Long Non-Coding RNA PVT1 Regulates the Proliferation and Metastasis of Human Breast Cancer Via the Wnt/B-Catenin Signalling Pathway

Basic research

Long non-coding RNA PVT1 regulates the proliferation and metastasis of human breast cancer via the Wnt/b-catenin signalling pathway

Nianqu Zhang1, Qing Li1, Shanmei Sun2

1Department of Breast Surgery, The First Affiliated Hospital of Shandong First Medical Corresponding author: University, Shandong Provincial Qianfoshan Hospital, Jinan, Shandong, China Shanmei Sun 2Department of Traditional Chinese Medicine, The First Affiliated Hospital of Department of Traditional Shandong First Medical University, Shandong Provincial Qianfoshan Hospital, Jinan, Chinese Medicine Shandong, China The First Affiliated Hospital of Shandong First Submitted: 25 March 2020 Medical University Accepted: 20 August 2020 Shandong Provincial Qianfoshan Hospital Arch Med Sci Jinan, Shandong DOI: https://doi.org/10.5114/aoms/126557 250014, China Copyright © 2021 Termedia & Banach Phone/fax: +86 531 8296 8900 E-mail: RosalindMurraynLxZ@ Abstract yahoo.com, [email protected] Introduction: The development of many human diseases has been implicated to be coupled by the dysregulation of long non-coding RNAs (lncRNAs). Considering this, the current study was aimed at identifying and then inves- tigating the molecular role of a specific lncRNA from a set of such genetic elements in regulating the developmental aspects of human breast cancer. Material and methods: The quantitative real-time polymerase chain reaction (qRT-PCR) method was used to deduce the gene expression levels. Prolifer- ation of cancer cells was determined by the cell counting kit 8 (CCK8). The evaluation of apoptotic cell death in breast cancer cells was made through the acridine orange/ethidium bromide (AO/EB) and annexin V-FITC staining protocols. Transwell assays were used to monitor cell migration and invasion. Results: Estimation of gene expression levels of a set of lncRNAs showed that lncRNA PVT1 is specifically overexpressed in the breast cancer tissues and cell lines. The downregulation of PVT1 in cancer cells negatively affected their proliferation rates, and cancer cells exhibited significantly lower viabilities due to induction of Bax/Bcl-2 signal arbitrated apoptotic cell death in the cancer cells. Moreover, the cancer cells showed significantly lower rates of migration and invasion when lncRNA PVT1 was repressed. The PVT1 repression-driven anti-cancer effects against the cancer cells were seen to be modulated through the Wnt/β-catenin signalling pathway. Conclusions: The results of this work are indicative of the prognostic role of lncRNA PVT1 in breast cancer. Also, the molecular targeting of PVT1 might prove to be a vital step against the progression of human breast cancer.

Key words: long non-coding RNA, breast cancer, apoptosis, anti-cancer, migration, invasion.

Introduction Human breast cancer is the most dominant cancerous malignancy among women [1]. Breast cancer is seen to affect about 1.7 million women on average annually globally [2]. The data suggest that in the USA alone approximately 1 in 8 women has to face breast cancer malignancy at some stage in her life, with highest incidence rate in the 55–64-year- old age group [3]. Breast cancer is also seen to cause the highest number Nianqu Zhang, Qing Li, Shanmei Sun of cancer-related deaths among women world- Material and methods wide [4]. Data about cancer-related mortality suggest that in 2013 the deaths resulting from breast Clinical specimens and maintenance of cell cancer accounted for about 15% of the total can- lines cer-related deaths [5]. The most important hurdle The clinical specimens pertaining to breast in combating the disease of breast cancer is poor cancer along with the surrounding normal tis- prognosis at the early stages of its incidence to- sues were obtained after obtaining informed con- gether with the lack of effective treatment mea- sent and under standard ethical guidelines from sures [6, 7]. Research studies of recent times have breast cancer patients being treated at the First laid profound stress on the vitality of the genetic Affiliated Hospital of Shandong First Medical Uni- factors regulating the progression of the develop- versity, Shandong Provincial Qianfoshan Hospital, ment of many dominant human disorders, includ- Jinan, Shandong, China. Immediately after their ing different types of cancer. These genetic factors collection, the tissue specimens were frozen in also include the category of long non-coding RNAs liquid nitrogen and kept at –80oC for future ex- (lncRNAs). lncRNAs have been shown to have tre- perimentation. The normal human breast cell line mendous potential to act as molecular prognostic (MB-157) and the cancer cell lines (MDA-MB-231, tools in the diagnosis of the human cancers, to- SK-BR-3, CAMA-1, and MDA-MB-436) were ob- gether with the possibility of the use of specific tained from ATCC, USA. DMEM growth medium lncRNA molecule targeting as an efficient antican- (Thermo Scientific) with 10% FBS was used for cer strategy against particular human cancers [8]. cell culturing, and a CO2 incubator was used to Among lncRNAs, lncRNA PVT1 has been shown to maintain the cell lines in an environment of 5% play a role in the development of different can- CO2 and 95% air at 37°C. The study was approved cers. For instance, lncRNA PVT1 has been shown by the research ethics committee of The First Af- to promote the angiogenesis and invasion of gas- filiated Hospital of Shandong First Medical Uni- tric cancer [9, 10]. Similarly, it has been shown to versity, Shandong Provincial Qianfoshan Hospi- regulate the development of triple-negative breast tal, Jinan, Shandong, China with approval number cancer through KLF5/β-catenin signalling [11]. In 6787FMU-2019. yet another study, lncRNA PVT1 has been shown to regulate the progression of human breast can- Transfection cer [12]. Nonetheless, the information on the reg- ulation of breast cancer growth and metastasis The transfection of cell lines was performed by via modulation of the Wnt/b-catenin signalling using Lipofectamine 2000 reagent. The downregu- pathway is still largely unknown. Considering lation of the PVT1 gene was made by transfecting this, the present study aimed to unravel the spe- the cancer cells by its RNA interference construct cific lncRNA molecule associated with the growth (si-PVT1) using si-NC as the silencing control. and progression of breast cancer together with its molecular characterization in regulating the Gene expression analysis proliferation and metastasis of breast cancer. The A TRIzol reagent (Thermo Scientific)-based results showed that lncRNA PVT1 is specifically method was used for isolation of total RNA from overexpressed in breast cancer tissues and cancer the tissue specimens and the cell lines. DNase I cell lines. Its downregulation led to a significant treatment was employed for removal of DNA con- decline in the proliferation rate and viability of tamination, and the RNA was then used for cDNA breast cancer cells. The reason behind this growth synthesis with the help of a miScript First Strand inhibitory effect was found to be the Bax/Bcl-2- cDNA synthesis kit (Qiagen). Using this cDNA as driven apoptotic induction in the cancer cells. a template, SYBR Green mix was used to perform Moreover, it was shown that the migration and the quantitative real-time expression analysis, and invasion of breast cancer cells declined severely the gene expression levels were ascertained by under the transcriptional repression of lncRNA employing the 2–ddct method. The human β-actin PVT1. The anti-cancer effects of downregulation gene was used as an internal control in the gene of lncRNA PVT1 against breast cancer cells were expression studies. A heat map was generated found to be propagated through the inhibition of from the expression values with the help of an on- the Wnt/β-catenin signalling pathway in the can- line software tool (www.heatmapper.ca). cer cells. Taken together, the results of this study indicate the molecular therapeutic potential of Assessment of cell proliferation lncRNA PVT1 to aid in the prognosis of breast cancer. Furthermore, it is also evident that targeting The SK-BR-3 breast cancer cells were transfect- of PVT1 might emerge as a vital step in the strat- ed with si-NC or si-PVT1 for 24 h and then about egy against the proliferation and advancement of 2 × 105 cells/well were cultured in 96-well plates human breast cancer in the future. for 0, 12, 24, 48, or 96 h at 37oC. Each well of the

2 Arch Med Sci Long non-coding RNA PVT1 regulates the proliferation and metastasis of human breast cancer via the Wnt/b-catenin signalling pathway

96-well plate was then supplied with 10 µl CCK8 Estimation of cell migration and invasion reagent from the Cell counting kit 8 (CCK8, Ther- The transwell assay method was performed for mo Scientific). The 37°C incubation was prolonged the estimation of the migration and invasion of for a further 4 h. Following the kit protocol, each SK-BR-3 cancer cells transfected with si-NC or si- sample was then processed for estimation of cell PVT1. For the estimation of the former parameter, proliferation rates by determining the absorbance the transwell lacked Matrigel but in the latter’s es- at 570 nm wavelength. timation, Matrigel-fitted transwell was used. The upper and lower chambers of the transwell plate Colony-forming assay were supplemented with the cancer cells and The viabilities of the SK-BR-3 cancer cells trans- growth medium, respectively. Culturing of the cells fected with si-NC or si-PVT1 was inferred through was done for 24 h at 37°C, after which the filter of their respective colony-forming potential. Briefly, the transwell chamber was taken and cells stick- a homogenized solution of about 200 cells was ing to its upper surface were cleared using cotton added to each well of the 6-well plate. The cells swabs. The cells from the lower surface were fixed were allowed to grow for 4 days at 37°C, after with 70% methanol and stained with 0.1% crystal which they were fixed with methanol, stained violet solution and then assessed under a light with crystal violet solution (0.1% w/v), and then microscope at 100× magnification to assess the observed for the colony formation under the light migration and invasion of the cancer cells. microscope. Statistical analysis AO/EB and annexin V-FITC/PI staining assays To minimize the experimental error, three repli- To confirm if downregulation of lncRNA PVT1 cates were used for every experimental set up. The inhbited the proliferation of the SK-BR-3 can- average and standard deviation were taken from cer cells, AO/EB and annexin V-FITC/PI staining the obtained values and final representation was assays were performed. Consequently, the cells given as the average ± standard deviation (SD). were added to 12-well plates at densities of 106 Student’s t-test and ANNOVA were performed cells/well. The cells were cultured at 37°C for 24 h. using GraphPad prism 7.0 software. The p-values Centrifugation was used to collect the cells, which ≤ 0.05 were taken as measure of a statistically sig- were fixed with 70% ethanol. The cells were then nificant difference between 2 experimental data- stained with acridine orange/ethidium bromide points. (AO/EB) or annexin V-FITC/PI solutions. The cancer cells stained with former staining mix were exam- Results ined under the fluorescent microscope and those PVT1 expression is downregulated in stained with the latter solution were assessed human breast cancer with the flow cytometric technique to determine the level of apoptotic cell death in breast cancer Among the different long non-coding RNAs cells. (lncRNAs) whose expression was analysed in different breast tissue specimens, only the lncRNA Western blotting PVT1 expression was specifically seen to be overexpressed in the breast cancer tissues, whereas The SK-BR-3 cancer cells transfected with si-NC the expression of all other lncRNAs did not reveal or si-PVT1 were lysed with the lysis buffer (20 mM any transcript level correlation (Figure 1 A). More- HEPES, 350 mM NaCl, 20% glycerol, 1% Nonidet over, the qRT-PR expression analysis of lncRNA P 40, 1 mM MgCl , 0.5 mM EDTA, 0.1 mM EGTA, 2 PVT1 in normal breast cell line (MB-157) and the 1 mM DTT, 1 mM PMSF, protease inhibitor cock- cancer cell lines (MDA-MB-231, SK-BR-3, CAMA-1, tail, and phosphatase inhibitor cocktail) to extract and MDA-MB-436) revealed that cancer cell lines the total cellular proteins, the concentrations exhibited significantly higher transcript levels of of which were calculated by the Lowry method. PVT1 (Figure 1 B). Amongst the cancer cell lines, From each protein extraction, about 40 µg pro- the SK-BR-3 cells possessed the highest expres- teins were separated on 10% SDS-PAGE. The gel sion of PT1 and thus were used for further experi- contents were transferred by blotting method to mental characterization. PVDF membranes. Primary antibody exposure was given overnight at 4°C. Then, the membranes Inhibition of SK-BR-3 cell viability upon were treated with secondary antibodies. Finally, PVT1 repression chemiluminescence reagent was used to visualize the protein bands for concentration estimations. Utilizing the RNA interference approach, the The protein expressions were standardized using lncRNA PVT1 was downregulated in the SK-BR-3 β-actin protein. cancer cells, and the same was confirmed by the

Arch Med Sci 3 Nianqu Zhang, Qing Li, Shanmei Sun

A B 7 * 6 –3 0 2 C1 C2 C3 C4 C5 C6 N1 N2 N3 N4 N5 N6 5 Column Z-Score CCAT2 4 3 DNM3OS 2 Relative expression Relative HF1A-AS1 1 0 KCNQ1DN MB-157 MDA- SK-BR-3 CAMA-1 MDA- MB-231 MB-436

PACERR C 1.2 TCL6 1.0 LINC00152 0.8

DLEU1 0.6

0.4 DSCAM-AS1

Relative expression Relative * 0.2 PVT1 0. si-NC si-PVT1 PLNCRNA-1

D 1.2 Figure 1. lncRNA PVT1 is overexpressed in breast cancer, 1.0 and its downregulation reduces the proliferation of breast cancer cells. A – qRT-PCR expression analysis of different 0.8 lncRNAs in normal and cancerous breast tissues presented * as a heat map; B – qRT-PCR expression analysis of lncRNA 0.6 PVT1 in normal (MB-157) and the cancer cell lines (MDA- 0.4 MB-231, SK-BR-3, CAMA-1, and MDA-MB-436); C – qRT- PCR expression analysis of lncRNA PVT1 in SK-BR-3 cancer Relative expression Relative 0.2 cells transfected with si-NC or si-PVT1; D – CCK8 kit assay 0. for the assessment of proliferation of SK-BR-3 cancer cells 0 12 24 48 96 transfected with si-NC or si-PVT1. Individual experiments Time [h] were performed in triplicate, and the values are presented si-NC si-PVT1 as mean ± SD (*p < 0.05) qRT-PCR method (Figure 1 C). The breast cancer SK-BR-3 cancer cells led to the induction of apop- cells transfected with si-NC or si-PVT1 constructs tosis of the cancer cells (Figures 3 A, B). The flow were processed for estimation of proliferation cytometry revealed that the percentage of apop- rates through a CCK8 kit-based method. The re- totic cells was 21.16% under PVT1 downregula- sults showed that downregulation of lncRNA PVT1 tion, while the control cells showed only 2.18% in breast cancer cells significantly reduced the cell apoptosis. The western blotting of Bax and Bcl-2 proliferation (Figure 1 D). Further, the downregu- proteins showed that downregulation of PVT1 in lation of PVT1 also led to a significant decline in cancer cells led to an increase of the former pro- the colony-forming potential of the breast cancer tein and a decrease in the concentration of the cells, and si-PVT1-transfected cells exhibited only latter (Figure 3 C). Thus, the results suggest that 47% of the number of colonies in comparison to transcriptional repression of PVT1 in breast can- the si-NC-transfected cancer cells (Figure 2). These cer cells induced Bax/Bcl-2-driven apoptotic cell effects are indicative of the therapeutic potential death. of lncRNA PVT1. SK-BR-3 cell migration and invasion Suppression of lncRNA PVT1 triggers declines under PVT1 transcriptional apoptosis in SK-BR-3 cells repression The AO/EB and annexin V-FITC/PI staining The migration and invasion of breast cancer assays revealed that the repression of PVT1 in cells transfected with si-NC or si-PVT1 was anal-

4 Arch Med Sci Long non-coding RNA PVT1 regulates the proliferation and metastasis of human breast cancer via the Wnt/b-catenin signalling pathway

si-NC si-PVT1 150

100

Colonies (%) Colonies 50

0 si-NC si-PVT1 Figure 2. Downregulation of lncRNA PVT1 decreases the viability of breast cancer cells. Analysis of the colony-forming ability of SK-BR-3 cancer cells transfected with si-NC or si-PVT1. Individual experiments were performed in triplicate, and the values are presented as mean ± SD (*p < 0.05) A si-NC si-PVT1

B si-NC si-NC 104 104 2.18% 21.16%

103 103

2 2

PI 10 PI 10

101 101

0.85% 18.40% 100 100 100 101 102 103 104 100 101 102 103 104 Annexin V Annexin V C si-NC si-PVT1 5

* Bcl-2 4

Bax 2

Relative expression Relative 1 Actin * 0 Bax Bcl-2 si-NC si-PVT1 Figure 3. Downregulation of lncRNA PVT1 induces apoptotic cell death in breast cancer cells. A – AO/EB staining procedure for the assessment of apoptosis of SK-BR-3 cancer cells transfected with si-NC or si-PVT1; B – Annexin V-FITC/PI staining procedure for the assessment of apoptosis of SK-BR-3 cancer cells transfected with si-NC or si- PVT1; C – Western blotting of Bax and Bcl-2 proteins from SK-BR-3 cancer cells transfected with si-NC or si-PVT1. Individual experiments were performed in triplicate

Arch Med Sci 5 Nianqu Zhang, Qing Li, Shanmei Sun ysed by the transwell assay. It was observed that a decline in the concentrations of the vital signal- repression of lncRNA PVT1 not only reduced the ling components of the Wnt/β-catenin signalling migration of cancer cells, but their invading poten- pathway, such as β-catenin, cyclin D1, and c-myc tial was also negatively affected to a significant (Figure 5). Together, the results show that the level (Figure 4). The percentage of cell migration anti-cancer effects of repression of lncRNA PVT1 and invasion declined to 32% and 25%, respec- against breast cancer are exerted through the tively, under PVT1 downregulation, which further blockage of the Wnt/β-catenin signalling pathway highlights the anti-cancer potential of lncRNA PT1 in cancer cells. against breast cancer. Discussion LncRNA PVT1 exerts its effects via Wnt/ The human genome is very complex, and more β-catenin signalling pathway than 90% of human DNA does not code for pro- The western blotting study of SK-BR-3 breast teins [13]. This major portion of human genome cancer cells transfected with si-NC or si-PVT1 was previously referred as “junk DNA” with ap- showed that repression of lncRNA PVT1 led to parently no known function [14]. However, the

si-NC si-PVT1 150

100 Migration

Migration (%) 50 * 50 µm 50 µm

0 si-NC si-PVT1

si-NC si-PVT1 150

100 Invasion

Invasion (%) 50 50 µm 50 µm *

0 si-NC si-PVT1 Figure 4. Downregulation of lncRNA PVT1 restricts the metastasis of breast cancer cells. Transwell assay for analysis of migration and invasion of SK-BR-3 cancer cells transfected with si-NC or si-PVT1. Individual experiments were performed in triplicate, and the values are presented as mean ± SD (*p < 0.05)

si-NC si-PVT1 1.2

1.0 β-catenin 0.8

0.6 Cyclin D1 0.4 * Relative expression Relative * 0.2 * c-myc 0 β-catenin Cyclin D1 c-myc si-NC si-PVT1 Actin

Figure 5. Downregulation of lncRNA PVT1 exerts anti-cancer effects through inhibition of Wnt/β-catenin signalling pathway. Western blotting of β-catenin, cyclin D1, and c-myc proteins from SK-BR-3 cancer cells transfected with si-NC or si-PVT1. Individual experiments were performed in triplicate

6 Arch Med Sci Long non-coding RNA PVT1 regulates the proliferation and metastasis of human breast cancer via the Wnt/b-catenin signalling pathway advancement in the genomic studies has led to progression by controlling the tumour-immune the understanding that the non-protein coding cycle in most of the nodes, including dendritic DNA is actively transcribed into a huge num- cells, T cells, and tumour cells [25]. Specifically, ber of genetically crucial RNA elements [15]. The abnormal Wnt/β-catenin signalling directly alters long non-coding RNAs (lncRNAs) also fall into the the number of regulators critical for the antitu- same category of RNA entities, which, although mor activities of T cells, especially effector T cells, not translated, serve important functions in the T helper cells, and regulatory T cells [26]. At the cellular development and play a role in cellular molecular level, the lncRNA PVT1 was shown to differentiation [16]. It has been noted that ln- target one of the critical signalling pathways, the cRNAs regulate the development of many human Wnt/β-catenin signalling pathway, to exert its reg- diseases and are critical to the advancement of ulatory role in breast cancer. The Wnt/β-catenin human cancer [17]. The dysregulation of lncRNAs signalling pathway is highly overexpressed in can- has been implicated to govern the advancement cer cells and enables them to proliferate at greatly of a number of human cancers including breast elevated levels through over-expression of crucial cancer [18, 19]. Thus, lncRNAs have been valued regulators like cyclin D1 and c-myc [27]. The di- to serve as prognostic biomarkers against human rect activation of c-myc by PVT1 has already been cancers [8]. In the present research work, the confirmed in previous reports [28]. Summing up, role of lncRNA plasmacytoma variant transloca- the results of this study indicate that the lncRNA tion 1 (PVT1) was ascertained in regulating the PVT1 regulates the growth, proliferation, and me- growth and progression of human breast cancer. tastasis of breast cancer in humans through the The PVT1 gene has been reported to play a role Wnt/β-catenin signalling pathway and thus en- in physiological as well as pathological process- lightens the possibility of molecular targeting of es [20]. The cancer-regulatory role of PVT1 is also PVT1 to more effectively restrict the advancement well established [21]. However, the regulatory of human breast cancer in the future. effects of PVT1 have not been elucidated in hu- In conclusion, the present study reveals the man breast cancer. Human breast cancer is the molecular therapeutic anticancer potential of most dominant female cancerous malignancy in lncRNA PVT1 against human breast cancer. The terms of both its incidence as well as the level intracellular targeting of PVT1 might prove effec- of mortality associated with this disorder [1, 4]. tive in better restricting the advancement of the The results of the present study showed that PVT1 breast cancer, which will invoke many future re- is specifically upregulated in breast cancer, which search studies. Future studies should be directed suggests its possible influence on the onset and to investigate the prognostic value of lncRNA PVT1 proliferation of this malignancy because it has and its effects on stemness markers. Additionally, been shown to be upregulated in other cancer in vivo studies need to be initiated to establish types in previous studies [22]. Surprisingly, the si- PVT as a therapeutic target for the treatment of lencing of PVT1 at the transcriptional level caused breast cancer. a sufficient decline in the proliferation and viability of breast cancer cells. Whether or not this Conflict of interest growth inhibition resulted from the induction of Authors declare that there are no conflicts of cell apoptosis, the assessment of nuclear viabili- interest. ty of cancer cells was made, and it was observed that downregulation of PVT1 activates the Bax/ Bcl2 apoptotic cell signal to induce apoptosis in References breast cancer cells. A similar mechanism of de- 1. Boyle P, Levin B. World cancer report 2008. IARC Press, cline in cancer cell proliferation has been reported International Agency for Research on Cancer 2008. in previous investigations of PVT1 [23]. PVT1 tran- 2. Tao Z, Shi A, Lu C, Song T, Zhang Z, Zhao J. Breast cancer: scriptional repression not only reduced the prolif- epidemiology and etiology. Cell Biochem Biophys 2015; eration of breast cancer cells, but the results indi- 72: 333-8. 3. Winters S, Martin C, Murphy D, Shokar NK. 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