Introduction solid tumor types, including breast cancer3,5. lncRNA

Topic: HealthAutism
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Last updated: September 7, 2019

IntroductionLong non coding RNAs ( lncRNAs) (<200 nt)  are heterogeneous group of transcripts that have crucial role  in cell biological processes including development, homeostasis, stem cell pluripotency, cell growth and apoptosis via modulating chromatin remodeling, transcription, post-transcriptional modifications and signal transduction.1-3 Recent findings have identified that lncRNAs are frequently deregulated in various diseases like cancer4. in addition, several lncRNAs have been recognized as a transcriptional regulation that associated with oncogenes and tumor suppressor in the variety of cancer cells1,2.Cancer stem cells (CSCs) are the small minority of tumorigenic cells that exhibit stem cell properties like self-renewal, pluripotency and high invasion and migration capacity.

CSCs have been distinguished and isolated from many solid tumor types, including breast cancer3,5. lncRNA and transcription factors like Oct4, Nanog and Sox2 are involved in fine-tune transcriptional regulation in keeping pluripotency state in ES cells. The expression level of Oct4, Nanog, and Sox2 transcription factors acting in embryonic stem cells (ESCs) have a strong correlation with CSCs5,6 LncRNA ES1 ( Linc01108) is one of the pluripotency lncRNAs that express in undifferentiated human embryonic stem cells and involves in pluripotency maintenance. ES1 as a member of pluripotency transcriptional network has neighboring Oct4, Nanog binding site.

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furthermore, ES1 was physically associated with SUZ12 and SOX2 and some nuclear factors to perform its regulation in stem cells7.Breast cancer prevalence and variation incidence through the world, indicate early detection is vital8. Therefore, with recognizing the genes and pathways underlying cancer cell would design better therapeutic and diagnostic the current study, we evaluated the role of ES1 in breast cancer. We found that ES1 overexpression in breast cancer tissues and in breast cancer cell lines is associated with histologic grade, p53 mutation, and ductal carcinoma. Further function experiments revealed that knockdown of lncRNA ES1 results in diminished cell proliferation, invasion, and increased apoptosis. Moreover, ES1 knockdown deregulated mRNA levels of EMT and cell cycle proteins in breast cancer cells. ES1 knockdown promotes some miRNAs expression in differentiation and EMT pathways.

Thus, our data clarify a novel role of ES1 as oncogene in breast tumorigenesis. Material and methodClinical specimens and data collectionA total of 45 paired tumor and adjacent non-tumor breast tissues were obtained from the Iran National Tumor Bank, Tehran, Iran between 2012 and 2015. All tissues had been histologically confirmed to be breast cancer.

clinicopathological characteristics of breast cancer patients were performed by the Iran National Tumor Bank that is shown in Table 2. All tissues were frozen in liquid nitrogen and stored at ?80°C. All human tissue samples were obtained with written informed consent from all subjects, and this project was approved by the Kerman Graduate University of Technology. Cell culturebreast cancer cell lines MCF7, MDA-MB-231, Skbr3; colorectal cancer cell lines HT-29, SW48, SW480, SW1116; prostate cancer cell lines LNCaP, PC3 ;  neural cancer cell lines U-87 mg, A172, SH-SY5Y; kidney cancer cell line HEK-293, cervical cancer cell line HELA;  gastric cancer cell line AGS; lung cancer cell line A549; hepatoma cancer cell line HepG2; Dental Pulp Stem Cell  line DPSC and  Pluripotent Embryonic Carcinoma NCCIT  were preserved in the Institute.

All cell lines except MDA-MB-231 were cultured in RPMI 1640 medium (Gibco, USA).MDA-MB-231 (Human Breast Cancer) were cultured in Dulbecco’s modified Eagle medium (DMEM). all media were supplemented with 10% FBS and antibiotics (penicillin/streptomycin 100 U/ml penicillin and 100 mg/ml streptomycin) and 10% fetal bovine serum at 37º C in a humidi?ed atmosphere of 5% CO2 incubator.

Passaging was routinely performed with 0.25% Trypsin/EDTA.RNA isolation, cDNA synthesis, and quantitative real-time PCR Total RNA was extracted from tissues or cultured cells using TRIzol reagent (Invitrogen, USA). Total RNA (1 ?g) was reverse transcribed to cDNA in a final volume of 20 ?l using random primers under standard conditions with using 1µg RNA, 200 U/µl MMLV reverse transcriptase (Fermentas, Lithuania). For each sample, a no-RT control was used in parallel to detect any potential contamination with genomic DNA. Specific primers were designed for ES1 variants and ?-actin (as an internal control) mRNAs using Gene Runner softwareWe performed real-time PCR analyses using SYBR Premix Ex TaqTM II (Takara, Japan) according to the manufacturer’s instructions on Rotor-Gene 6000 instrument (Corbett Life Science, Sydney, Australia).

Results were normalized to the expression of ?-actin, and data were collected based on the comparative cycle threshold (CT) (2???CT) method. Specific primer sequences are listed in table1. Differentiation of NCCIT cell line by trans-RA (retinoic acid) TreatmentThe differentiation process was done according to the previous study with brief modification. NCCIT cell line was cultured in DMEM/F12 medium (Invitrogen, USA) containing 10% fetal bovine serum (Invitrogen, USA) and 100 U/mL penicillin and streptomycin (Invitrogen, USA) at 37°C in a humidified atmosphere with 5% CO2. The differentiation was induced by adding 10 ?M trans-RA (Sigma-Aldrich, St. Louis, MO) every two days for up to 14 days. The expression level of some genes involved in stem cell determination including Oct-4, Nanog and Nucleostemin was assessed before and after RA treatment.

 RNA interferenceMDA-MB-231 and Skbr3 cell lines were seeded in twelve -well plates, then 24 h later they (40-60% confluency) were transfected with specific siRNAs using Lipofectamine 2000(Invitrogen, USA) according to the manufacturer’s instructions. We purchased ES1 siRNAs (si-LINC01108) and scrambled negative control siRNA (si-NC) from Dharmacon. Cells were harvested for qRT-PCR or flow cytometry analysis 48 h after transfection. Senescence-associated ?-galactosidase stainingSenescence ?-galactosidase activity at pH 6.0, the standard biomarker of cellular senescence  cells were fixed with 4% paraformaldehyde  and 0.2 % glutaraldehyde in PBS for 10 min, washed with PBS, and stained with X-gal staining solution (1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2 at pH 6.0) at 37C for 12-16 h.

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