Pim-1 mRNA Research Articles

Effects of PIM1 Gene on Proliferation, Apoptosis and JAK2/STAT3 Signaling Pathway of Acute Myeloid Leukemia U937 Cells

To investigate the effects of the serine/threonine kinase family member 1 (PIM1) gene on the proliferation and apoptosis of acute myeloid leukemia (AML) U937 cells, and the regulation effect on Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Bone marrow mononuclear cells from newly diagnosed adult AML patients and patients with iron deficiency anemia were collected and PIM1 mRNA expression was detected by RT-qPCR. AML cell line U937 cells were divided into U937 group (U937 cells were cultured normally), Si-PIM1 group (U937 cells were transfected with low expression adenovirus vector containing PIM1 mRNA), Si-NC group (U937 cells were transfected with low expression adenovirus vector without PIM1 mRNA), coumermycin A1 (CoA1) group (JAK2 activator CoA1 was added to U937 cells at a concentration of 20 μmol/L), and Si-PIM1+CoA1 group (U937 cells were transfected with adenoviral vector containing low expression of PIM1 mRNA and added with CoA1 at a concentration of 20 μmol/L). After culture for 24 h, the expressions of PIM1 mRNA and protein, JAK2/STAT3 pathway, cell cycle and apoptosis-related proteins in U937 cells were detected by RT-qPCR and Western blot, the cell proliferation activity was detected by MTT assay, and flow cytometry was used to detect cell cycle changes and apoptosis rate. The PIM1 mRNA expression level in bone marrow mononuclear cells in AML patients was higher than that in patients with iron deficiency anemia (P < 0.05). Compared with U937 group, PIM1 mRNA and protein, phosphorylated JAK2 (p-JAK2)/JAK2, phosphorylated STAT3 (p-STAT3)/STAT3, Cyclin D1, cyclin-dependent kinase 2 (CDK2) protein, cell proliferation activity, S phase and G 2/M phase proportions were decreased in Si-PIM1 group (all P < 0.05), while p27, Caspase-3 protein, G0/G1 phase proportion and apoptosis rate were increased (all P < 0.05). However, the changes of above indicators in CoA1 group were just opposite to those in Si-PIM1 group, indicating that CoA1 could reverse the effect of Si-PIM1 on U937 cells. There were no significant differences in above indexes of U937 cells between U937 group, Si-PIM1+CoA1 group and Si-NC group (P >0.05). Knockdown of PIM1 gene expression can inhibit U937 cell proliferation and promote apoptosis, in order to alleviate ALM process, which may be related to the inhibition of JAK2/STAT3 pathway activation.

Regulation of cagA-Helicobacter on gastric PIM2 expression in gastric cancer.

The association between infection with cagA-positive H. pylori and an elevated susceptibility to gastric cancer has been firmly established. PIM2 is known to be overexpressed in various types of cancers; however, the specific mechanism by which cagA influences the regulation of PIM2 expression in gastric cancer remains unidentified at present. A mutant NCTC11637ΔcagA strain of H. pylori and the eukaryotic expression vector pcDNA-cagA were constructed for evaluating PIM2 expression levels in gastric cancer cells (HGC27, SGC7901, and AG) co-cultured with the NCTC11637 and NCTC11637ΔcagA strain, as well as pcDNA-cagA and the empty vector pcDNA3.1 (+). Co-culturing gastric cancer cells with NCTC11637 significantly increased PIM2 expression levels (P< 0.001) compared to the negative control group. Additionally, the expression of PIM2 in cells co-cultured with NCTC11637 was higher than that co-cultured with NCTC11637ΔcagA (P< 0.001). Furthermore, successful construction of the eukaryotic expression vector pcDNA-cagA resulted in a significant increase in PIM2 mRNA expression levels after its transfection into gastric cancer cells compared to the control group after 48 hours. The findings indicate that H. pylori/cagA A could be one of the key factors in regulating PIM2 expression levels, potentially influencing the progression of H. pylori-related Gastric Cancer.

HGF Regulation in the Tumor-Supportive Microenvironment Combined with Pan-PIM Kinases Inhibition in Malignant Cells As a Multi-Target Approach for Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), persistent clonal proliferation of malignant myeloid cells results in high severity and low cure potential. Resistance to currently available chemotherapeutics and disease relapse remains an important concern worldwide. Several molecular targets play key roles in impairing therapeutic response, including PIM-kinases, a serine/threonine kinase family associated with the progression of hepatocyte growth factor (HGF)-driven cancers through its receptor, MET. We reported that enhanced HGF secretion by bone marrow (BM) mesenchymal stromal cells (MSC) correlated with low levels of the serine protease inhibitor kunitz-type2 (SPINT2) in AML (Roversi et al., 2019), which are affected by immunosuppressive properties of the tumor microenvironment (TME). However, the interplay between SPINT2, HGF, and PIM-kinases in the TME is yet to be elucidated and could represent an important multi-target therapeutic strategy in hematological cancers. Aims: Given the importance of HGF to AML prognosis and progression, we analyzed the interplay between SPINT2, HGF, and pan-PIM kinases by evaluating the gene expression and the effects of a novel pan-PIM kinase inhibitor, PIM447 (Novartis), in leukemia cells and in the TME. Methods: SPINT2, HGF, PIM1, PIM2 and PIM3 mRNA expression data were obtained from OHSU (2018) AML study and TCGA (cBio Portal). Correlation analysis was obtained by Spearman's coefficient (r). A total of 5 healthy donors (HD) and 5 de novo AML patients BMMSC (CD90+CD105+CD73+CD45-CD34-CD31-HLA-DR-) were treated with 5-Azacitidine (Aza), and evaluated for SPINT2 gene expressions. Myeloid (OCI-AML3, MOLM13, U937) and lymphoblastic (RS4;11, Jurkat) leukemia cells were treated with increasing concentrations of PIM447, and the 101nd apoptosis rates were determined. Peripheral blood and BM mononuclear cells (PBMC/BMMC) from HD were tested for cytotoxicity. Leukemia cells were also examined for reactive oxygen species (ROS), nitric oxide (NO) production, and mitochondrial membrane potential (ΔΨm). To verify PIM447 action onto an immunosuppressive TME, cocultures of leukemia cells and monocytes or monocyte-derived macrophages (MDMs) were performed. To understand the interplay between SPINT2 and PIM-kinases, cocultures of SPINT2-overexpressed MSC and leukemia cells, were performed. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests ( P&amp;lt;.05). Results: The analysis of gene expression performed in MC from 188 AML patients (age median: 57.5 years [range: 18-88]) showed that levels of HGF correlated positively with PIM1, PIM2, and PIM3 ((r) =.8727;.9094;.9093, respectively, P&amp;lt;.0001) and negatively with SPINT2 (r=-.06402). We also verified that SPINT2 mRNA expression was upregulated in AML-BMMSC ( P&amp;lt;.05) after Aza treatment when compared to HD-BMMSC, suggesting that this gene is methylated in AML. Our in vitro results showed PIM447 treatment (2-20μM) yielded reduced cell viability and increased apoptosis in leukemia cells [IC50 range: 12.2-22.2μM, P&amp;lt;.01] without affecting HD cells. PIM447 reduced ROS, NO production, and ΔΨm (all P&amp;lt;.0001) in all cell lines. PIM447 treatment of macrophages differentiated from HD-PBMC revealed a shorter population of M2 macrophages (HLA-DR-CD206+) ( P&amp;lt;.001) and an increased population of M1 macrophages (HLA-DR+CD80+) ( P&amp;lt;.05). Moreover, PIM447 promoted apoptosis of leukemia cells co-cultured with MDMs ( P&amp;lt;.0001). Interestingly, co-culture analyses of SPINT2-overexpressed MSC enhanced PIM447 cytotoxic activity towards leukemia cells (P&amp;lt;.05) and reduced cell-cell adhesion by diminishing CD49b, CD49d, and CD49e (P&amp;lt;.05), suggesting an interplay between SPINT2 and pan-PIM kinases Conclusion: Collectively, our data identify a novel pan-PIM kinase inhibitor, PIM447, which effectively induces leukemia cell apoptosis through reducing MET receptor activation as well as acts on their tumor-supportive microenvironment by inducing a phenotypic shift from immunosuppressive M2 to anti-tumor M1 macrophages. PIM447 also interplays with SPINT2/HGF pathway in the TME, reducing AML progression. These findings suggest that a pivotal strategy to treat AML could be a multi-target therapy, acting both on leukemia cells and in the tumor microenvironment. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

The Mechanism of Resveratrol on Acute T-Lymphocyte Leukemia through IL-7-Mediated JAK / STAT Signaling Pathway

To explore the roles and relative mechanism of resveratrol against T-ALL through detecting the signaling molecules in IL-7 and JAK/STAT pathway. In vitro experiments, Molt4 cells were divided into 3 groups, including the control group, the DMSO group and resveratrol-treated group (Res group). The control group cells without any treatment, the DMSO group cells treated with 0.05% DMSO for 48 hours, the Res group cells treated with 200 μmol/L resveratrol for 48 hours. In vivo experiments, female C57BL/6J mice (6-8 weeks) were randomly divided into the control group, the T-ALL model group (T-ALL group), and Res treatment group (Res group). The control group mice treated with 0.05% DMSO by intragastric treatment, the T-ALL group mice treated with 0.05% DMSO by intragastric treatment, and the Res group mice treated with 10 mg/ml resveratrol. Expression of IL-7, IL-7R and Pim1 mRNA in the cells and mice spleen tissues were detected by RT-qPCR. Cell proliferation ability was detected by CCK-8. The expression of JAK1, JAK3, STAT5, phosphorylated JAK1 (p-JAK1), phosphorylated JAK3 (p-JAK3), phosphorylated STAT5 (p-STAT5) and Pim1 were detected by Western blot. ELISA was used to detect the IL-7 and IL-7R in the cells and mice serum of each groups. Resveratrol could inhibit the proliferation ability of Molt4 cells, decrease the relative levels of p-JAK1, p-JAK3, p-STAT5, Pim1 protein, and the expression levels of Pim1, IL-7 and IL-7R mRNA in cells and mice spleens, reduce the IL-7 and IL-7R in Molt4 cells and mice serum. Resveratrol may inhibite IL-7-medicated JAK/STAT signaling pathway to reduce the expression of target protein Pim1 to further exert its anti-T-ALL effects.

Decreased expression of miR-3135b reduces sensitivity to 5-fluorouracil in colorectal cancer by direct repression of PIM1.

5-Fluorouracil (5-FU)-based chemotherapy is the conventional treatment approach for patients with colorectal cancer (CRC). However, de novo and acquired resistance to 5-FU are frequently observed during treatment, which eventually lead to patients succumbing to the disease. Accumulating data have revealed an association of CRC resistance to 5-FU with aberrant expression of microRNAs (miRs). In the present study, Cell Counting Kit-8 was performed to measure cell viability, flow cytometry was performed to detect cell apoptosis, reverse transcription-quantitative PCR was conducted to measure proviral integration site for Moloney murine leukemia virus 1 (PIM1) and miR-3135b expression, western blotting was conducted to measure PIM1 expression. Microarray data analysis indicated that the level of miR-3135b expression was decreased in patients with recurrent CRC that were treated with 5-FU when compared with non-recurrent cases. Overexpression of miR-3135b increased the sensitivity of CRC cells to 5-FU treatment. Moreover, PIM1 was identified as a target gene of miR-3135b using bioinformatics analysis, reverse transcription-quantitative PCR and western blotting. The direct interaction between these two targets was confirmed by luciferase reporter assays. Notably, PIM1 overexpression compensated the effect of miR-3135b in CRC cells. Furthermore, an inverse correlation between PIM1 mRNA expression levels and miR-3135b expression was observed in clinical samples. Therefore, the present study identified miR-3135b as a novel regulator of 5-FU sensitivity in CRC.

PIM2 promotes lung adenocarcinoma cell migration by regulating XIAP/NF-κB pathway

Background and objective: Proviral insertion site in Moloney murine leukemia virus (PIM)2 functions as a serine/threonine kinase to participate in regulating cell proliferation and cell cycle. PIM2 has been shown to be elevated in the lung cancer cell lines. This study was performed to investigate the role of PIM2 in lung adenocarcinoma cell growth. Mateial and methods: Expression level of PIM2 in lung adenocarcinoma tissues and cells was detected by qRT-PCR (quantitative Reverse Transcription PCR) and western blot. The over-expression and knockdown of PIM2 were separately established by employing pcDNA and siRNA to explore the effects on the cell viability, apoptosis, invasion and migration. The downstream pathways were evaluated by western blot assay. Results: Lung adenocarcinoma tissues and cells showed an elevation of both PIM2 mRNA and protein expression. Knocking down PIM2 decreased the cell viability and promoted the apoptosis, which can be reversed by pcDNA-mediated over-expression of PIM2. PIM2 silencing suppressed the promotional effect of over-expression of PIM2 on cell invasion and migration through increasing IκBα expression and decreasing the X-linked inhibitor of apoptosis protein (XIAP), p65 and IκBα phosphorylation. While, over-expression of PIM2 showed opposite effect on IκBα and XIAP expression or p65 and IκBα phosphorylation. Conclusion: PIM2 can not only suppress lung adenocarcinoma cell apoptosis but also promote cell migration and invasion depending on XIAP/NF-κB signaling pathway.

Circular RNA CircITGA7 Promotes Tumorigenesis of Osteosarcoma via miR-370/PIM1 Axis.

Many studies have shown that there are many circular RNA (circRNA) expression abnormalities in osteosarcoma (OS), and this abnormality is related to the development of osteosarcoma. But at present, it is unclear as to what circITGA7 has in the OS and what it does. In this study, qRT-PCR was used to detect the expression of circITGA7, miR-370, and PIM1 mRNA in OS tissues and cells. The CCK-8 assay was used to detect the effect of circITGA7 on cell proliferation. Later, the transwell assay was used to detect cell migration and invasion. The dual-luciferase reporter assay confirmed the existence of the targeting relationship between circITGA7 and miR-370, and miR-370 and PIM1. We found that circITGA7 was upregulated in OS tissues and cell lines. Knockdown of circITGA7 weakened the cell's ability to proliferate and metastasize. Furthermore, we observed that miR-370 was negatively regulated by circITGA7, while PIM1 was positively regulated by it. A functional assay validated that circITGA7 promoted OS progression via suppressing miR-370 and miR-370 affected OS proliferation and migration via PIM6 in OS. In summary, this study shows that circITGA7 promotes OS proliferation and metastasis via miR-370/PIM1.

Pim-1 Protects Retinal Ganglion Cells by Enhancing Their Regenerative Ability Following Optic Nerve Crush.

Provirus integration site Moloney murine leukemia virus (Pim-1) is a proto-oncogene reported to be associated with cell proliferation, differentiation and survival. This study was to explore the neuroprotective role of Pim-1 in a rat model subjected to optic nerve crush (ONC), and discuss its related molecules in improving the intrinsic regeneration ability of retinal ganglion cells (RGCs). Immunofluorescence staining showed that AAV2-Pim-1 infected 71% RGCs and some amacrine cells in the retina. Real-time PCR and Western blotting showed that retina infection with AAV2-Pim-1 up-regulated the Pim-1 mRNA and protein expressions compared with AAV2-GFP group. Hematoxylin-Eosin (HE) staining, γ-synuclein immunohistochemistry, Cholera toxin B (CTB) tracing and TUNEL showed that RGCs transduction with AAV2-Pim-1 prior to ONC promoted the survival of damaged RGCs and decreased cell apoptosis. RITC anterograde labeling showed that Pim-1 overexpression increased axon regeneration and promoted the recovery of visual function by pupillary light reflex and flash visual evoked potential. Western blotting showed that Pim-1 overexpression up-regulated the expression of Stat3, p-Stat3, Akt1, p-Akt1, Akt2 and p-Akt2, as well as βIII-tubulin, GAP-43 and 4E-BP1, and downregulated the expression of SOCS1 and SOCS3, Cleaved caspase 3, Bad and Bax. These results demonstrate that Pim-1 exerted a neuroprotective effect by promoting nerve regeneration and functional recovery of RGCs. In addition, it enhanced the intrinsic regeneration capacity of RGCs after ONC by activating Stat3, Akt1 and Akt2 pathways, and inhibiting the mitochondrial apoptosis pathways. These findings suggest that Pim-1 may prove to be a potential therapeutic target for the clinical treatment of optic nerve injury.

A New Compound with Increased Antitumor Activity by Cotargeting MEK and Pim-1.

SummaryFeedback circuits are one of the major causes underlying tumor resistance. Thus, compounds that target one oncogenic pathway with simultaneously blocking its compensatory pathway will be of great value for cancer treatment. Here, we develop a new MEK inhibitor designated as KZ-02 that exhibits unexpectedly higher cytotoxicity than its starting compound AZD6244, a well-known MEK inhibitor, in colorectal cancer (CRC). Subsequent kinase selectivity study identified Pim-1 as an additional cellular target for KZ-02. Further studies showed that AZD6244 and Pim-1 1 (a Pim-1 inhibitor) have a synergistic effect on CRC suppression. Mechanistic study revealed that MEK inhibition by AZD6244 leads to increased Pim-1 expression, which could be a general mechanism behind the compromised cell-killing activity of MEK inhibitors. KZ-02, despite increasing Pim-1 mRNA expression, simultaneously promotes Pim-1 proteasomal degradation. Therefore, we uncover a new MEK inhibitor KZ-02 with significantly enhanced antitumor activity by co-targeting MEK and Pim-1.

High Expression of NEK2 and PIM1, but Not PIM3, Is Linked to an Aggressive Phenotype of Bronchopulmonary Neuroendocrine Neoplasms

Dysregulations of the NEK2 and PIM1-3 kinase signaling axes have been implicated in the pathogenesis of several cancers, including those with a neuroendocrine phenotype. However, their impact on bronchopulmonary neuroendocrine neoplasms (BP-NENs) has not been investigated. The aim of this pilot study was to determine mRNA and protein levels of NEK2, PIM1, and PIM3 in a group of 49 patients with BP-NENs: 11 typical carcinoids, 5 atypical carcinoids, 11 large cell neuroendocrine carcinomas, 22 small cell lung carcinomas (SCLC). The expression was measured using TaqMan-based RT-PCR and immunohistochemistry. NEK2 and PIM1 mRNA levels were higher in the SCLC patients than in the other BP-NEN groups (p < 0.001). There was an association between NEK2 mRNA and protein expression (p = 0.023) and elevated NEK2 mRNA levels were related to reduced survival in BP-NEN patients (p = 0.015). Patients with higher PIM1 protein expression had also diminished survival comparing with those with weak or no PIM1 expression (p = 0.037). Elevated NEK2 and PIM1 expression were related to aggressive tumor phenotype and indirectly affected the overall survival of BP-NEN patients. Our pilot study supports the need for future investigation of the biological function of NEK2 and PIM1 in BP-NEN transformation to verify the clinical value of our findings.

PIM1 inhibitor SMI-4a attenuated lipopolysaccharide-induced acute lung injury through suppressing macrophage inflammatory responses via modulating p65 phosphorylation

PIM kinase is involved in the cellular processes of growth, differentiation and apoptosis. However, the role of PIM1 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains largely unknown. A trend of PIM1 in the lung tissue of LPS-induced ALI at different time points was detected. Histology, wet/dry (W/D) ratio, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and survival rate analyses were performed when mice received the PIM1 inhibitor SMI-4a intratracheally 3 h before LPS administration. Cytokine production in vivo and in vitro was measured after SMI-4a pretreatment. NF-κB subunit p65 expression in nuclei and phosphorylation at Ser276 in lung tissues or cells were detected by Western blot analysis. The results showed that PIM1 mRNA and protein were upregulated in the lung tissue of LPS-induced ALI. The PIM1 inhibitor SMI-4a markedly improved the survival rate after lethal LPS administration, reduced the severity of lung edema, attenuated the histologic injuries of the lung tissue and reduced the counts of infiltrated inflammatory cells in the BALF. The PIM1 inhibitor SMI-4a suppressed the production of cytokines in LPS-treated RAW264.7 cell supernatants and BALF. Furthermore, LPS administration upregulated the levels of nuclear p65 and phosphorylated p65 (p-p65) at Ser276, whereas pretreatment with the PIM1 inhibitor SMI-4a reduced p65 upregulation in the nucleus and p-p65 at Ser276. Taken together, these data indicate that the PIM1 inhibitor SMI-4a may serve as a promising therapeutic strategy for LPS-induced ALI by suppressing macrophage production of cytokines via a reduction of p65 activities.

Role of Pim1 Gene Overexpression in Pathogenesis of Acute Myeloid Leukemia

To investigate the effect of Pim1 expression up-regulation on cell proliferation, apoptosis, chemotaxis and angiogenesis in acute myeloid leukemia (AML) cell line U937, and to explore the possible molecular mechanism involved, finally to estimate the Pim1 expression in primary AML cells. GFP-tagged plasmid for Pim1 overexpression and an empty vector plasmid were constructed, and then a stable Pim1 expressed U937 cell line and a control virus-infected U937 cell line were established by a lentiviral vector system. After confirming Pim1 overexpression in U937 cells, proliferation and apoptosis are determined by CCK-8 Kit and flow cytometry respectively. Transwell chemotaxis assay was used to measure the effect of Pim1 overexpression on AML cells. Flow cytometry and confocal microscopy were applied to detect the influence of Pim1 overexpression on phosphorylated CXCR4 (pCXCR4) and its location. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression of angiogenesis and adhesion related genes in AML primary cells. The lentivirus-infected AML cell line with Pim1 overex-pression and the control virusinfected AML cell line were established successfully. The Pim1 overexpression could enhance the proliferation and inhibit the cell apoptosis, moreover accompnied with the increasing expression of cyclin D1, phosphorylated BAD (pBAD) and pCXCR4. After SDF-1 α stimuli, Pim1 overexpression induced AML cell chemotaxis accompanied with p-CXCR4 expression and calcium influx increment. Pim1 overexpression has no effect on angiogenesis. Pim1 mRNA expression was significantly higher in AML patients than the healthy people. Pim1 plays an important role in the pathogenesis of AML, which not only promotes AML cell proliferation and inhibition of apoptosis, but also enhances the chemotactic ability of leukemia cells, which closely relates with Pim1 phosphorylation of CXCR4 and the increase of intracellular calcium ion influx signals.

Site-Specific Cleavage of RNAs Derived from the PIM1 3'-UTR by a Metal-Free Artificial Ribonuclease.

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.

Pim-1 Expression in Rat Retina and its Changes after Optic Nerve Crush.

Pim-1 is a proto-oncogene which has been discovered to involve in cell proliferation, differentiation, and survival. In this study, we observed the expression of Pim-1 in neonatal and adult rat retina and the changes in rat retina following optic nerve crush (ONC) in order to explore the relationship between Pim-1 and the survival of retinal ganglion cells (RGC). We discovered that Pim-1 was distributed mainly in retinal pigment epithelial cells (RPE) and retinal ganglion cell layer (GCL) in normal newborn rats, and it appeared in RPE, cone rod cell layer and GCL in normal adult rats by immunohistochemistry. Our double immunofluorescent staining of Pim-1 and γ-synuclein further confirmed that Pim-1 was localized in 80% of RGC. Moreover, we found that the amount of Pim-1 mRNA and protein in adult rat retina was transiently increased after ONC and then decreased 2 weeks after ONC, and the expression level was lower than that of neonatal rat retina under all conditions. We also discovered that Pim-1 expression in GCL detected by immunohistochemistry was upregulated at Day 1 and Day 3 after ONC, but downregulated at Day 14 after ONC when the survival of RGC was decreased and the apoptotic cells in GCL were increased by hematoxylin-eosin staining, immunohistochemistry, and TUNEL detection. We suggest that the overexpression of Pim-1 in the RGC is related to the optic nerve repair while the low expression of Pim-1 in RGC may be associated with apoptosis and weak intrinsic regeneration ability of RGC. Anat Rec, 301:1968-1976, 2018. © 2018 Wiley Periodicals, Inc.

MiR-486 inhibited osteosarcoma cells invasion and epithelial-mesenchymal transition by targeting PIM1.

Osteosarcoma is the most common malignant tumor of bone with high recurrent rate. miR-486 was downregulated and acted as a tumor suppressor in plenty of tumors. The purpose of this study was to explore how miR-486 worked in osteosarcoma on cell invasion and EMT. miR-486 was low expressed in osteosarcoma while PIM1 was overexpressed, and it had negative correlation between miR-486 and PIM1. miR-486 upregulation or PIM1 downregulation could inhibit osteosarcoma cell invasion and EMT. Meanwhile, miR-486 mediated PIM1 expression through binding to PIM1 mRNA 3'-UTR. PIM1 could reveal partial function of miR-486 on osteosarcoma invasion. In addition, miR-486 low expression or PIM1 overexpression predicted poor prognosis of osteosarcoma patients. miR-486 regulated osteosarcoma cell invasion and EMT through targeting to PIM1. miR-486 low expression or PIM1 overexpression predicted poor prognosis of osteosarcoma patients. The newly identified miR-486/PIM1 axis provides novel insight into the pathogenesis of osteosarcoma.

PO-144 Role of increased expression of brain and acute leukaemia, cytoplasmic (BAALC) in acute myeloid leukaemia (AML) DNA damage repair pathways

IntroductionHepatocellular Carcinoma (HCC) is the sixth most frequent cancer and the second leading cause of cancer related death worldwide, with a poor five year survival and increasing incidence in the Western World. The family of oncogenic serine/threonine kinases comprises three members: Pim1, Pim2 and Pim3. While Pim1 has been the major focus in oncological research and therapeutic exploration so far, Pim2 is overexpressed in HCC as well and has been suggested to be relevant in HCC tumorigenesis.Material and methodssiRNA-mediated knockdown of Pim2 cells was determined on (mRNA) and protein level (Western blot). Biological effects of Pim2 knockdown in HCC were explored by investigating cell proliferation (WST-1-assay), cell cycle distribution (flow cytometry), apoptosis (flow cytometry, Caspase activity-assay), and colony formation (clonogenic assay). Changes in downstream signalling pathways leading to the observed biological effects after Pim2 knockdown were explored on mRNA as well as on protein level. The therapeutic potential of a Pim2 was assessed in an in vivo s.c. xenograft mouse model using polymeric nanoparticles based on polyethyleneimine (PEI) as siRNA-delivery platform for systemic treatment.Results and discussionsKnockdown of Pim2 using siRNAs lead to strong reduction in Pim2 mRNA and protein levels. This resulted in various cellular effects, including the inhibition of proliferation, increased apoptosis, as well as altered cell cycle distribution and reduced colony formation. On the molecular level, Pim2 knockdown led to changed expression and/or phosphorylation of key proteins involved in the regulation of cell cycle (cyclin B1) and apoptosis (Survivin, Bad). In the in vivo xenograft mouse model, systemic delivery of PEI/siPim2 nanoparticles resulted in profound tumor-inhibitory effects.ConclusionOur results highlight the relevance of the oncogenic serine/threonine kinase Pim2 in HCC. Counteracting its overexpression by siRNA therapeutics, or specifically inhibiting Pim2 on the protein level, might represent novel treatment strategies in HCC therapy.

Expression of silk/threonine kinase 1 in glioma and its effect on proliferation of U87MG cells and its mechanism

Objective To investigate the expression of serine/threonine kinase 1 (PIM1) in glioma and its effects on proliferation of glioma cell line U87MG and the possible mechanism. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of PIM1 in 28 cases of glioblastoma specimens and 15 cases of traumatic brain specimens. The expression of PIM1 in U87MG cells was knocked down by RNA interference technique, and cells were divided into short hairpin RNA (shRNA) negative control group and shRNA interference group. The proliferation of U87MG cells was examined by cell counting kit-8 (CCK-8) assay. By Western blotting, the expression of PIM1, protein kinase B (Akt), and phosphorylated protein kinase B (p-Akt) proteins was detected in glioma tissues. Results The expression of PIM1 mRNA (1.59±0.13) was significantly higher in glioma tissues than that in normal brain tissue (0.42±0.09, P=0.010). The proliferation of U87MG cells in shRNA interference group at 72 h (0.462±0.014) was significantly reduced as compared with that in shRNA negative group (0.826±0.034, P=0.008). The knockdown of PIM1 could lead to changes in Akt signaling pathway. Conclusion The PIM1 could promote the proliferation of U87MG cells by Akt signaling pathway. Key words: Serine/threonine kinase 1; Glioma; Cell proliferation

PIM-1 mRNA expression is a potential prognostic biomarker in acute myeloid leukemia

BackgroundHigh expression of proviral integration site for Moloney murine leukemia virus-1 (PIM-1), a serine/threonine kinase, is associated with many cancers. The main purpose of this study were to investigate that the correlation between PIM-1 mRNA levels and clinicopathologic features and its clinical significance in acute myeloid leukemia (AML).MethodsqRT-PCR was performed for 118 de novo AML and 20 AML complete remission patients and 15 normal individuals. All statistical analysis were performed using Graphpad Prism5 software.ResultsWe observed that expression of PIM-1 mRNA was higher in AML patients than in healthy individuals and in complete remission AML patients (P = 0.0177). Further, high PIM-1 mRNA levels were more associated with high-risk FLT3+ AML patients than the FLT3− group (P = 0.0001) and were also associated with clinical factors such as risk stratification (P = 0.0029) and vital status (P = 0.0322). Kaplan–Meier survival analysis indicated that PIM-1 mRNA expression correlated with overall survival (OS), disease free survival (DFS), and relapse rate (RR) in AML patients. Most importantly, the high PIM-1-expressing patients took longer to achieve complete remission than the low expression group (P = 0.001). In addition, the complete remission rate was significantly lower in the high PIM-1 group (P = 0.0277) after induction therapy.ConclusionsAbove results suggest that PIM-1 mRNA levels may be an independent prognostic factor in AML.

The role of Pim-2 in apoptotic signal transduction pathway of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors. The objective was to investigate the role of serine/threonine kinase Pim-2 in apoptosis signal transduction pathway, because there is little study about its contribution to apoptosis in hepatocellular carcinoma.Methods: The Pim-2 gene and protein expression were examined by qRT-PCR, Western blot and immunohistochemical stain in HCC tissues and normal liver tissues. The plasmid pCI-neo-Pim2 was transfected into human hepatoma cell line SMMC7721 by lipofectamine. Total RNAs were extracted from SMMC7721 cell in logarithm growth phase. The mRNA expression of Pim-2, Akt-1 (protein kinase B), 4E-BP1 (translation repressor of mammalian target of rapamycln), SOCS-1 (repressor of cytokine), Bad（Bcl-xL/Bcl-2 associated death promoter, Bim（Bc1-2 interacting mediator of cell death）and Puma (p53 upregulated modulator of apoptosis) were identified by qRT-PCR. The cell cycle of post-transfected SMMC7721 cells was assessed by flow cytometry.Results: Pim-2 expression was enhanced in HCC. In post-transfected SMMC7721 cells, Pim-2 mRNA expression was up-regulated, level of Bad mRNA was attenuated, furthermore, the transcription level of Akt-1, SOCS-1, 4E-BP1, Bim and Puma gene wasn’t variety. Up-graulated Pim-2 can’t cause distinct change of cell cycle or apoptosis in hepatoma cell.Conclusions: The serine/threonine kinase Pim-2 plays an import role in the development of HCC, Pim-2 dependent maintenance of cell size and survival correlated with its ability to maintain down-regulated expression of the BH3 protein Bad. Pim-2 is not a trigger in cell-autonomous survival or inhibiting apoptosis in hepatocellular carcinoma. Pim-2 is a redundancy pathway of survival signaling.

PIM1 gene silencing inhibits proliferation and promotes apoptosis of human esophageal cancer cell line Eca-109.

We aimed to study the effect of PIM1 gene silencing on the proliferation and apoptosis of human esophageal cancer cell line Eca109. Cultured Eca109 cells were transfected with the recombinant plasmids in mediation of Lipofectamine TM 2000 Reagent. The Eca109 cells in logarithmic growth phase were collected and assigned into three groups: the PIM1 siRNA group (stably transfected with PIM1-shRNA plasmids), the negative control (NC) group (transfected with vacant plasmids), and the blank group (Eca109 cells without any transfection). The PIM1 mRNA expression was determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cell cycle was analyzed by flow cytometry. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8). Cell apoptosis was assessed by Annexin V-FITC/PI double-staining and TUNEL assays. The PIM1 mRNA expression of Eca109 cells in the PIM1 siRNA group was significantly lower than that in the NC and blank groups. Compared with the NC and blank groups, the viability and proliferation of the Eca109 cells in the PIM1 siRNA group were significantly decreased at 48 h, 72 h and 96 h after transfection. The cell growth inhibition rate of the PIM1 siRNA group was higher than that of the NC and blank groups after transfection. Furthermore, the apoptotic rate of the PIM1 siRNA group was also higher than that of the NC and blank groups. In conclusion, our preliminary findings suggest that PIM1 gene silencing could inhibit proliferation and promote apoptosis of esophageal cancer cells.