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LINC00483 Is Regulated by IGF2BP1 and Participates in the Progression of Breast Cancer

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LINC00483 Is Regulated by IGF2BP1 and Participates in the Progression of Breast Cancer European Review for Medical and Pharmacological Sciences 2021; 25: 1379-1386 LINC00483 is regulated by IGF2BP1 and participates in the progression of breast cancer Y.-S. QIAO1, J.-H. ZHOU2, B.-H. JIN3, Y.-Q. WU1, B. ZHAO4 1Second Department of Oncology, Chifeng Municipal Hospital, Chifeng, China 2Department of Nutrition, Changhai Hospital, Second Military Medical University, Shanghai, China 3Department of Nephrology, Chifeng Municipal Hospital, Chifeng, China 4Department of Nuclear Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China Abstract. – OBJECTIVE: To explore the role Key Words: of long intergenic non-coding ribonucleic acid Breast cancer, LINC00483, IGF2BP1, Proliferation. 483 (LINC00483) in the development of breast cancer (BC) and its possible mechanism of ac- tion. PATIENTS AND METHODS: LINC00483 ex- pression level in BC tissues and cell lines was Introduction detected via quantitative Reverse Transcrip- tion-Polymerase Chain Reaction (qRT-PCR). The Breast cancer (BC) remains a worldwide pub- association between LINC00483 expression and lic health problem. According to the Global Can- survival rate of BC patients was analyzed us- cer Statistics 2018, there were about 2.1 million ing Kaplan-Meier survival analysis. The bind- new cases (11.6% of total new cases of cancer) and ing relation between LINC00483 and insulin-like growth factor 2 mRNA-binding protein 1 (IG- 627 thousand deaths of BC (6.6% of total deaths F2BP1) was verified via RNA immunoprecip- of cancer) in 2018 around the world. BC is the itation (RIP) and RNA pull-down assays. The most common cancer in females, accounting for expression of IGF2BP1 in BC patients was de- 24.0% of female carcinoma, and also the most im- termined using qRT-PCR. Moreover, the role of portant cause of cancer death, which accounts for LINC00483 on the proliferative ability of BC cells 15.0% of the total female cancer deaths1. With the was detected via cell counting kit-8 (CCK8) and development of BC screening and treatment, the 5-Ethynyl-2’-deoxyuridine (EdU) assays. Wheth- survival time of about 80% of patients with local- er LINC00483 exerts its effects under the regula- 2,3 tion of IGF2BP1 was verified via reversal assay. ized lesions can be prolonged . RESULTS: The results of qRT-PCR showed BC has an extremely complicated pathogene- that LINC00483 had a significantly high expres- sis. It has been proved that BC is the result of the sion in BC tissues and corresponding cell lines, combination of various factors, such as personal and it rose with the increase in tumor stage, lifestyle, environment, and genetic and reproduc- which was higher in patients with metastasis. 4 CCK8/EdU assay revealed that the proliferative tive factors . According to follow-up studies, early ability of MDA-MB-231 and MCF-7 cell lines was screening and diagnosis of invasive BC can signifi- enhanced by overexpression of LINC00483. It cantly improve the 5-year survival rate. In develop- was confirmed by RIP and pull-down assays ing countries, however, the rates of early diagnosis that IGF2BP1 could bind to LINC00483, and and effective treatment are low, and the prognosis the expression of LINC00483 was significantly of cancer patients is generally poor. Therefore, it is promoted after up-regulation of IGF2BP1. It was of great importance to actively search for effective found via qRT-PCR that the expression of IG- F2BP1 evidently rose in BC patients, which was biomarkers and apply them to early diagnosis, mo- positively related with the expression level of lecular typing and individualized treatment of BC LINC00483. The results of reversal assay man- for the precise prevention and treatment. ifested that the function of LINC00483 on cell Non-coding ribonucleic acids (ncRNAs), a new proliferation was regulated by IGF2BP1. class of transcripts, are encoded by genomes, but CONCLUSIONS: LINC00483 has a significant- most of them cannot be translated into protein. In ly higher expression in BC tissues than that in spite of this, ncRNAs are important participants para-carcinoma tissues, and its effect of pro- 5 moting proliferation of BC cells may be regulat- in various physiological functions . Particularly, ed by IGF2BP1. long ncRNAs (lncRNAs), with more than 200 nt in Corresponding Author: Bo Zhao, MM; e-mail: [email protected] 1379 Y.-S. Qiao, J.-H. Zhou, B.-H. Jin, Y.-Q. Wu, B. Zhao length, play a key role in regulating chromatin dy- lection (ATCC; Manassas, VA, USA). They were namics, gene expression, growth, differentiation and maintained with Dulbecco’s Modified Eagle’s development6. It has been fully recognized that more Medium (DMEM; Invitrogen, Carlsbad, CA, than 80% of the human genome possesses biological USA) containing 10% fetal bovine serum (FBS; functions and encodes lots of ncRNAs7. Thousands Invitrogen, Carlsbad, CA, USA), 100 U/mL pen- of lncRNAs have abnormal expressions or muta- icillin and 100 μg/mL streptomycin (Invitrogen, tions and play a crucial role in the occurrence and Carlsbad, CA, USA) with 5% CO2 at 37°C. development of various cancers8. Some researches have shown that they are related to the occurrence Cell Transfection and development of BC9. It is reported that ITGA7 is The 3’-UTR sequence of insulin-like growth down-regulated in BC tissues compared with that in factor 2 mRNA-binding protein 1 (IGF2BP1) or a normal ones. In vitro experiments indicated that the mutant sequence with the predicted target sites was migration and invasion of BC MDA-MB-231 and inserted into the KpnI and SacI sites of pGL3 pro- BT-549 cell lines can be significantly inhibited by moter vector. LINC00483, IGF2BP1 overexpression ITGA7 knockdown10. Moreover, the expression of plasmids, IGF2BP1 small-interfering RNA and long intergenic non-coding RNA 52 (LINC00052) negative control sequences were synthesized by is positively correlated with the HER3/ErbB3 level Shanghai GenePharma Co., Ltd. (Shanghai, China) in BC cells. In vitro and in vivo assays have suggest- based on the sequence issued by National Center for ed that the overexpression of LINC00052 promotes Biotechnology Information.. The cells were planted growth of BC cells and enhances HER3-mediated onto 6-well plates and were transfected with 100 ng downstream signal transduction11. of LINC00483 or IGF2BP1 overexpression plas- LncRNAs are likely to play a crucial role in mids, and IGF2BP1 small-interfering RNA (50nM) BC. In the present study, it was newly found that by using Lipofectamine 3000 (Invitrogen, Carlsbad, LINC00483 had a significantly high expression in CA, USA). The differences in transfection efficien- BC tissues and cell lines, and it could greatly pro- cy were normalized by co-transfecting a Renilla lu- mote the proliferation of BC cells. This study aims ciferase vector pRLSV40 (5 ng). to explore the role of LINC00483 in the development of BC and further explore its regulatory mechanism, Cell Counting Kit-8 (CCK8) Assay which was correlated with insulin-like growth fac- CCK8 assay was performed using CCK8 pro- tor 2 mRNA-binding protein 1 (IGF2BP1). liferation assay kits (KeyGEN Biotech, Nanjing, China). After transfection, the cells were cultured in an incubator for 24 h. After the medium in the Patients and Methods wells was discarded, the cells were washed once with phosphate-buffered saline (PBS), digested Clinical Samples with trypsin and neutralized with complete me- A total of 32 cases of BC tissues and corre- dium. After centrifugation at 1000 rpm for 3 min, sponding para-carcinoma tissues were surgically the cell precipitate was collected, resuspended with resected from BC patients in Chifeng Municipal complete medium and counted. Then, they were in- Hospital from May 2017 to June 2019. None of oculated into a 96-well plate (1000-4000 cells/well), the patients underwent radiotherapy and chemo- with 3 replicates in each group. At 1, 2 and 3 d, the therapy before operation. The fresh tissue sam- original medium was replaced with 100 μL of fresh ples collected were immediately stored in liquid complete medium in each well, and 10 μL of CCK8 nitrogen for a long time. The pathological classifi- reagent was added into each well, followed by cul- cation was made and graded by two independent ture in the incubator for 4 h. At last, the absorbance experienced pathologists according to the World was measured at 450 nm using a microplate reader, Health Organization Classification. Sample col- and the tumor cell proliferation curves were plotted. lection was reviewed and approved by the Ethics Committee of Chifeng Municipal Hospital, and 5-Ethynyl-2’-Deoxyuridine (EdU) Assay all patients signed the informed consent. The EdU incorporation assay was applied by using the Cell-LightTM EdU imaging detecting Cell Culture kit following the manufacturer’s instructions (Ri- BC cell lines (MDA-MB-231, BT-549 and boBio). Cells in the logarithmic growth phase were MCF-7) and normal breast cells (MCF-10A) were seeded into a 96-well plate (4×104 cells/well) and purchased from the American Type Culture Col- cultured until normal growth. EdU solution was 1380 LINC00483 promotes occurrence and development of breast cancer diluted into the culture medium at 1000:1 (reagent The primer sequences are as follows: LINC00483 A), and prepared into an appropriate amount of 50 (F: 5’-GCTGAACCGGAACAGGACAT-3’, R: μM EdU medium. According to the instructions 5’-CCAGTTCACAGCAACTCAC-3’). Glyceral- of kits (RiboBio, Guangzhou, China), the cells dehyde 3-phosphate dehydrogenase (GAPDH) (F: were fixed and stained with Apollo and DNA dye. 5’-GAAGAGAGAGACCCTCACGCTG-3’, R: Eventually, representative photograph were taken 5’-ACTGTGAGGAGGGGAGATTCAGT-3’). IG- by Zeiss Axiophot Photomicroscope (Carl Zeiss, F2BP1 (F: 5’-CAGGAGATGGTGCAGGTGTTT Oberkochen, Germany). ATC C-3’, R: 5’-GTTTGCCATAGATTCTTCCCT- GAG C-3’). RNA Immunoprecipitation (RIP) Whether LINC00483 can interact with or bind Statistical Analysis to IGF2BP1 was detected via RIP. According to the Statistical Product and Service Solutions protocol of EZ-Magna RIP kits (Millipore, Billerica, (SPSS) 17.0 software package (SPSS Inc., Chica- MA, USA), the cells were lysed with complete RIP go, IL, USA) was used for statistical analysis.
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