LncRNA MALAT1/miR-181a-5p affects the proliferation and adhesion of myeloma cells via regulation of Hippo-YAP signaling pathway.

Multiple myeloma (MM) is a plasma cells malignant proliferative disease, especially in aged people. LncRNAs have been considered as important regulators in MM. This research was to study the effect of LncRNA MALAT1 on the proliferation and adhesion of myeloma cells and whether Long non-coding RNAs MALAT1(LncRNA MALAT1) plays its regulative role through Hippo-YAP signaling pathway by targeting miR-181a-5p. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was used to detect the LncRNA MALAT1/miR-181a-5p expression and improve the transfection efficiency. Western blot analysis was used to analyze the expression of proliferation and apoptosis related proteins and Hippo-Yes-associated protein (YAP) signaling pathway related proteins. Cell proliferative ability and cell apoptosis were respectively determined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis. ELISA assay was for the determination of adherence factors. Immunohistochemistry was to detect the expression of proliferation and adhesion related proteins. LncRNA MALAT1 targeting gene was determined by Dual-luciferase reporter assay. LncRNA MALAT1 was increased in MM cells and LncRNA MALAT1 interference could inhibit cell proliferation and promote cell apoptosis with the changes in the related proteins. Also, LncRNA MALAT1 interference could inhibit cell adhesion through Hippo-YAP signaling pathway. MiR-181a-5p was demonstrated to be a target of LncRNA MALAT1 and miR-181a-5p overexpression could also regulate the changes in cellular behavior in accordance with the LncRNA MALAT1 interference. In addition, LncRNA MALAT1 interference could decrease the expression of miR-181a-5p and inhibit the growth of tumor. In conclusion, this study showed that LncRNA MALAT1 interference inhibited the proliferation and adhesion of myeloma cells by the up-regulation of miR-181a-5p through activating the Hippo-YAP signaling pathway.

Changes of Apolipoprotein M Gene Expression During the Cell Differentiation and Apoptosis Induced by Simvastatin in Combination with All-Trans Retinoic Acid in Human Promyelocytic Leukemia Cell Line NB4.

We examined the effect of simvastatin (SV) alone and in combination with all-trans retinoic acid (ATRA) on proliferation, differentiation, apoptosis and apolipoprotein M (apoM) expression in the human promyelocytic leukemia cell line NB4. The NB4 cells were incubated with 10 µM Simvastatin (10SV) and 0.5 µM ATRA alone or in combination, taking NB4 cells without any treatment as normal controls. The cells of different groups were collected at 24, 48 and 72 h post-incubation for further detection. Their morphological changes were observed after Wright stain. MTT method was used to assay the growth inhibition rate and flow cytometry to detect CD11b expression level and the early stage apoptosis ratio. Real-time quantitative reverse transcriptase-polymerase chain reaction was used to detect the apoM gene expression levels. As expected 0.5 µM ATRA did not affect proliferation or apoptosis, strongly induced differentiation and decreased apoM expression. 10SV inhibited proliferation, increased apoptosis, induced differentiation and increased apoM expression in a time-dependent manner. The addition of ATRA to SV did not increase the effect of SV on proliferation and apoptosis, but increased the effect of SV on differentiation. And completely abrogated the effect of SV on apoM expression. Together these results show that SV has anti-leukemic properties by itself and that combined therapy may have a place in the current anti-leukemic arsenal.

Suppressive effects of simvastatin on the human acute promyelocytic leukemia NB4 cell line through the regulation of the nuclear factor-κB signaling pathway.

The present study examined the effects of simvastatin on the proliferation, apoptosis and gene expression levels involved in the nuclear factor-κB (NF-κB) signaling pathway in the human acute promyelocytic leukemia NB4 cell line by methyl thiazolyl tetrazolium assay, flow cytometry and the Human NF-κB Signaling Pathway RT2 Profiler™ PCR Array profiles. The results showed that simvastatin significantly inhibited proliferation and induced apoptosis of the NB4 cells in a time- and dose-dependent manner. Changes were noted in the expression levels of 56 genes involved in the NF-κB signaling pathways in the NB4 cells treated with 15 µm simvastatin at 48 h post-incubation, among which, 47 genes were downregulated and 9 were upregulated. In conclusion, simvastatin potentially inhibits the proliferation and induces the apoptosis of NB4 cells through the regulation of the expression levels of genes involved in the NF-κB signaling pathway.

[Effects of simvastatin on PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1].

To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (SV) on proliferation, apoptosis and the PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1. SHI-1 cells were incubated with different concentrations of SV (5, 10, 15 µmol/L). Otherwise, SHI-1 cells without any treatment were used as control. Cells in different groups were collected at 24, 48 and 72 h after incubation for further detection. MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the early stage apoptosis ratio. The human PI3K-AKT Signaling Pathway RT(2) Profiler(TM) PCR Array was used to detect the expression of 84 genes involved in PI3K-AKT signaling. The results indicated that the SV inhibited the proliferation and inducted the apoptosis of SHI-1 cells in time- and dose-dependent manners significantly. The growth inhibition rates of SHI-1 cells treated with 15 µmol/L SV for 24, 48 and 72 h were 26.82, 47.09 and 63.92, respectively; and their early stage apoptosis ratios were 5.75, 13.25 and 15.59, respectively. Compared with the control group, expression levels of 39 genes were changed in the group of 15 µmol/L SV at 48 h, among them 26 genes were down-regulated and 13 genes were up-regulated. It is concluded that the SV can inhibit proliferation and induce apoptosis of SHI-1 cells, and the mechanism may be associated with the changes of gene expression level in PI3K-AKT signaling pathway regulated by SV.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.