DOI: https://doi.org/10.21203/rs.3.rs-15500/v1
Objectives Breast cancer (BC) is one of the most ordinary fatal cancers. Recent studies have identified the vital role of long noncoding RNAs (lncRNAs) in the development and progression of BC. In this research, lncRNA TTN-AS1 was studied to identify how it functioned in the metastasis of BC.
Methods TTN-AS1 expression of tissues was detected by RT-qPCR in 56 BC patients. Wound healing assay and transwell assay were used to observe the biological behavior changes of BC cells through gain or loss of TTN-AS1. In addition, luciferase assays and RNA immunoprecipitation (RIP) assay were performed to discover the potential targets of TTN-AS1 in BC cells.
Results TTN-AS1 expression level in BC samples was higher than that of adjacent ones. Besides, cell migrated ability and cell invaded ability of BC cells were inhibited after TTN-AS1 was silenced. Cell migrated ability and cell invaded ability of BC cells were promoted after TTN-AS1 was overexpressed. In addition, miR-140-5p was upregulated after silence of TTN-AS1 in BC cells, while miR-140-5p was downregulated after overexpression of TTN-AS1 in BC cells. Furthermore, luciferase assays and RNA immunoprecipitation assay (RIP) showed that miR-140-5p was a direct target of TTN-AS1 in BC.
Conclusion Our study uncovers a new oncogene in BC and suggests that TTN-AS1 could enhance BC cell migration and invasion via sponging miR-140-5p, which provides a novel therapeutic target for BC patients.
Breast cancer(BC) is the most frequently malignancy diagnosed and the second-leading cause of cancer-related death in female in the world[1]. It is reported that 246,660 new cases were diagnosed of BC which accounts for 29% of all cancers in women in America in 2016. Moreover, 40,450 cases were estimated to die due to BC in the same year[2]. Despite tremendous advances have been made in the diagnosis and therapeutic management of BC for the last decades, the prognosis for patients with BC remains poor due to the high rate of metastasis[3]. Therefore, it is urgent to have a better understanding of molecular mechanism of pathogenesis in BC and improve the poor prognosis for BC patients.
Most of the genome is transcribed into noncoding RNA (ncRNA) molecules that do not coding proteins. Long noncoding RNAs (lncRNAs) are those transcriptions longer than 200 nucleotides and have been recently reported to exploit multiple modes of action in regulating gene expression and development of cancers. For example, by sponging miR-27b-3p, lncRNA KCNQ1OT1 facilitates cell proliferation and cell invasion in the progression of non-small cell lung cancer via modulating the expression of HSP90AA1[4]. By acting as a sponge to miR-101-3p, lncRNA SPRY4-IT1 promotes the progression of bladder cancer via upregulating the expression of EZH2[5]. LncRNA PVT1 promotes glucose metabolism, cell motility, cell proliferation and tumor progression in osteosarcoma by modulation of miR-497/HK2 axis[6]. LncRNA MEG8 enhances epigenetic induction of the epithelial-mesenchymal transition in pancreatic cancer cells[7].
However, the clinical role and underlying mechanisms of TTN-AS1 in the development of BC remain unexplored. In the present study, we performed function and mechanism assays to explore whether TTN-AS1 functioned in the metastasis of BC.
Patients and clinical samples
56 cases of BC tissues and their adjacent tissues were collected from patients who received surgery at First Affiliated Hospital of Anhui Medical University between 2015-2018. Written informed consent was achieved before surgical resection. No radiotherapy or chemotherapy was performed before the surgery. All tissues were saved immediately at –80°C. An Institutional Review Board of First Affiliated Hospital of Anhui Medical University approved the protocols for using those tissues for our research.
Cell culture
Human BC cell (MCF-7, LCC9, T-47D, SKBR3) and normal human breast cell line (MCF-10A) were got from the American Type Culture Collection (ATCC, USA). Culture medium consisted of 10% fetal bovine serum (FBS; Invitrogen Life Technologies, USA), Dulbecco’s Modified Eagle Medium (DMEM) as well as 100 U/mL penicillin/streptomycin (Sigma, St. Louis, MO, USA). Besides, cells were cultured in an incubator containing 5% CO2 at 37˚C.
Cell transfection
Specific short-hairpin RNA (shRNA; BiosettiaInc., San Diego, CA, USA) against TTN-AS1 was synthesized. Negative control shRNA was also synthesized. TTN-AS1 shRNA (sh-TTN-AS1) and negative control (control) were then used for transfection in LCC9 BC cells. 48h later, RT‑qPCR was used to detect transfection efficiency in these cells. Besides, lentivirus (BiosettiaInc., San Diego, CA, USA) against TTN-AS1(TTN-AS1) was synthesized and then used for transfection in SKBR3 BC cells. Empty vector was used as control.48h later, RT‑qPCR was used to detect transfection efficiency in these cells.
RNA extraction and RT-qPCR
Total RNA was extracted from cultured BC cells or patients’ tumor tissues by using TRIzol reagent (Takara Bio, Inc., Shiga, Japan) and then reverse-transcribed to cDNAs through reverse Transcription Kit (TaKaRa, Japan). Thermocycling conditions was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, a total of 35 cycles. Following are the primers using for RT‑qPCR: TTN-AS1, forwards 5′-TCCTTAGGCATCACCTAGCC-3′ and reverse 5′-GATGGAGGAAGTAGAGTCATTGG-3′; β-actin, forward 5'-CCAACCGCGAGAAGATGA-3' and reverse 5'-CCAGAGGCGTACAGGGATAG-3'.
Scratch wound assay
1.0 × 104 cells were seed into a 6-well plate. Three parallel lines were made on the back of each well. After growing to about confluent of 90 %, cells were scratched with a pipette tip and cultured in a medium. Cells were photographed under a light microscope after 48 h. Each assay was independently repeated in triplicate.
Transwell assay
8μm pore size insert was provided by Corning (Corning, New York, USA). 4 ×104 cells in 150µL serum-free DMEM were transformed to top chamber of the insert coated with or without 50 µg Matrigel (BD, Bedford, MA, USA). The bottom chamber was filled with DMEM and FBS. 48h later, the top surface of chambers was immersed for 10 min with precooling methanol and was stained in crystal violet for 30 min.
Luciferase assay and RNA immunoprecipitation (RIP) assay
DIANA LncBASE Predicted v.2 was used to predict the potential target microRNAs and fragment sequences containing TTN-AS1 reaction sites. The TTN-AS1 3′-UTR wild-type (WT) sequence named TTN-AS1-WT was 5′-CUUUUCCAUCCUUAAACCACUU-3′ and the mutant sequence of TTN-AS1 3′-UTR missing the binding site with miR-140-5p named TTN-AS1-MUT was 5′-CUUUUCCAUCCUUUUUGGUGAU-3′. Luciferase reporter gene assay kits (Promega, Madison, WI, U.S.A.) were used to detect the luciferase activity of BC cells. The luciferase reporter gene vector was constructed, and BC cells were transfected. For RIP assay, Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was performed according to the protocol. Then RT- qPCR was used to detect co-precipitated RNAs. Treated BC cells were collected and lysed using RIP lysis buffer containing protease inhibitor and RNase inhibitor. Cells were incubated with the RIP buffer containing magnetic beads coated with Ago2 antibodies (Millipore). IgG acted as a negative control (input group). After incubation for 2 h at 4°C, co-precipitated RNAs were isolated and measured by RT-qPCR analysis.
Statistical analysis
All statistical analyses were performed by GraphPad Prism 5.0. The difference between two groups were compared by independent-sample t test. The statistically significance was defined as p<0.05.
TTN-AS1 expression level in BC tissues and cells
Firstly, TTN-AS1 expression was detected via RT-qPCR in 56 patients’ tissues and 4 BC cell lines. As a result, TTN-AS1 was significantly upregulated in BC tissue samples (Figure 1A). TTN-AS1 expression level in BC cells was higher than that of MCF-10A (Figure 1B).
Silence of TTN-AS1 inhibited cell migration and invasion in LCC9 BC cells
In this study, we chose LCC9 BC cell lines for the silence of TTN-AS1. Then TTN-AS1 expression was detected by RT‑qPCR (Figure 2A). Moreover, results of wound healing assay showed that silence of TTN-AS1 significantly inhibited the ability of cell migration in BC cells (Figure 2B). The outcome of transwell assay also revealed that the number of migrated cells was remarkably decreased after TTN-AS1 was silenced in BC cells (Figure 2C). The number of invaded cells was remarkably decreased after TTN-AS1 was silenced in BC cells (Figure 2D).
Overexpression of TTN-AS1 promoted cell migration and invasion in SKBR3 BC cells
In this study, we chose SKBR3 BC cell lines for the overexpression of TTN-AS1. Then TTN-AS1 expression was detected by RT‑qPCR (Figure3A). Moreover, results of wound healing assay showed that overexpression of TTN-AS1 significantly promoted the ability of cell migration in BC cells (Figure 3B). The outcome of transwell assay also revealed that the number of migrated cells was remarkably increased after TTN-AS1 was overexpressed in BC cells (Figure 3C). The outcome of transwell assay also revealed that the number of invaded cells was remarkably increased after TTN-AS1 was overexpressed in BC cells (Figure 3D).
The interaction between miR-140-5p and TTN-AS1 in BC
DIANA LncBASE Predicted v.2 (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2%2Findex-predicted) was used to find the miRNAs that contained complementary base with TTN-AS1. We selected miR-140-5p as it contained binding area of TTN-AS1(Figure 4A). RT-qPCR results showed that miR-140-5p was upregulated in sh-TTN-AS1 group compared with control group (Figure 4B). Meanwhile, miR-140-5p was downregulated in TTN-AS1 group compared with empty vector group (Figure 4C). Furthermore, results of luciferase assay showed that luciferase activity was significantly reduced through co-transfection of TTN-AS1-WT and miR-140-5p, while no significant changes of luciferase activity were observed through co-transfection of TTN-AS1-MUT and miR-140-5p (Figure 4D). In addition, RIP assay identified that TTN-AS1 and miR-140-5p were significantly enriched in Ago2-containing beads compared to input group (Figure 4E).
LncRNAs can regulate gene expression through multiple mechanisms, mostly depending on subcellular localization and the nature of molecular interactors (DNA, RNA, and proteins). The interaction between lncRNA–miRNA functional networks has drawn much attention recently. By targeting miR-873, lncRNA NRF modulates programmed necrosis and myocardial injury during ischemia and reperfusion[8]. Through negatively regulating miR-200b/a/429, lncRNA ILF3-AS1 enhances cell proliferation, cell migration and invasion in melanoma[9]. By acting as a molecular sponge for miR-200s, depletion of lncRNA ZEB1-AS1 significantly suppresses cell proliferation and call migration in osteosarcoma[10]. LncRNA PCAT-1 facilitates cell invasion and metastasis in hepatocellular carcinoma via miR-129-5p-HMGB1 signaling pathway by directly binding to miR-129-5p[11].
Researches have proved that altered expression of many lncRNAs are associated with the progression of BC closely. For example, downregulation of lncRNA snaR inhibits the proliferation, migration, and invasion of BC cells and may be a potential treatment for triple-negative BC[12]. LncRNA OR3A4 facilitates cell proliferation and cell migration in BC through inducing epithelial-mesenchymal transition[13]. LncRNA linc-ITGB1 functions as an oncogene in BC by inducing cell cycle arrest[14]. LncRNA CAMTA1 enhances cell proliferation and cell mobility in BC by targeting miR-20b[15].
TTN-AS1 is a novel lncRNA which has reported to promote cell proliferation and cell migration in cervical cancer via sponging miR-573[16]. In our study, TTN-AS1 was upregulated in BC tissues. Besides, silence of TTN-AS1 inhibited cell migration and invasion in BC cells, while overexpression of TTN-AS1 promoted cell migration and invasion in BC cells. Above results indicated that TTN-AS1 promoted metastasis of BC and might act as an oncogene.
To further identify the underlying mechanism of how TTN-AS1 affects BC cell migration and invasion, we predicted and picked miR-140-5p as the potential binding microRNAs of TTN-AS1 through bioinformatic analysis and experimental verification. MiR-140-5p is dysregulated in various tumors. In addition, miR-140-5p has been reported to serve as a tumor suppressor in some tumors. For example, miR-140-5p inhibits cell proliferation and cell migration in gastric cancer via regulation of YES1[17]. By targeting fibroblast growth factor 9 and transforming growth factor β receptor 1, miR-140-5p depresses tumor growth and metastasis in hepatocellular carcinoma[18]. MiR-140-5p suppresses tumor growth and cell metastasis in cervical cancer by targeting insulin like growth factor 2 mRNA binding protein 1[19]. Moreover, miR-140-5p has also been reported to inhibit the invasion and angiogenesis of BC by targeting vascular endothelial growth factor-A (VEGFA)[20].
In the present study, miR-140-5p expression could be upregulated after knockdown of TTN-AS1. Meanwhile, miR-140-5p expression could be downregulated after overexpression of TTN-AS1. Moreover, miR-140-5p could directly bind to TTN-AS1 through a luciferase assay. MiR-140-5p was significantly enriched by TTN-AS1 RIP assay. All the results above suggest that TTN-AS1 might promote metastasis of BC via sponging miR-140-5p.
Above data identified that TTN-AS1 was remarkably upregulated in BC patients. Besides, TTN-AS1 could facilitate cell migration and invasion in BC through sponging miR-140-5p. These findings suggest that TTN-AS1 may contribute to therapy for BC as a candidate target.
Consent for publication
Yes
Availability of data and material
Yes if request.
Competing interests
no
Funding
no
Authors' contributions
Yusheng Li made this manuscript, and Fan Wang help revise this work.
Acknowledgments
We thank Department of department of Oncology in the First Affiliated Hospital of Anhui Medical University for their contributions to our study. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
The research has been carried out in accordance with the World Medicial Association Declaration of Helsinki. All subjects provided written informed consent.
Disclosure
All authors declare no conflict of interest.
[1]Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015; 65: 5-29.
[2]Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016; 66: 7-30.
[3]Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11-30.
[4]Dong Z, Yang P, Qiu X, Liang S, Guan B, Yang H, Li F, Sun L, Liu H, Zou G, Zhao K. KCNQ1OT1 facilitates progression of non-small-cell lung carcinoma via modulating miRNA-27b-3p/HSP90AA1 axis. J Cell Physiol 2018.
[5]Liu D, Li Y, Luo G, Xiao X, Tao D, Wu X, Wang M, Huang C, Wang L, Zeng F, Jiang G. LncRNA SPRY4-IT1 sponges miR-101-3p to promote proliferation and metastasis of bladder cancer cells through up-regulating EZH2. Cancer Lett 2017; 388: 281-291.
[6]Song J, Wu X, Liu F, Li M, Sun Y, Wang Y, Wang C, Zhu K, Jia X, Wang B, Ma X. Long non-coding RNA PVT1 promotes glycolysis and tumor progression by regulating miR-497/HK2 axis in osteosarcoma. Biochem Biophys Res Commun 2017; 490: 217-224.
[7]Terashima M, Ishimura A, Wanna-Udom S, Suzuki T. MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells. J Biol Chem 2018; 293: 18016-18030.
[8]Wang K, Liu F, Liu CY, An T, Zhang J, Zhou LY, Wang M, Dong YH, Li N, Gao JN, Zhao YF, Li PF. The long noncoding RNA NRF regulates programmed necrosis and myocardial injury during ischemia and reperfusion by targeting miR-873. Cell Death Differ 2016; 23: 1394-1405.
[9]Chen X, Liu S, Zhao X, Ma X, Gao G, Yu L, Yan D, Dong H, Sun W. Long noncoding RNA ILF3-AS1 promotes cell proliferation, migration, and invasion via negatively regulating miR-200b/a/429 in melanoma. Biosci Rep 2017; 37.
[10]Liu C, Pan C, Cai Y, Wang H. Interplay Between Long Noncoding RNA ZEB1-AS1 and miR-200s Regulates Osteosarcoma Cell Proliferation and Migration. J Cell Biochem 2017; 118: 2250-2260.
[11]Zhang D, Cao J, Zhong Q, Zeng L, Cai C, Lei L, Zhang W, Liu F. Long noncoding RNA PCAT-1 promotes invasion and metastasis via the miR-129-5p-HMGB1 signaling pathway in hepatocellular carcinoma. Biomed Pharmacother 2017; 95: 1187-1193.
[12]Lee J, Jung JH, Chae YS, Park HY, Kim WW, Lee SJ, Jeong JH, Kang SH. Long Noncoding RNA snaR Regulates Proliferation, Migration and Invasion of Triple-negative Breast Cancer Cells. Anticancer Res 2016; 36: 6289-6295.
[13]Liu G, Hu X, Zhou G. Long non-coding RNA OR3A4 promotes proliferation and migration in breast cancer. Biomed Pharmacother 2017; 96: 426-433.
[14]Yan M, Zhang L, Li G, Xiao S, Dai J, Cen X. Long noncoding RNA linc-ITGB1 promotes cell migration and invasion in human breast cancer. Biotechnol Appl Biochem 2017; 64: 5-13.
[15]Lu P, Gu Y, Li L, Wang F, Yang X, Yang Y. Long Noncoding RNA CAMTA1 Promotes Proliferation and Mobility of the Human Breast Cancer Cell Line MDA-MB-231 via Targeting miR-20b. Oncol Res 2018; 26: 625-635.
[16]Chen P, Wang R, Yue Q, Hao M. Long non-coding RNA TTN-AS1 promotes cell growth and metastasis in cervical cancer via miR-573/E2F3. Biochem Biophys Res Commun 2018; 503: 2956-2962.
[17]Fang Z, Yin S, Sun R, Zhang S, Fu M, Wu Y, Zhang T, Khaliq J, Li Y. miR-140-5p suppresses the proliferation, migration and invasion of gastric cancer by regulating YES1. Molecular Cancer 2017; 16: 139.
[18]Hao Y, Feng F, Ruimin C, Lianyue Y. MicroRNA-140-5p suppresses tumor growth and metastasis by targeting transforming growth factor β receptor 1 and fibroblast growth factor 9 in hepatocellular carcinoma. Hepatology 2013; 58: 205-217.
[19]Su Y, Xiong J, Hu J, Wei X, Zhang X, Rao L. MicroRNA-140-5p targets insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) to suppress cervical cancer growth and metastasis. Oncotarget 2016; 7: 68397-68411.
[20]Lu Y, Qin T, Li J, Wang L, Zhang Q, Jiang Z, Mao J. MicroRNA-140-5p inhibits invasion and angiogenesis through targeting VEGF-A in breast cancer. Cancer Gene Ther 2017; 24: 386-392.