USA).

4.8 Western blot analysis

Protein lysates were separated using sodium dodecyl sulfate polyacrylamide electrophoresis gels and transferred to polyvinylidene fluoride membranes (Merck KGaA), the membranes were blocked with 5% BSA at room temperature for 1 hour. Corresponding primary antibodies, including Vinculin (Bioss, bsm541448, 1:5000), Ras (CST, 8832, 1:1000), MEK (CST, 4694, 1:1000), p-MEK (CST, 9154, 1:1000), ERK (CST, 4695, 1:1000), p-ERK (CST, 4370, 1:1000), overnight at 4°C. The membranes were washed thrice with Tris-buffered saline and Tween-20 for 10 minutes and incubated with secondary antibodies for 2 hours at room temperature. The protein bands were examined with Immobilon Western Chemiluminescent reagent (Merck KGaA) and images were captured with a chemiluminescence imaging system (Tanon 5200, Shanghai, China).

4.9 RNA Immunoprecipitation (RIP)

RIP assay was performed using Magna RIPTM Kit (17-700, Millipore Corporation, USA) according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were harvested and lysed with RIP lysis buffer. The magnetic beads were washed three times with RIP wash buffer, followed by incubation with 5 µg anti-THBS1 or anti-IgG antibodies for 30 mins at room temperature. Then, cell lysates were incubated with magnetic beads at 4°C overnight. Subsequently, the incubated samples were washed 6 times with RIP wash buffer. Finally, The immunoprecipitated RNAs were eluted and analyzed by RT-PCR.

4.10 Transwell assays

After transfection for 48 hours, the cells were seeded in 6.5 mm transwell chambers with 8 μm pores (3422, Corning Incorporated, New York, USA). The upper chambers were filled with 200 μL of transfected cells (8×10⁴ cells) with the medium of 0.1%FBS, and the lower chambers were filled with 500 μL of complete medium. After 24 hours of incubation, the non-migratory cells that remained on the upper surface were removed with a cotton swab. The migrated cells on the lower surface of the membrane insert were fixed with 4% paraformaldehyde in PBS and stained with 0.1% crystal violet. The number of cells in the lower chamber was counted with light microscopy.

4.11 Luciferase activity analyses

The wild-type (AC112721.1-WT) or mutant-type (AC112721.1-MUT) fragment and the 3’ untranslated region (UTR) of C2CD2L (C2CD2L-WT or C2CD2L-MUT) were cloned into the pmeI and XbaI sites of the PmirGLO vector (Promega). Both constructs were verified by sequencing. 293T cells were cultured in 12-well plates, and were co-transfected with 50 nM control mimic or miR-491-5p mimic, 2µg either wild-type vector or mutant vector using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, the relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer’s instructions. Values represent the mean±standard deviation of three experiments from three independent assays.

4.12 Animal experiments

All protocol was approved by the Animal Ethics Committee of the Kunming University of Science and Technology (PZWH（dian）K2024-0001), all experiments were performed in accordance with relevant guidelines and regulations. Four-week-old female null mice were purchased from Hunan SJA Laboratory Animal Co.，Ltd. 1×10⁶MDA-MB-231 cells that transfected with lentivirus (sh-NC or sh-AC112721.1) were injected subcutaneously. Tumor volumes were measured every 2 days, the volume was calculated using the following formula: V=0.5×length×width². After 4 weeks, mice were sacrificed by cervical dislocation and the tumor weigh was examined.

4.13 RNA-sequencing

Total RNAs were extracted from MDA-MB-231 cells which were transfected with si-AC112721.1 or si-NC. The integrity of RNA was tested by Agilent 2100 bioanalyzer, and the library was built using NEBNext® Ultra™ Directional RNA Library Prep Kit (Illumina, USA). Each library was sequenced on Illumina Novaseq 6000 platform following the vendor’s recommended protocol by Novogene Technology Co., Ltd (Beijing, China).

Differential Expression Gene (DEG) analysis steps: First, the raw gene expression data is preprocessed, including data cleaning, normalization, and filtering. Then, samples are grouped according to different experimental conditions. Subsequently, statistical methods are used to compare the differences in gene expression levels between different groups. To control the false discovery rate caused by multiple hypothesis testing, p-values are usually corrected. Finally, for the identified DEGs, biological interpretation is conducted. This may include gene function annotation, pathway analysis, gene ontology analysis, etc.

4.14 RNA pulldown

The biotinylated probes targeting AC112721.1 were synthesized by GuangZhou Lab Biotechnology Co.,Ltd. MDA-MB-231 cells were lysed with RIP buffer. The supernatant was incubated with biotinylated probes at 4℃ overnight. Streptavidin-linked magnetic beads were added to the mixture and incubated at room temperature for 1h. The captured proteins were eluted and analyzed by mass spectrometry.

4.15 Mass spectrometry (MS)

For mass spectrometry, the proteins were subjected SDS-PAGE gel, the different band was cut and identified by mass spectrometry,. The mass spectrometer was used to acquire raw mass spectrometry data. Proteome Discoverer 1.4 (Version 1.4.0.288, Thermo Fisher) was used to convert the RAW files into MGF format mass spectrometry files. The MGF format mass spectrometry files and protein search library were input into ProteinPilot™ Software 4.5 (version 1656, AB Sciex) for mass spectrometry analysis. The built-in parameter settings in

ProteinPilot™ Software 4.5 were as follows:

Items	Parameters
Detected Protein Threshold [Unused ProtScore (Conf)] >	0.05 (10.0%)
Paragon™ Algorithm	4.5.0.0, 1654
Annotations Retrieved from UniProt	Yes
Sample Type	identification
Cys. Alkylation	MMTS
Digestion	Trypsin
Instrument	Orbi MS, Orbi MS/MS
ID Focus	Biological modifications
Search Effort	Thorough
FDR Analysis	Yes
User Modified Parameter Files	No

The search database used in this experiment is Homo sapiens: https://www.uniprot.org/proteomes/UP000005640. After subtracting the control group data from the identified protein data of the experimental group, the remaining differentially expressed proteins were considered as target binding proteins.

The exact sequence of AC112721.1 used for RNA-pulldown was shown in supplementary Table 2.

4.16 Prediction of AC112721.1 target miRNAs

Firstly, R language Corrplot R package was used to analyze the correlation between all differentially expressed genes and AC112721.1, then the miRNAs associated with HAGLROS were obtained (| R2 | > 0.3 & p < 0.05). Secondly, prediction of AC112721.1-related miRNAs using cis-trans regulation method[13]. Thirdly, Using the cor.test function in R language, we calculated the correlation between AC112721.1 and all related miRNAs using Pearson correlation coefficient. Finally, 6 miRNAs with the highest correlation with AC112721.1 were obtained, including hsa-miR-24-1-5p, hsa-miR-3926-1, hsa-miR-483-5p, hsamiR-489-5p, hsa-miR-491-5p, hsa-miR-6788-5p.

4.17 The potential targets of miR-491-5p

We used four online databases to predict the potential targets of miR-491-5p, including TargetScan, RNA22, PolymiRTs and MiRBase. The target genes we screened were simultaneously predicted by at least three databases.

4.18 Statistical analyses

Data were shown as the mean ± standard deviation (SD). All statistical analyses were conducted using SPSS version 20 software (IBM Corp., Armonk, USA), GraphPad Prism 7 (GraphPad Software, La Jolla, USA), and R language (version 3.3.1; http://www.r-project.org/; R Foundation). A value of p < 0.05 was considered statistically significant. All experiments were performed at least thrice with triplicate samples.

Ethics approval and consent to participate

20 breast cancer samples and 20 paracancerous tissues were collected from 30 patients who underwent surgery at the First People's Hospital of Yunnan Province. The protocol was approved by the Ethical Committee of First People's Hospital of Yunnan Province (No. KMUST-MEC-016). The informed consent was obtained from all human subjects. The animal experiments were approved by the Animal Ethics Committee of the Kunming University of Science and Technology (PZWH（dian）K2024-0001). Research on human subjects adheres to the Declaration of Helsinki principles.

Conflicts of Interest: The authors declare no potential conflicts of interest.

Data Availability

The original contributions presented in the study are included in the article/supplementary files. Further inquiries can be directed to the corresponding author.

Funding

This work was supported by Yunnan High-level Personnel Training Support Program (YNWR-QNBJ-2020-243) and Yunnan Province Science and Technology Program, China (grant number 202401AT070372).

Authors' contributions

PM, WT and MS contributed to the design and conception of the study. PM, SK, HL and ML conducted experiments. YZ collected the clinical samples. HY and JP performed the statistical analysis. PM, HY and MS wrote the first draft of the manuscript. All authors read and approved the submitted the submitted version.

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No competing interests reported.

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A novel lncRNA AC112721.1 promotes the progress of triple-negative breast cancer by directly binding to THBS1 and regulating the miR-491-5p/C2CD2L axis

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