ABSTRACT
Objective To explore the effect of Linc00460 on the aerobic glycolysis of breast cancer (BC) cells through sponge adsorption of miR-320a. Methods The qRT-PCR method was used to detect Linc00460 and miR-320a expression levels in normal breast epithelial cell line MCF-10A and five BC cell lines. The effect of interfering Linc00460 on miR-320a expression was detected by qRT-PCR. The double luciferase reporter gene experiment was used to analyze the targeting relationship between miR-320a and Linc00460. In addition, the si-Linc00460 and miR-320a inhibitor were co-transfected into MDA-MB-231 cells, and the expression level of miR-320a in the cells was detected by qRT-PCR; cell proliferation ability was measured by the MTT method; glucose uptake rate was detected by 2-NBDG method; the content of lactic acid in the cell supernatant was detected by colorimetric method; the key enzymes of glycolysis was detected by the enzyme activity kit; and the expression levels of the key proteins in the glycolysis pathway were detected by Western blot. Results Linc00460 was highly expressed in five BC cell lines, while miR-320a was lowly expressed as compared with MCF-10A cells. The expression of miR-320a in MDA-MB-231 cells significantly increased after interfering with Linc00460. The double luciferase reporter gene experiment confirmed that miR-320a and Linc00460 could target binding. Interfering with the expression of Linc00460 could inhibit MDA-MB-231 cells proliferation (all P=0.000), reduce the rate of cellular glucose uptake and the content of lactic acid in the cell supernatant (all P=0.000), inhibit the activities of PFK, PK, and LDH enzymes (all P=0.000), and downregulate the protein expression levels of PFKM, GLUT1, and LDHA (all P=0.000). Meanwhile, inhibition of miR-320a could reverse the inhibitory effect of si-Linc00460 on the proliferation and glycolysis of MDA-MB-231 cells (all P=0.000 or 0.001). Conclusion Linc00460 might adsorb miR-320a, consequently leading to upregulation of PFKM expression, thereby promoting aerobic glycolysis in BC cells.
ABSTRACT
Objective:To explore the effect of aquaporin 4 (AQP4) regulated by miR-320a on a cell model of Alzheimer′s disease.Methods:A rat adrenal pheochromocytoma cell line (PC12) was induced into neurons using nerve growth factor (NGF). The morphology of the PC12 cells and the neurons was observed, and ubiquitin carboxy terminal hydrolase L1 (Uch-L1) and neurofilament protein (NFP) were detected. Levels of microtubule-associated protein (MAP2) and AQP4 target genes were related to the mRNA expression of NFP to determine the neuron-inducing effect. The neurons were then randomly divided into a control group (given no treatment), an miR-320a mimic transfection group (cultured by adding 50nmol/L miR-320a as a mimic agent), an miR-320a inhibitor group (cultured by adding 50nmol/L miR-320a as an inhibitor), an Aβ treatment group (cultured by adding Aβ), an Aβ+ miR-320a mimic group (cultured by adding both 50nmol/L miR-320a and Aβ), and an Aβ+ miR-320a inhibitor group (also cultured by adding Aβ, but with 50nmol/L miR-320a as an inhibitor). Cell activity was measured by the CCK8 method. Reverse-transcription polymerase chain reactions were used to detect the relative expression of the target gene miR-320a, AQP4, B-cell bcl2-associated X (Bax), and B-cell bcl-2 (Bcl-2) mRNA. Western blotting was employed to detect the relative expression of AQP4, Bax and Bcl-2 proteins.Results:After PC12 was induced by 50μg/L NGF, the expression of Uch-L1 genes in the induced neuron was significantly down-regulated compared with the PC12. The expressions of NFP, MAP2 and AQP4 genes were significantly up-regulated, and the relative expressions of MAP2 and AQP4 proteins increased significantly. Compared with the control group, the apoptosis and cell activity of neurons in the treatment group increased, the mRNA and protein expressions of miR-320a, AQP4, bcl-2, AQP4 and Bcl-2 decreased significantly, and the mRNA and protein expressions of Bax increased significantly. Compared with the Aβ-treated group, the cell activity of the Aβ+ Mir-320a mimic group increased significantly, the mRNA and protein expressions of miR-320a, AQP4 and Bcl-2 increased significantly, and the mRNA and protein expressions of Bax decreased significantly. Compared with the Aβ+ miR-320a mimic group, the cell activity of the Aβ+ miR-320a inhibitor group decreased significantly, the mRNA and protein expressions of miR-320a, AQP4 and Bcl-2 decreased significantly, and the mRNA and protein expressions of Bax increased significantly.Conclusion:miR-320a can up-regulate AQP4 expression in a cell model of Alzheimer′s disease, reduce apoptosis and increase the cell survival rate.
ABSTRACT
Objective The aim of this study was to investigate the expression of miR-320a and cephalospermoma syndrome protein(CYLD)in patients with gastric cancer and its relationship with clinicopathological characteristics and prognosis. Methods A total of 460 patients with gastric cancer underwent tumor resection in our hospital from March 2013 to November 2014 were enrolled. Tumor tissues,non-tumor gastric mucosa tissues and normal tissues were collected. The expression of miR-320a and CYLD at levels of mRNA and protein were detected by Real-Time PCR,immunohistochemistry and Western blotting. The relationship between the expression of miR-320a and CYLD,and the clinicopathological features&prognosis of gastric cancer patients was analyzed. Results The relative expression of miR-320a and CYLD at the mRNA level in tumor tissues was(0. 37 ± 0. 09),(0. 91 ± 0. 23),and the relative expression in non-tumor tissues was(0. 86 ± 0. 15),(1. 56 ± 0. 42),respectively. The relative expression of miR-320a and CYLD mRNA in tumor tissues was significantly different from non-tumor tissues(t=60. 078,29. 113,P=0. 000),the positive ex-pression rate of CYLD protein in tumor tissues was 43. 48% when compared to 73. 91% in non-tumor tissues. The difference was statistically significant(χ2=86. 624,P=0. 003). The expression level of miR-320a was significantly associated with the diameter of the tumor and lymph node metastasis(χ2=25. 859 and 13. 742,P<0. 05). The expression of CYLD was also significantly associated with the TNM stage and degree of tumor differentiation(χ2=37. 725 and 59. 323,P<0. 05). The median survival of patients with low miR-320a expression(20. 36 months,95% CI:19. 252 ~21. 462 months) and those with high miR -320a expression(28. 29 months,95% CI:27.158~29.412 months)were statistically significant(χ2=87.967,P<0.001).The median survival of patients with CYLD negative expression(17. 70 months,95% CI:16. 599~18. 796 months)and those with CYLD positive expression(26. 74 months,95% CI:25. 474~27. 997 months)were statistically significant(χ2=109. 887,P<0. 001);The median survival of patients with the co-expressed miR-320a and CYLD was(29. 01 months,95% CI:26. 831~28. 946 months)and those with the non-co-expressed miR-320a and CYLD(17. 13 months,95% CI:17. 214~19. 568 months)were statistically significant(χ2=117. 680,P<0. 001). There showed a positive correlation between the expression of miR-320a and CYLD at mRNA level(r=0. 607,P<0. 001);miR-320a at the low expression,CYLD at the negative expression,TNM staging,lymph node metastasis and degree of tumor differen-tiation were independent risk factors for the prognosis of gastric cancer(HR=1. 939,2. 180,1. 561,1. 719,1. 608,95% CI:1. 141~3.295,1.252~3.796,1.014~2.403,1.115~2.650,1.097~2.357,respectively)(P<0.05).Conclusion The expressions of miR-320a and CYLD in tumor tissues of patients with gastric cancer is significantly decreased,which is related to the occurrence and development of diseases,and poor prognosis. It is a potential target for diagnosis and treatment of gastric cancer.