LncRNA PGM5-AS1 regulates cell functions and acts as a potential biomarker to predict prognosis in breast cancer

Abstract

Background

The aggressive proliferation and spread of breast cancer contributes to a dismal clinical outcome. The present study was to investigate the function and underlying mechanism of the long non-coding RNA (lncRNA) PGM5-AS1 in the modulation of breast cancer.

Methods

Quantitative real time-polymerase chain reaction was utilized to assess the levels of PGM5-AS1 and miR-18a-3p. The prognostic significance was evaluated through Kaplan–Meier survival analysis and multivariate Cox regression analysis. Cell viability in breast cancer cells was measured utilizing a cell counting kit-8 kit. Cell migration and invasion were investigated using a transwell assay. The targeted regulatory interaction between PGM5-AS1, miR-18a-3p, and TGFBR3 was validated via dual luciferase reporter gene assay.

Result

Levels of PGM5-AS1 were low in both breast tissue and cancer cell lines. This reduction in expression was linked to various clinical characteristics and a reduced overall survival rate in breast cancer patients. Upregulation of PGM5-AS1 expression noticeably inhibited proliferation, migration, and invasion in breast cancer cells. PGM5-AS1 regulated breast cancer development by controlling the miR-18a-3p/TGFBR3 axis.

Conclusion

The lncRNA PGM5-AS1/miR-18a-3p/TGFBR3 axis is considered a potential genetic target for the development of breast cancer treatments.

Introduction

For the year 2020, global statistics indicate that there was an alarming increase in the number of new breast cancer (BC) diagnoses, totaling 2.26 million cases. Tragically, this was accompanied by 685,000 BC-related fatalities. As a result, BC has surpassed other types of cancer to become the “world's largest cancer” in terms of impact. Furthermore, data reveal that the incidence and mortality rates associated with BC are showing a troubling upward trend annually.^1,2 Treatments for BC have continued to improve in recent years. However, more than 50% of patients will have distant metastasis of cancer cells after treatment, which is life-threatening and has a poor prognosis. ³ Therefore, elucidating the metastasis and recurrence mechanism of BC from the molecular perspective and exploring new molecular targets of BC are of great significance for improving the treatment and outcome for individuals with BC.

Long non-coding RNAs (lncRNAs) are characterized as RNAs exceeding 200 nucleotides in length. ⁴ Accumulating evidence suggests that alterations in the expression of lncRNAs are implicated in the initiation, development, and metastatic potential of tumors.^5–7 Numerous lncRNAs have been demonstrated to contribute to the onset of BC, including the lncRNA SNHG1 that enhances M2-like polarization of macrophages and aids in the progression and spread of BC. ⁶ In a study on lncRNA identification of novel biomarkers for BC, a number of differentially expressed lncRNAs were identified, among which lncRNA PGM5-AS1 was reduced in expression within BC specimens and had diagnostic significance for BC. ⁸ However, the prognostic value of PGM5-AS1 in BC and its role in BC cells have not been elucidated.

According to extensive research, a group of lncRNAs (including RMRP, ⁹ MIR200CHG, ¹⁰ and DANCR ¹¹ ) is capable of altering the aggressive behavior of cancer cells by acting as miRNA sponges, thereby regulating the expression of target genes. We queried downstream miRNAs regulated by PGM5-AS1 through an online database. It has been documented that miR-18a-3p is implicated in the progression of various diseases, such as diabetic cardiomyopathy, ¹² osteoarthritis, ¹³ polycystic ovary syndrome. ¹⁴ One investigation revealed that the level of miR-18a-3p was elevated in the tumor specimens of individuals suffering from BC. ¹⁵ In addition, we also queried the target genes regulated by miR-18a-3p through an online database. Type III TGF-β receptor (TGFBR3) plays a role in a variety of cancers, such as lung, ¹⁶ stomach, ¹⁷ and esophageal squamous cell carcinoma. ¹⁸ One study found that the downregulation of TGFBR3 expression was associated with poor prognosis in BC patients. ¹⁹ However, the association between PGM5-AS1 and miR-18a-3p/TGFBR3 axis remains to be explored.

The objective of this research was to examine the expression of PGM5-AS1 in cases of BC and its clinical value for the prognosis of BC patients and to further explore its regulatory mechanism.

Materials and methods

Patients and sample collection

A sample of 148 individuals suffering from BC who were undergoing mastectomy at Zibo Central Hospital from June 2017 to May 2019 were included in the research. Following the removal of the tumor, the tumor and its neighboring tissues were frozen using liquid nitrogen, and the specimens were promptly stored at −80°C. Fasting blood samples of the researchers were also collected, serum was taken after centrifugation and stored at −80°C for later use. All patients were followed up for a period of 5 years and their prognosis was recorded. The Ethics Committee of Zibo Central Hospital approved the study, and all enrolled patients provided written informed consent. In addition, this experiment is based on the Herkey Declaration.

Cell culture and transfection

Cancerous breast cell lines (MCF-7, BT549, SKBR3, MDA-MB-231) and a normal breast cell line (MCF-10A) were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were thawed, preserved at ultra-low temperatures, and cultivated in DMEM medium supplemented with 10% fetal bovine serum (Gibco, Billings, MT, USA) and subsequently placed in a cell incubator set at 37°C with an atmosphere of 5% CO₂ for the purpose of cell passage.

The PGM5-AS1 overexpression vector (oe-PGM5-AS1) and negative control (oe-NC) were obtained from Transheep Bio (Shanghai, China). Furthermore, the miR-18a-3p mimics/mimics non-coding sequences and miR-18a-3p inhibitor/inhibitor non-coding sequences, were obtained from GenePharma (Shanghai, China). The BC cells were infected with the mentioned agents through Lipofectamine 2000-assisted transfection (Thermo Fisher Scientific, Waltham, MA, USA).

Quantitative real time-polymerase chain reaction analysis

The tumor sample was weighed, and RNA extraction was performed using Trizol agent (Thermo Fisher Scientific). Subsequently, the extracted RNA was converted to complementary (c) DNA through reverse transcription with the PrimeScript RT reagent Kit including genomic DNA Eraser (Takara Bio Inc., Shiga, Japan). The cDNA synthesis process involved incubation at 37°C for 15 min, a brief heat denaturation at 85°C for 5 s, and then storage at 4°C. Quantitative polymerase chain reaction (qPCR) was performed using the THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan) and the LightCycler 480 Real-Time PCR system (Roche, Shanghai, China). The temperature cycling routine began with a 2-min dwell at 94°C, preceded by 30 repeated cycles including a 30-s incubation at 94°C, a 30-s interval at 56°C, and a 60-s extension at 72°C. Primer sequences are exhibited as follows:

PGM5-AS1 forward: 5′-GACTATGTTGTGAGCCTGCG-3′.

PGM5-AS1 reverse: 5′-AAAAGGGGAGGGGCAATACA-3′.

MiR-18a-3p forward: 5′-TCGGCAGGACUGCCCUAAGUG-3′.

MiR-18a-3p reverse: 5′- CTCAACTGGTGTCGTGGA-3′.

GAPDH forward: 5′-GGGAAACTGTGGCGTGAT-3′.

GAPDH reverse: 5′-GAGTGGGTGTCGCTGTTGA-3′.

U6 forward: 5′-CTCGCTTCGGCAGCACA-3′.

U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′.

Cell counting kit-8 proliferation assay

Following the manufacturer's recommended protocol, the cell counting kit-8 (CCK-8) cell proliferation kit (Jiangsu KaiJi Biological, China) was used to measure cell growth. Cells were added into 96-well plates at a density of 5 × 10³ per well, with five replicate wells established for each group. After a 24-h transfection period, a 10% CCK-8 solution was added to each well in the dark and the mixture was further incubated at 37°C for an additional 2 h. The optical density (OD) of each well at a wavelength of 450 nm was then measured using a microplate reader. The OD at this initial time point was recorded as 0 h, and subsequent OD values were determined at 24, 48, and 72 h. The mean OD values from multiple wells within each group were calculated, and a line graph was generated with time on the x-axis and OD values on the y-axis to visualize the proliferation trend.

Transwell assay

The capacity for migration and invasion in BC cell lines was assessed by employing the transwell system (Corning Inc., NY, USA), following the manufacturer's recommended protocol. In short, cells were seeded into the upper compartment of a transwell filter plate, which was either treated with or without a basement membrane matrix coating to separately evaluate their invasive and migratory capabilities. Thereafter, the upper and lower chambers of the transwell were filled with DMEM medium devoid of serum and DMEM medium supplemented with 10% fetal bovine serum, respectively. A density of 10⁵ BC cells was introduced into the upper chamber and allowed to incubate for a duration of 48 h under standard conditions. After the incubation period, the cells in the lower chamber were stained with 0.1% crystal violet solution and examined microscopically. The quantities of cells in five randomly chosen fields of view were then tallied to quantify the migratory and invasive potential of the cells.

Dual-luciferase reporter assay

The interaction between PGM5-AS1, TGFBR3, and miR-18a-3p was confirmed via a dual-luciferase reporter assay system (Promega, Madison, WI, USA). The 3′UTR of the PGM5-AS1 and TGFBR3, both wild type (WT) and mutant (MUT), were respectively inserted into pmirGLO vector (Promega, Madison, WI, USA). These constructs were then introduced into BC cells using Lipofectamine 2000 (Thermo Fisher Scientifc, Inc., Waltham, MA). Subsequently, BC cells transfected with the WT or MUT of the PGM5-AS1 and TGFBR3 gene were concurrently transfected with miR-18a-3p mimics or mimic NC, miR-18a-3p inhibitors or inhibitor NC.

Statistical analysis

The data were presented as mean ± standard deviation. The comparison between the two groups was analyzed using the T-test, and the difference between the multiple groups was analyzed using one-way or two-way analysis of variance. Statistical evaluation was performed using GraphPad Prism 9.0 software. The χ² test was employed to assess the correlation between PGM5-AS1 expression and the pathological parameters of BC patients. A Kaplan–Meier analysis was conducted to investigate the correlation between PGM5-AS1 expression and overall survival of BC patients. Cox regression analysis was utilized to determine the contributing factors affecting the prognostic status of BC patients. A P-value below 0.05 was deemed statistically significant.

Results

PGM5-AS1 was differentially expressed in tumor tissues of BC patients and affects the prognosis of patients

The findings demonstrated that compared to the adjacent normal tissues, the level of PGM5-AS1 was significantly decreased in BC tumors represented in Figure 1(a). The classification was conducted according to the levels of PGM5-AS1. The overall survival rate was notably poorer in patients showing low PGM5-AS1 expression compared to those demonstrating high expression (Log-rank test P < 0.001, refer to Figure 1(b)). Furthermore, multivariate Cox regression analysis identified PGM5-AS1 level, tumor node metastasis stage, degree of differentiation, and axillary lymph node metastasis as prognostic factors in BC patients (Figure 1(c)).

Figure 1.

The level of PGM5-AS1 in patients with BC, which has prognostic value for BC. ^***P＜ 0.001. (a) The PGM5-AS1 levels in BC tissues were quantified via qRT-PCR (n = 148). (b) Kaplan–Meier analysis was employed to examine the correlation between PGM5-AS1 expression and overall survival of patients with BC. (c) Forest map of multivariate regression analysis of BC prognosis.

PGM5-AS1 was significantly correlated with clinicopathological features of BC

The association of PGM5-AS1 expression levels with the clinicopathological characteristics of BC was investigated using the Chi-square test. Based on the meaning PGM5-AS1 expression in tumor specimens, patients with BC were divided into two distinct categories: one with high expression and another with low expression of PGM5-AS1. Research indicated that reduced PGM5-AS1 expression in tumor cells was correlative with a higher proportion of patients having stage III–IV tumors, less differentiated tumors, estrogen receptor positivity, and presence of axillary lymph node metastasis (Table S1).

Overexpression of PGM5-AS1 inhibited the proliferation, migration, and invasion of BC cells

To investigate the impact of PGM5-AS1 on BC cells, we examined the levels of PGM5-AS1 across various BC cell lines (Figure 2(a)) and chose two cell lines (MCF-7 and SKBR3) that exhibited the most pronounced alterations in expression for further study. The results from the quantitative real time-polymerase chain reaction (qRT-PCR) revealed that the transfection of oe-PGM5-AS1 markedly increased the expression of PGM5-AS1 in both MCF-7 and SKBR3 cells (Figure 2(b)). Thereafter, we examined the effects of overexpression of PGM5-AS1 on cell proliferation, migration, and invasion. Figures 2(c) to (f) demonstrated that overexpression of PGM5-AS1 noticeably inhibited the capabilities of MCF-7 and SKBR3 cells to proliferate, migrate, and invade.

Figure 2.

Effects of overexpression of PGM5-AS1 on the function of BC cells. *P ＜ 0.05, **P ＜ 0.01, ^***P ＜ 0.001; n = 3. (a) The expression of PGM5-AS1 in BC cell lines was quantified via qRT-PCR. (b) The transfection efficiency of oe-PGM5-AS1 was quantified via qRT-PCR. (c and d) CCK-8 assay was employed to assess cell proliferation after PGM5-AS1 overexpression. (e and f) The transwell assay was employed to assess the capacity of cell migration and invasion following PGM5-AS1 overexpression.

miR-18a-3p is targeted by PGM5-AS1 in BC cells

miR-18a-3p expression was up-regulated in BC patients (Figure 3(a)). Furthermore, Pearson correlation analysis revealed a significant negative correlation between the altered expression patterns of these two factors within the tumorous samples (Figure 3(b)). Bioinformatics prediction website lncRNASNP2 (https://guolab.wchscu.cn/lncRNASNP/#!/) indicated that an interaction may exist between PGM5-AS1 and miR-18a-3p, as illustrated in Figure 3(c). The results from dual luciferase reporter gene experiments revealed that the activity of luciferase was notably enhanced in BC cells co-transfected with both PGM5-AS1-WT and miR-18a-3p inhibitors, and conversely, it was significantly reduced in cells co-transfected with PGM5-AS1-WT and miR-18a-3p mimics, as shown in Figure 3(d) and (e). These findings implied that miR-18a-3p was capable of binding directly to the 3′-UTR of PGM5-AS1.

Figure 3.

miR-18a-3p is a target of PGM5-AS1. **P ＜ 0.01, ***P ＜ 0.001. (a) The miR-18a-3p level in BC tissues were quantified via qRT-PCR (n = 148). (b) PGM5-AS1 was negatively correlated with miR-18a-3p. (c) The sequence of the binding site between PGM5-AS1 and miR-18a-3p was forecasted utilizing lncRNASNP2. (d and e) The specific interaction between PGM5-AS1 and miR-18a-3p was investigated using a dual luciferase reporter gene assay (n = 3).

PGM5-AS1 regulates the proliferation, migration, and invasion of BC cells by regulating mir-18a-3p

To determine whether PGM5-AS1 influences BC cell function through the modulation of miR-18a-3p expression, we conducted transfection experiments using oe-PGM5-AS1 or oe-PGM5-AS1 + miR-18a-3p mimic in the cells and subsequently assessed their proliferative, migratory, and invasive capabilities. We found that overexpression PGM5-AS1 markedly decreased the levels of miR-18a-3p, whereas the combination of oe-PGM5-AS1 and miR-18a-3p mimic significantly increased miR-18a-3p expression (Figure 4(a)). CCK-8 assays revealed that miR-18a-3p mimic reversed the decrease of cell proliferation induced by PGM5-AS1 knockdown, as depicted in Figure 4(b) and (c). Additionally, transwell assays indicated that miR-18a-3p mimic partially offset the effects of PGM5-AS1 overexpression on cell migration and invasion (Figure 4(d) and (e)).

Figure 4.

miR-18a-3p mimic partially counteracts the effects of oe-PGM5-AS1 on cellular function. *P ＜ 0.05, **P ＜ 0.01, ^***P ＜ 0.001; n = 3. (a) miR-18a-3p level was quantified via qRT-PCR. (b and c) Cell proliferation was assessed using the CCK-8 assay. (d and e) The migration and invasion capability of cells was assessed by the transwell assay.

miR-18a-3p regulates the expression of TGFBR3

The expression of TGFBR3 was down-regulated in the tissues of BC patients (Figure 5(a)), and the level of TGFBR3 is positively correlated with the expression level of PGM5-AS1 (Figure 5(b)) and negatively correlated with the expression level of miR-18a-3p (Figure 5(c)). miRDB database (https://mirdb.org/) was used to predict the binding site of TGFBR3 and miR-18a-3p, as shown in Figure 5(d). The results of dual luciferase reporter gene assay showed that the activity of luciferase was significantly enhanced in BC cells co-transfected with TGFBR3-WT and miR-18a-3p inhibitors, while in contrast, the activity of luciferase was significantly decreased in cells co-transfected with TGFBR3-WT and miR-18a-3pmimics (Figure 5(e) and (f)). At the same time, we also detected TGFBR3 expression levels in cells with miR-18a-3p inhibitors and miR-18a-3p mimics. The results showed that TGFBR3 expression was elevated in BC cells transfected with miR-18a-3p inhibitors, while TGFBR3 expression was decreased in cells transfected with miR-18a-3p mimics (Figure 5(g)).

Figure 5.

TGFBR3 is a target of miR-18a-3p. *P ＜ 0.05, **P ＜ 0.01, ^***P ＜ 0.001. (a) The TGFBR3 level in BC tissues were quantified via qRT-PCR (n = 148). (b) PGM5-AS1 was positively correlated with TGFBR3. (c) miR-18a-3p was negatively correlated with TGFBR3. (d) The sequence of the binding site between TGFBR3 and miR-18a-3p was forecasted utilizing miRDB database. (e and f) The specific interaction between TGFBR3 and miR-18a-3p was investigated using a dual luciferase reporter gene assay (n = 3). (g) The TGFBR3 level in BC cells were quantified via qRT-PCR (n = 3).

Discussion

lncRNAs are dysregulated in BC and are increasingly being recognized as pivotal regulators in the disease's progression. ²⁰ PGM5-AS1 has the capacity to either enhance or inhibit the development of cancer within tumors. The compelled overexpression of PGM5-AS1 inhibited the proliferation of prostate cancer cells and promoted their apoptosis, as evidenced by the curtailed growth of xenograft tumors in nude mice. ²¹ The upregulation of PGM5-AS1 markedly diminished the proliferative and migratory capabilities of bladder cancer cells in vitro, enhanced apoptosis, and attenuated tumor development in vivo. ²² Our research indicated that PGM5-AS1 expression is reduced in BC tissue samples, The pattern of expression aligns with findings from prior research. ⁸ Furthermore, lower levels of PGM5-AS1 expression are associated with poorer patient prognosis. It was widely recognized that increased proliferation rates were linked to the accelerated progression of tumors. Despite contemporary progress in medical science, the phenomenon of metastasis continues to be the principal culprit in BC-related mortalities. ²³ PGM5-AS1 exerted its tumor-suppressing function by suppressing the proliferation, migration, and invasion of BC cells.

Studies have shown that miR-18a-3p is altered in numerous types of cancer. The levels of miR-18a-3p are markedly elevated in gastric cancer linked to Helicobacter pylori, which enhances the proliferation and migration of cancerous cells. ²⁴ The expression of miR-18a-3p was substantially elevated in patients with nasopharyngeal carcinoma compared to the healthy control group. ²⁵ miR-18a-3p promotes the proliferation, invasion, and migration of liver cancer cells. ²⁶ The findings of this research indicated that the level of miR-18a-3p was elevated in the cancerous breast tissues of patients suffering from BC, thus corroborating the outcomes of earlier investigations. ¹⁵

Earlier research has elucidated the potential mechanisms by which lncRNAs function, which are intricately linked to the cellular localization of these RNAs. ⁴ For instance, lncRNAs situated in the cytoplasm could act as miRNA sponges, preventing the impact of miRNAs. ²⁷ Our investigation utilized the bioinformatics prediction tool to forecast that miR-18a-3p is capable of binding to the 3′-UTR of PGM5-AS1. Additionally, we observed that the application of miR-18a-3p mimics the partially restored function of BC cell lines that had been affected by PGM5-AS1 overexpression. Such data implicated PGM5-AS1 in the growth and metastasis of BC, potentially through the modulation of miR-18a-3p expression.

The silencing of TGFBR3 expression has been shown to perturb the typical processes of tissue formation and development. ²⁸ Furthermore, the absence of TGFBR3 has been linked to the development of breast invasive ductal carcinoma and various other forms of epithelial malignancies, reinforcing the notion that TGFBR3 functions as a tumor suppressor mechanism. ²⁹ Similar results were found in this study, with decreased expression of TGFBR3 in BC tissues. In addition, TGFBR3 specifically binds to miR-18a-3p.

Previous studies have reported that lncRNA HOTAIR affect the growth, migration, invasion, and epithelial-mesenchymal transformation of BC cells. ³⁰ Other studies have found that lncRNA PRBC affects the autophagy of BC cells. ³¹ lncRNA DIO3OS also affects chemotherapy resistance of BC cells. ³² lncRNA UBA6-AS1 regulates oxidative stress responses in BC. ³³ In conclusion, lncRNAs could affect the progression of BC through multiple mechanisms. In future studies, we will conduct more experiments to study the effect mechanism of PGM5-AS1 on BC and provide more experimental data for the clinical application of PGM5-AS1. Of course, this study also has some other limitations, such as insufficient sample size, and future studies need to include more patients to provide more convincing data. In addition, there may be more than one molecular axis regulated by PGM5-AS1; more molecular mechanisms need to be explored and verified by many experiments, so this is what we need to strive for in future studies.

Conclusion

The investigation implied that lncRNA PGM5-AS1 was expressed at lower levels in BC specimens and cell lines, and its expression level was indicative of BC patient survival outcomes. In addition, lncRNA PGM5-AS1 appeared to regulate BC development by controlling the miR-18a-3p/TGFBR3 axis, thus impacting the disease's pathogenesis. The lncRNA PGM5-AS1/miR-18a-3p axis is considered a potential genetic target for the development of BC treatments.

Supplemental Material

sj-docx-1-jbm-10.1177_03936155251325846 - Supplemental material for LncRNA PGM5-AS1 regulates cell functions and acts as a potential biomarker to predict prognosis in breast cancer

Supplemental material, sj-docx-1-jbm-10.1177_03936155251325846 for LncRNA PGM5-AS1 regulates cell functions and acts as a potential biomarker to predict prognosis in breast cancer by Lei Xiang, Yueqing Yin, Linna Kong, Pengfei Li, Huihui Zhang, Wenqing Bian and Zhaoxu Wu in The International Journal of Biological Markers