[MiR-217 Targeting PI3K/Akt Pathway Enhances Sensitivity of Adriamycin to Acute Myeloid Leukemia].

OBJECTIVE: To investigate the effects of miR-217 on proliferation and adriamycin sensitivity of acute myeloid leukemia (AML) cells. METHODS: The mimic NC and miR-217 mimic vectors were constructed and transfected into HL-60 cells, and transfection efficiency was detected by qPCR. The cells were treated with different concentrations of adriamycin for 24 h and 48 h. CCK-8 assay was used to detect the chemical sensitivity of adriamycin and screen the optimal concentration and time of adriamycin treatment. Cells were divided into control group, mimic NC group, miR-217 mimic group, adriamycin group and miR-217 mimic+adriamycin group. Apoptosis was detected by flow cytometry, and the expressions of miR-217, PI3K and Akt3 were detected by qPCR. Western blot was used to detect the expression of PI3K/Akt pathway proteins PI3K, Akt3 and apoptosis proteins Bcl-2, Bax, and double luciferase was used to verify the relationship between miR-217 and Akt3. RESULTS: MiR-217 mimic could enhance the sensitivity of HL-60 cells to adriamycin. The optimal concentration and treatment time of adriamycin were 160 ng/ml and 48 h, respectively. Compared with control group, apoptosis rate, miR-217 and Bax protein levels were significantly increased in miR-217 mimic and adriamycin groups (P < 0.01), while Bcl-2 protein, PI3K, Akt3 mRNA and protein levels were significantly decreased (P < 0.01). Compared with adriamycin group, apoptosis rate, miR-217 and Bax protein levels were significantly increased in miR-217 mimic+adriamycin group (P < 0.01), while Bcl-2 protein, PI3K, Akt3 mRNA and protein levels were significantly decreased (P < 0.01). Dual luciferase assay showed that there was a targeted regulatory relationship between miR-217 and Akt3. CONCLUSION: MiR-217 regulates the PI3K/Akt pathway targeting Akt3, inhibits cell proliferation, promotes cell apoptosis and enhances the sensitivity of adriamycin to AML cells.

[Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells].

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.

miR-93-5p promotes insulin resistance to regulate type 2 diabetes progression in HepG2 cells by targeting HGF.

Insulin resistance is a common feature of type 2 diabetes mellitus (T2DM). However, the mechanisms underlying insulin resistance are not completely understood. The present study aimed to investigate the effect of microRNA (miR)­93­5p on insulin resistance in T2DM cells. Human hepatocellular carcinoma (HCC; HepG2) cells were cultured in medium with high glucose content (30 mM glucose) to establish an in vitro insulin­resistant cell model (IR group). Glucose consumption and glycogen synthesis assays were performed to assess glucose consumption and glycogen synthesis, respectively. By performing immunoprecipitation assays, the abundance of the Met­insulin receptor complex was detected in HepG2 cells. miR­93­5p and hepatocyte growth factor (HGF) mRNA expression levels were measured via reverse transcription­quantitative PCR, and HGF protein expression levels were measured via western blotting. A dual­luciferase reporter assay was conducted to investigate the interaction between miR­93­5p and HGF. Cell Counting Kit­8, BrdU and caspase­3 activity assays were performed to evaluate cell viability, proliferation and apoptosis, respectively, in insulin­resistant HepG2 cells following transfection with small interfering RNA­HGF, HGF overexpression vector, miR­93­5p mimic or miR­93­5p inhibitor. The results demonstrated that miR­93­5p expression was significantly increased and HGF expression was significantly decreased in HCC tissues isolated from patients with or without T2DM compared with adjacent healthy tissues isolated from patients without T2DM. Compared with the IR group, miR­93­5p overexpression significantly increased cell proliferation, glucose consumption and glycogen synthesis, but significantly inhibited apoptosis in insulin­resistant HepG2 cells. By contrast, compared with the IR group, HGF overexpression significantly inhibited cell proliferation, glucose consumption and glycogen synthesis, but significantly enhanced cell apoptosis in insulin­resistant HepG2 cells. Following co­transfection with HGF overexpression vector and miR­93­5p mimic, miR­93­5p mimic­mediated induction of HepG2 cell proliferation, glucose consumption and glycogen synthesis in insulin­resistant HepG2 cells was inhibited. Collectively, the results of the present study indicated that miR­93­5p enhanced insulin resistance to regulate T2DM progression in HepG2 cells by targeting HGF.

[EFFECT OF α-LIPOIC ACID IN INHIBITING OXIDATIVE STRESS AND PROMOTING DIABETIC WOUND HEALING BY SUPPRESSING EXPRESSION OF miR-29b IN MICE].

OBJECTIVE: To investigate the effect of α-lipoic acid on the oxidative stress of wound tissues and diabetic wound healing in mice with diabetic feet. METHODS: Sixty male C57BL/6J mice weighting 200-300 g were randomly divided into model group (control group, n=15), α-lipoic acid-treated model group (n=15), miR-29b mimic group (n=15), and miR-29b mimic negative control group (NC group, n=15). All animals received intraperitoneal injection of streptozocin to establish the diabetic model. Then, a full thickness wound of 5 mm×2 mm in size was created at 4 weeks after modeling. All mice were administrated with high-sugar-fat-diet. At the same day after modeling, α-lipoic acid-treated model group was continuously given intravenous injection of 100 mg/(kg·d) α-lipoic acid for 14 days; miR-29b mimic group and NC group received the tail intravenous injection of lentiviral vector for miR-29b mimic and miR-29b mimic negative control (a total of 2×107 TU), respectively, with the treatment of α-lipoic acid. The wound healing was observed and wound area was measured at 7 and 14 days. The wound tissues were harvested to detect the levels of superoxide dismutase (SOD) and glutathione (GSH) using xanthine oxidase method and 5, 5-dithiobis-2-nitrobenzoic acid staining method at 14 days. At the same day, 7, and 14 days after modeling, the relative miR-29b expression in wound tissues from control and α-lipoic acid-treated model groups was detected by real-time fluorescence quantitative PCR. RESULTS: All mice survived to the experiment end. The wound healing was faster in α-lipoic acid-treated group than control group. At 7 and 14 days, the relative wound area and miR-29b expression level were significantly lower, while the contents of SOD and GSH were significantly higher in α-lipoic acid-treated group than control group (P<0.05). In addition, miR-29b mimic group had significantly increased relative wound area and significantly decreased the contents of SOD and GSH when compared with NC group at 7 and 14 days (P<0.05). CONCLUSIONS: α-lipoic acid could inhibit oxidative stress and promote diabetic wound healing by suppressing expression of miR-29b in mice.

[Prognostic analysis of chronic myeloid leukemia with Cox model and the Sokal, Hasford score].

This study was aimed to analyze the prognostic factors of the patients with chronic myeloid leukemia (CML). Survival curve and survival rate of 204 patients with CML were estimated with Kaplan-Meier method and Logrank respectively. Univariate and multivariate analysis of prognostic factors carried out by Cox's regression model. The Sokal and Hasford score were used to discriminate the relative risk of Hu and IFN group. The results showed that among the 204 patients, the median survival time was 50 (32-65) months, and 5 year survival rate was 32.3% (95% CI, 23.7%-42.6%). The median survival times of IFN and Hu group were 56 (41-67) and 41 (19-56) months, and 5 year survival rates were 45.4% (95% CI, 37.5%-54.2%) and 26.8% (95% CI, 21.6%-33.3%) (P < 0.001) respectively. From the Cox stepwise regression model, Ph chromosome negative, high LDH, low Hct, percentage of peripheral blood basophils > or = 10%, marrow blasts + promyelocytes > or = 10% and presence of nucleated RBCs was associated with poor prognosis, and the treatment also played an important role in CML. According to the Sokal score, the high, intermediate and low risk rates of Hu group were 72.9%, 21.5% and 5.6%, the median survival time reached 34 (23-49) months, 43 (32-58) and 50 (38-62) months respectively; while censored by the Hasford score, the high, intermediate and low risk rates of IFN group were 17.6%, 25.1% and 57.3% respectively, the median survival time was 44 (33-57), 56 (45-70) and 66 (52-76) months respectively. It is concluded that Ph chromosome, concentration of LDH, percentage of Hct, peripheral blood basophils, marrow blasts, promyelocytes, presence of nucleated RBCs and treatment are the most important prognostic factors for CML. The Sokal score can not discriminate the relative risk of Hu group well, while the Hasford score can discriminate the relative risk of IFN group.