Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms.@*METHODS@#The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot.@*RESULTS@#The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway.@*CONCLUSION@#LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

Effect of Carvacrol on the Biological Behavior of Leukemia Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of carvacrol on the biological behavior of leukemia cells and its regulation to circ-0008717/miR-217 molecular axis.@*METHODS@#Human acute lymphoblastic leukemia cells Molt-4 were cultured in vitro, and different concentrations of carvacrol were added to the cells. si-NC and si-circ-0008717 were transfected into Molt-4 cells (si-NC group, si-circ-0008717 group). pcDNA, pcDNA-circ-0008717, anti-miR-NC, anti-miR-217 were transfected into Molt-4 cells and then added to carvacrol-treated cells (carvacrol+pcDNA group, carvacrol+pcDNA-circ-0008717 group, carvacrol+anti-miR-NC group, carvacrol+anti-miR-217 group). MTT, plate clone formation experiment, and flow cytometry were used to detect the viability of the cell, colony formation number, and apoptosis rate of cells, respectively. The RT-qPCR method was used to detect the expression levels of circ-0008717 and miR-217. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ-0008717 and miR-217.@*RESULTS@#After carvacrol treatment, the cell viability decreased significantly (r=-0.9405), expression level of circ-0008717 decreased (r=-0.9117), colonies formed number decreased (r=-0.9256), while the cell apoptosis rate increased (r= 0.8464), and the expression level of miR-217 increased (r=0.9468). Compared with the si-NC group, the expression level of miR-217 in si-circ-0008717 group increased (P＜0.001), the cell apoptosis rate increased (P＜0.001), while cell viability decreased (P＜0001), the number of colonies formed decreased (P＜0.001). Compared with the carvacrol+pcDNA group, the cell viability of the carvacrol+pcDNA-circ-0008717 group increased (P＜0.001), the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0.001). circ-0008717 could target miR-217. The cell viability of the carvacrol+anti-miR-217 group increased (P＜0.001), and the number of colonies formed increased (P＜0.001), while the cell apoptosis rate decreased (P＜0001) as compared with the carvacrol+anti-miR-NC group.@*CONCLUSION@#Carvacrol can promote the expression of miR-217 by down-regulating the expression of circ-0008717, thereby reducing the proliferation and cloning ability of leukemia cells and promoting cell apoptosis.

Long non-coding RNA LINC01268 promotes cell growth and inhibits cell apoptosis by modulating miR-217/SOS1 axis in acute myeloid leukemia

The aim of this study was to evaluate the pathogenic role of newly identified long non-coding (lnc)-RNA LINCO1268 in acute myeloid leukemia (AML), and investigate its therapeutic potential. The expression level of LINC01268 in AML was measured by quantitative PCR (qPCR). The viability, cell cycle progression, and apoptosis of AML cells were measured by CCK-8 assay and flow cytometry, respectively. The interaction between LINC01268 and miR-217 were predicted by the miRDB website, and then verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The relationship between miR-217 and SOS1 was predicted by TargetScan website, and verified by luciferase reporter assay. LINC01268 was significantly upregulated by 1.6 fold in bone marrow samples of AML patients, which was associated with poor prognosis. LINC01268 was also significantly upregulated in AML cells. LINC01268 knockdown inhibited viability and cell cycle progression but promoted apoptosis of AML cells. Furthermore, LINC01268 functioned as a ceRNA via competitively binding to miR-217, and SOS1 was identified as a target of miR-217. Moreover, LINC01268 positively regulated SOS1 expression to promote AML cell viability and cell cycle progression but inhibited apoptosis via sponging miR-217. LINC01268 promoted cell growth and inhibited cell apoptosis through modulating miR-217/SOS1 axis in AML. This study offers a novel molecular mechanism for a better understanding of the pathology of AML. LINC01268 could be considered as a potential biomarker for the therapy and diagnosis of AML.

MiR-217 regulates colorectal cancer cell proliferation partly through ERK1/2 signal pathway / 医学研究生学报

Objective A variety of miRNAs have been found to be involved in the occurrence and development of colorectal cancer. This paper aim to investigate the clinical and biological relevance of miR-217 and the pathway by which miR-217 may be involved in progression in colorectal cancer. Methods According to the diameter of tumor, the tumor was divided into tumors>5 cm(n=22) and tumors≤5 cm(n=28); 18 cases of colorectal cancer I-II specimens and 32 of III-IV patients; according to median expression of miR-217, the specimens were divided into high expression group and low expression group. The expression of miR-217 in 50 colon cancer patients’ carcinoma and adjacent tissues was determined by qRT-PCR. Cells were grouped: overexpression control Group (transfected control mimic) and miR-217 overexpression group(transfected miR-217 mimic), low expression control group((transfected control inhibitor) and miR-217 low expression group(transfected miR-217 inhibitor), miR-217&ERK1/2 low expression group(transfected miR-217 inhibitor+U0126). MiR-217 and HCT116 cells were over expressed in SW480 cells,and miR-217 was knocked down. A series of biological function assays were performed to assess cell viability (cck-8 assay), clony formation ability (clony formation assay), proliferation (edu assay), Changes in ERK1/2 expression were measured at protein level, and the relationship between miR-217 and ERK1/2 in colorectal cancer cells was explored by relevant rescue experiments. Results Compared with colorectal adjacent noncancerous tissues, the expression of miR-217 was significantly decreased in carcinoma tissues(-1.360±0.645 vs 2.244±0.168, P<0.01); the expression of miR-217 in tumors with a diameter >5cm was significantly lower than that of tumors with a diameter ≤5cm(-1.718±0.272 vs -0.587±0.288, P<0.01); the expression of miR-217 in stage I-II colorectal cancer tissues was significantly higher than that stage III-IV(-0.413±0.330 vs -01.463±0.230, P<0.05); the expression of miR-217 in the miR-217 overexpression group was significantly higher than miR-217 overexpression control group(15.120±0.522 vs 1.004±0.003, P<0.01), and the number of clone formation was significantly less than that of the overexpression control group(199.30±15.62 vs 439.70±18.91, P<0.01) . In HCT116 cells, the expression of miR-217 in the miR-217low expression group was significantly lower than miR-217 low expression control group(0.2070±0.021 vs 1.006±0.003, P<0.01), and the number of clone formation was significantly higher than that control group(237.30±12.14 vs 117.00±7.00, P<0.01) . When miR-217 overexpressed in SW480, the protein expression of ERK1/2 decreased; when miR-217 was inhibited in HCT116, the protein expression of ERK1/2 increased. The number of colons in ERK1/2 low expression group was significantly lower than that miR-217&ERK1/2 low expression group(221.70±12.73 vs 108.00±5.51) , the difference was statistically significant(P<0.01). Conclusion Low expression of miR-217 is observed in both colorectal cancer tissues and cells, and miR-217 can affect tumor cell proliferation in the progression of colorectal cancer partly by inhibiting the expression of ERK1/2.

Regulation of miR-217 on the proliferation of mouse adult pancreatic stem cells / 中国药科大学学报

@#To further evaluate the effect of miR-217 in the proliferation of mouse adult pancreatic stem cells, we firstly transfected adult pancreatic stem cells with miR-217 mimics and studied the effect of miR-217 on proliferation through Western blot and immunofluorescence. Results showed that during the proliferation of adult pancreatic stem cells, miR-217 inhibited the protein expression of Ki-67 and Cyclin D1, which are related to cell propagation. As well as that, to investigate the target genes of miR-217 and their conserved sites bound by the seed region of miR-217, we used bioinformatic algorithms to find a potential target of miR-217 and verified by dual-luciferase activity assay. Surprisingly, dual-luciferase activity assay revealed that miR-217 could decrease PMIR-REPORT-Sirt1-3′UTR luciferase activity and Sirt1 is a direct target of miR-217. Finally, we verified the function of Sirt1 in the proliferation of pancreatic stem cells. Overexpression of miR-217 in pancreatic stem cells inhibited the level of Sirt1 in protein level but not in mRNA level. Furthermore, activator of Sirt1 played positive effect on colony formation ability and cell proliferation and inhibitor of Sirt1 showed the opposite function. In conclusion, miR-217 inhibits the proliferation of mouse adult pancreatic stem cells through Sirt1 and decreased expression of miR-217 to contribute to the pancreatic stem cells development.

Identification of microRNA-217 targeted gene ANLN in pancreatic cancer PANC1 cells / 中华胰腺病杂志

Objective To identify the miR-217 targeted gene ANLN by experiment.Methods Bioinformatic algorithms were used to predict the potential targets of miR-217.Then,ANLN binding with miR217 and mutant ANLN (mutANLN) sequence were designed and synthesized,and their amplified fragments were inserted into plasmid psiCHECK-2,and recombinant plasmid psiCHECK-2-ANLN and psiCHECK-2-mutANLN were reconstructed.The two recombinant plasmids were co-transfected into pancreatic cancer cell line PANC1 with miR-217,miR-217 inhibitor,NC,NC inhibitor by liposome,respectively.Dual luciferase reporter system was used to determine the luciferase activity,and Western blot was used to measure the expression of ANLN protein.Results The luciferase activities of psiCHECK-2-ANLN,psiCHECK-2-ANLN +miR-217,psiCHECK-2ANLN + miR-217 inhibitor,psiCHECK-2ANLN + NC,psiCHECK-2-ANLN + NC inhibitor were 2.221 ± 0.188,0.769 ± 0.061,3.764 ± 0.371,2.265 ± 0.201,2.242 ± 0.018,and the difference among these groups was statistically significant (F =77.405,P ＜0.001),but the difference among psiCHECK-2ANLN group,psiCHECK-2-ANLN + NC group and psiCHECK-2-ANLN + NC inhibitor group was not statistically significant.However,luciferase activities of psiCHECK-2-ANLN + miR-217 group were significantly decreased when compared with other 3 groups,and luciferase activity of psiCHECK-2-ANLN +miR-217 inhibitor group were significantly increased when compared with other 4 groups (all P ＜0.001).Luciferase activities of groups transfected with psiCHECK-2-mutANLN was not significantly different (P =0.053).The expression of ANLN protein in PANC1 with psiCHECK-2-ANLN + miR-217 co-transfection was significantly down-regulated when compared with that with psiCHECK-2-ANLN transfection alone.Conclusions ANLN is one of the direct target genes of miR-217 in PANC1 cells.