| nnual incidence rate is about 3-8 people per 100,000 population.Currently,the most commonly used glioma is the grading system developed by the World Health Organization(WHO).According to this grading system,gliomas are classified into grade 1(the lowest degree of malignancy,the best prognosis)to grade 4(the highest degree of malignancy and the worst prognosis).Because of its high invasiveness,difficult surgical resection,and most patients are not sensitive to radiotherapy and chemotherapy,the prognosis is not satisfactory.Gliomas are due to the fact that,like other tumors,the abnormal molecular mechanisms involved in the development of tumors,for this reason,to grasp the changes in the molecular mechanisms involved in the development of glioma,will be the treatment of glioma providing new directions and thus improving patient survival and prognosis.Human genomic sequence data indicates that most transcripts are non-coding ribonucleic acids(RNAs)and only 2% of transcripts encode proteins.At present,research on microRNAs(miRNAs)has been extensive,and miRNAs have been confirmed to have certain regulatory effects in the development and progression of many cancers.Recent studies have also shown that long noncoding RNAs(lncRNAs)play an important role in normal development and in many diseases,including many cancers.Multiple lncRNAs have been shown to be highly expressed in glioma tissues and cell lines and are associated with poor prognosis in glioma patients.The competitive endogenous RNA(ceRNA)mechanism is one of the known regulatory mechanisms of lncRNA.In glioma,some lncRNAs have been shown to regulate miRNA expression through ceRNA mechanism,which in turn affects glioma growth and Changes in possible signal pathways associated with invasion.Polo-like kinase 1(PLK1)is a serine/threonine protein kinase that is a key regulator of mitosis in cell cycle-related processes such as mitosis entry,centrosome maturation,mitotic spindle assembly,sisters Chromatid cohesion and cytokinesis play a key role.PLK1 is involved in the regulation of cell cycle and growth of various tumors,including breast cancer,renal cell carcinoma and prostate cancer.Based on the results of the microarray,we found that lncRNA ENST00000413528 is overexpressed in glioma tissues and has a complementary pairing region with miR-593-5p,while miR-593-5p also shares the same complementary pairing with the 3'UTR region of PLK1 mRNA.region.In combination with the ceRNA regulation mechanism of lncRNA,we hypothesized that lncRNA ENST00000413528 may regulate PLK1 through miR-593-5p to regulate glioma cells.To this end,we further verified the expression of lncRNA ENST00000413528 in gliomas,and observed the effects of lncRNA ENST00000413528 on the proliferation and apoptosis of glioma cells at the cellular level,and initially explored its role in regulation.Molecular mechanism.Methods1.This subject collected the cancer tissues and adjacent normal tissue specimens of 19 glioma patients who underwent neurosurgery from the First Affiliated Hospital of Zhengzhou University from January 2017 to April 2018,including 3 cancer tissues and The lncRNA gene chip was detected in the adjacent tissues,and the remaining 16 samples were extracted and purified by TRIzol method in human glioma and adjacent normal tissue samples.Fluorescence real-time quantitative PCR was used to detect lncRNA ENST00000413528 and miR-in the specimens.The relative expression of 593-5p.2.The down-regulated siRNA and si-NC,miR-593-5p mimic and negative control(miR-NC)of lncRNA ENST00000413528 used in this experiment were designed and synthesized,and siRNA(si-)was applied according to the experimental design.NC,si-Lnc)and miR-593 mimics or miR-NC were transfected into glioma LN229 cells.3.The proliferation of LN229 cells was down-regulated by the expression of lncRNA ENST00000413528 by CCK-8 assay.4.The apoptosis of LN229 cells was down-regulated by the expression of lncRNA ENST00000413528 by Annexin V-FITC/PI apoptosis assay kit.5.Bioinformatics analysis of the presence of complementary pairing regions between lncRNA ENST00000413528 and miR-593-5p,construction of wild-type and mutant recombinant reporter vectors of lncRNA ENST00000413528,which were co-transfected with miR-593 mimics or miR-NC into glia In the tumor cell LN229,it was confirmed by Western blot and dual luciferase reporter experiments that lncRNA ENST00000413528 negatively regulates the expression of miR-593-5p.6.Further combined with bioinformatics analysis,the dual luciferase reporter assay was used to verify the targeted regulation of miR-593 and PLK 1.7.The effects of down-regulation of lncRNA ENST00000413528 and upregulation of miR-593-5p on glioma cells were examined using CCK-8 and Annexin V-FITC/PI apoptosis assay kits.8.Statistical analysis was performed by SPSS 21.0 software,P < 0.05 was statistically significant.Results1.The results of LncRNA microarray analysis showed that microarrays of 3 human glioma tissues and adjacent normal tissues showed 22 differentially expressed lncRNAs,including 13 down-regulated and 9 up-regulated.Among them,the expression level of lncRNA ENST00000413528 was significantly higher than that of adjacent tissues(P<0.05).2.The results of the CCK-8 experiment showed that compared with the NC group and the Blank group,the expression of the down-regulated lncRNA ENST 00000413528 decreased at OD490 on days 2,3,4 and 5,and the difference was statistically significant(P < 0.05).3.Flow cytometry(Annexin V-FITC/PI)showed that compared with NC group and Blank group,the number of apoptosis of glioma cells expressing lncRNA ENST 00000413528 was significantly higher than that of NC group and Blank group.The difference was statistically significant(P < 0.05).4.The dual luciferase reporter assay demonstrated that lncRNA ENST0000041 3528 has a targeted regulatory effect on miR-593-5p.5.The miR-593-5p was negatively regulated by targeting to the 3'UTR end of PLK1 by a dual luciferase reporter assay.6.It was demonstrated by CCK-8 and flow cytometry(Annexin V-FITC/PI)that down-regulation of ENST00000413528 and up-regulation of miR-593-5p have consistent proliferation inhibition and apoptosis-promoting effects on glioma cells.ConclusionLncRNA ENST00000413528 is highly expressed in glioma cells and may regulate the proliferation and apoptosis of glioma cells through the miR-593-5p/PLK1 pathway. |
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