Study On The Dryness Characteristics And Carbohydrate Metabolism Of Human Breast Cancer Stem Cells Regulated By LIN28B

Objective:Cancer stem cells (CSCs) are small subpopulation of cells within tumors which exhibit stem cell properties of self-renewal and differentiation. CSCs are supposed to account for resistance to both radiation and chemotherapy, tumor relapse and metastasis. It was been proved that CD44high cells of lung cancer displayed high tumorigenesis. As few as 300 cells with this phenotype were able to form tumors, whereas 3×104 cells with alternate phenotype or 5×105 mixed cells failed to form tumors in mouse xenograft transplantation. LIN28 initially discovered in C. elegans is highly conserved RNA binding protein and has been found to play important roles in cell growth and tissue development. LIN28 has two isoforms in humans known as LIN28A and LIN28B. LIN28 overexpressed in many types of cancer has been linked to tumor initiation and metastasis. microRNAs are 21-25 nucleotide (nt) long, non-coding RNAs that regulating target genes’post-transcription by directly binding their 3 -untranslated region.Although LIN28 has been characterized to repress the expression of let-7 by binding to the precursors and blocking their maturation, which target genes regulating cell metabolism and sternness, LIN28B also regulates other miRNAs including miR-150. In this study, we focus on the regulation of LIN28B on breast cancer stem cells and miRNAs.Methods:Firstly we tested the expression of stemness related genes including SOX2, OCT4, NANOG and LIN28B in aggressive trip-negative breast cancer cell MDA-MB-231 and low metastatic ability breast cancer cell MCF-7 in both mRNA and protein level by qPCR or western blot. Then LIN28B was instantaneously overexpressed in MCF-7 cells by transfecting p-CMV6-LIN28B plasmid. miRNAs with different expression were discovered by RNA sequencing in MCF-7-LIN28B cells. Furthermore, we established MCF-7 cell line constantly overexpressing LIN28 by virus transfection (MCF-7-LIN28B) and then tested the expression of sternness related genes in both mRNA and protein level, the proportion of CD44high CD24low by flow cytometry and the ability of tumor forming in vitro. Finally we detected the proportion of CD44high CD24low and the ability of tumor forming in vitro in MCF-7 cell line after transfecting miRNA mimic which expressed significantly diverse.Results:1. The expression of LIN28 was dramatically increasing in MDA-MB-231 cells.2. The expression SOX2, OCT4 and NANOG were upregulated significantly in MCF-7-LIN28B cells.3. The proportion of CD44high CD24low and the ability of tumor forming were promoted in MCF-7-LIN28B cells.4. The expression of miR-92b-5p was upregulated and the expression of miR-4521, miR-149-3p and miR-92a-1-5p were down regulated in MCF-7-LIN28B cells.5. The proportion of CD44high CD24low increased and the ability of tumor forming was enhanced in MCF-7 after transfecting miR-92b-5p mimic.Conclusion:It is suggested that LIN28B maintain the sternness of cancer stem cells via regulating miR-92b-5p in MCF-7 breast cancer cell line.Objective: Glucose is tiie most important energy resource for cells. Normal differentiated cells convert glucose to C〇2 and H2 O via tricarboxylic acid cycle in the condition of abundant oxygen supply and metabolize glucose by glycolysis only in hypoxia. In contrast to normal differentiated cells, cancer cells mostly rely on glycolysis to generate the energy essential fbr cellular processes even in presence of oxygen,which is termed aerobic glycolysis,a phenomen on known as "Warburg effect,,. Hanahan and Weinberg concluded reprogramming energy metabolism as one of ten hallmarks of cancer in 2012. The unique metabolism of cancer has been generally emphasized and its role in tumor initiation and development has also become the focus. Although it is said that CSCs would change metabolic pathway in order a adjust to the microenvirenment,the metabolic state of glucose in CSCs is still unclear. It is suggested that metabolic state plays an important role in self-renewal and glycolysis is of critical importance to maintain sternness of CSCs. Vermeersch et al. found ther were numerous metabolic changes between ovarian cancer cells(OCCs) and ovarian cancer stem cells(OCSCs)using mass spectrometry and OCSCs were more dependent on glycolysis. Our research focus on the metabolic state of human breast cancer stem cells and the mechanism link of LIN28 in this cellular process and the relationship between glucose metabolism and sternness of cancer stem cells,which will offer a new perspective of tumor therapy.Methods:Firstly, we enriched cancer stem cell in MDA-MB-231 by tumorsphere culture in vitro and filtewd tumospheres by 70μm cell strainer. A metabolic Oux analyzer was used to determine the oxygen consumption rates of mitochondrial :respiratory and extracellular acidification rates of glycolysis. The protein expression of glycolysis related key genes PKM2, LDHA and HIF-la were detected by western blot. Secondly, LIN28 B gene was knocked out in MDA-MB-231 cells by TALEN technology and glucose uptake was analyzed in both wild type cells and LIN28 B knocked out in 11 s in MDA-MB-231 cells by now cytometry with 2-NBDG Lastly,LDHA activity of tumospheres which diameter was over 70|xm and adherent cells were detected by LDHA activity assay kit.Cell viability was analyzed by cell counting kit in MDA-MB-231 cells and HI299 cells with different concentrations of oxamate,one of LDHA inhibkors. The self-renew ability of MDA-MR-231 was analyzed by tumorsphere fbrming culture in vitro with different oxamate concentrations.Results:1. Glycolytic phenotype is more apparent and oxidative phosphorylatio打 is reduced in breast cancer stem cells apart from glycolysis related genes PKM2,LDHA and HIF-la are upregulated.2.LIN28 B promotes the glucose uptake and THe expression of PKM2,LDHA and HIF-la in MDA-MB-231 cell line.3.LIN28 B increases aerobic glycolysis and inhibits mkochondrial oxidative phosphorylation in MDA-MB-231 cell line.4.LIN28 B directly binds to the mRNA of LDHA and HIF-1a.5.LDHA activity is increased in breast cancer stem cells and the ability of tumorsphere forming in vitro is reduced when LDHA activity is inhibked.Conclusions: Breast cancer stem cells(BCSCs) mainly depend on glycolysis and the sternness of BCSCs is reduced when glycolysis is inhibUed; LIN28 B upregulates the expression of LDHA and HIF-la by binding to their mRNA directly and promotes glycolysis of breast cancer cells.