Growth-inhibitory and apoptosis-inducing effects of andrographolide on acute lymphoblastic leukemia cells

AIM To explore the effect of andrographolide (AND) on the growth and apoptosis of acute lymphoblastic leukemia （ALL） cells. METHODS CCK-8 assay was used to assess the viability of human acute lymphoblastic leukemia CEM-C1 cells treated with AND for 12 h, 24 h and 48 h. The cell morphological changes were observed by Wright-Giemsa staining. The cell apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide (PI) staining, and the cell cycle was examined by flow cytometry with PI staining. The expression levels of apoptosis-related proteins were examined by Western blot. The mitochondrial membrane potential (MMP) of the cells was determined by JC-1 assay. RESULTS The results of CCK-8 assay indicated that AND inhibited the viability of CEM-C1 cells in a dose- and time-dependent manner. After administration of AND for 24 h, CEM-C1 cells shrank, the cytoplasm turned red and the cell numbers were significantly reduced. Incubation of AND for 24 h resulted in G₂-phase arrest and apoptosis. Treatment with AND for 24 h increased the protein levels of cleaved caspase-3, cleaved caspase-7 and Bax, and down-regulated Bcl-2 in the CEM-C1 cells (P<0.05). The ratios of cleaved caspase-3/caspase-3, cleaved caspase-7/caspase-7 and Bax/Bcl-2 were elevated with the increase in the concentration of AND. Collapsed MMP in CEM-C1 cells was observed after AND administration for 24 h. Treatment with AND in vivo suppressed the growth of the xenograft tumor and increased the protein level of cleaved caspase-3. CONCLUSION Andrographolide exerts growth-inhibitory and apoptosis-inducing effects on ALL cells both in vitro and in vivo.     

Long non-coding RNA and diabetes mellitus

Long non-coding RNAs (lncRNAs) are a group of RNAs, which are longer than 200 nucleotides without containing functional open reading frame and cannot encode protein. The study of lncRNA will help to understand the multi-level expression regulatory network of the body, and is expected to provide the basis of prediction, diagnosis and treatment of complex diseases. Although the functions and mechanism of lncRNA remain unclear, some studies indicate that lncRNA is involved in the development of diabetes mellitus, and those lncRNAs may be new diagnostic markers and therapeutic targets.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

Effect of transcription factor BACH2 over-expression on viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes

AIM To investigate the effect of over-expression of BTB and CNC homology 2 （BACH2） on the viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes CCRF-CEM. METHODS CCRF-CEM cells were divided into 3 groups： control group, empty vector group, and BACH2 over-expression group. The BACH2 over-expression vector was transfected into CCRF-CEM cells of BACH2 over-expression group by liposome transfection method. The difference in mRNA expression of BACH2 between CCRF-CEM cells and peripheral blood mononuclear cell （PBMC） was detected by qPCR. CCK8 assay was performed to evaluate the viability of CCRF-CEM cells. Flow cytometry was used to analyzed the apoptosis of CCRF-CEM cells. The protein expression of BACH2 and cyclin D3 in the CCRF-CEM cells was observed by immunofluorescence staining. The protein expression of cyclin D3, Bcl-2 and caspase-3 was determined by Western blot. RESULTS The mRNA expression of BACH2 in CCRF-CEM cells was significantly lower than that in PBMC （P<0.05）. Compared with control group, BACH2 over-expression significantly suppressed the viability,increased the apoptotic rate and caspase-3 expression, and decreased the expression of cyclin D3 and Bcl-2 in CCRF-CEM cells （P<0.05）. CONCLUSION BACH2 expression is decreased in T lymphocytes of human acute lymphoblastic leukemia. Over-expression of BACH2 inhibited the viability of human acute lymphoblastic leukemia T lymphocyte and induced apoptosis.     

Long non-coding RNA TTTY15 expression in osteosarcoma and its effect on viability and invasion ability of osteosarcoma cells

AIM:To observe the expression of long noncoding RNA TTTY15 in osteosarcoma tissues and cell lines and to explore its effect on the viability and invasion ability of osteosarcoma cell lines. METHODS:qPCR was used to detect the expression of TTTY15 in 11 cases of osteosarcoma and its adjacent tissues. The mRNA levels of TTTY15 in osteosarcoma cell lines (143B, Saos2, MG-63, U2OS and HOS) and human osteoblast cell line hFOB1.19 were also tested. TTTY15 was down-regulated after transfected with small interfering RNA in MG-63 cells, the cell line with the highest level of TTTY15. The effect of TTTY15 knockdown on the viability of MG-63 cells was measured by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The effect of TTTY15 knockdown on the cell invasion ability was detected by Transwell assay. The levels of miR-216b-5p and FOXM1 mRNA were detected by qPCR, and the changes of the related proteins were determined by Western blot. RESULTS:Compared with the adjacent tissues, the expression of TTTY15 increased in the osteosarcoma tissues (P<0.01). Compared with the human osteoblast cell line, the expression of TTTY15 increased in the osteosarcoma cell lines (P<0.05), and the level of TTTY15 in the MG-63 cells was the highest (P<0.01). After knockdown of TTTY15 expression in the MG-63 cells, the cell viability was decreased (P<0.05), cell cycle progression was inhibited (P<0.01), and the cell invasion ability was decreased (P<0.01). The expression of miR-216b-5p was increased (P<0.01) and the expression of FOXM1 mRNA was decreased (P<0.01). The protein expression of FOXM1, CDK4, cyclin D1, MMP-2 and N-cadherin was decreased, while the protein expression of E-cadherin was increased (P<0.05). CONCLUSION:The expression of TTTY15 is increased in the osteosarcoma tissues and cell lines. The low expression of TTTY15 inhibits the cell viability and invasion ability of osteosarcoma cells. The possible mechanism is that the knockdown of TTTY15 expression results in the increase in miR-216b-5p expression and the down-regulation of FOXM1 expression.     

Effect of long non-coding RNA-671 on proliferation and invasion of esophageal squamous-cell carcinoma cells

AIM: To detect the expression of long non-coding RNA-671 (lnc671) in esophageal squamous-cell carcinoma cell lines and to investigate the effect of lnc671 on the malignant phenotype of esophageal squamous-cell carcinoma cells. METHODS: The level of lnc671 in the esophageal squamous-cell carcinoma cells was detected by RT-qPCR. Specific lnc671 small interfering RNA (siRNA) used to explore the effects of lnc671 on proliferation, colony formation, invasion and migration abilities of esophageal squamous-cell carcinoma cells. RESULTS: The database of GEPIA analysis showed that increased expression of lnc671 was associated with shorter survival in the patients of esophageal cancer (P<0.05). Compared with normal immortalized esophageal epithelial cells, lnc671 was highly expressed in a variety of esophageal squamous-cell carcinoma cell lines. lnc671 knock-down significantly inhibited the growth, colony formation ability, migration and invasion abilities of esophageal squamous-cell carcinoma cells(P<0.01). CONCLUSION: The expression of lnc671 is increased in various esophageal squamous-cell carcinoma cell lines. Knock-down of lnc671 expression inhibits the malignant phenotype of esophageal squamous-cell carcinoma cells.     

Effect of CFTR on viability and apoptosis of acute leukemia cells and its related mechanisms

AIM: To investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in acute myeloid leukemia (AML) and its effect on the biological function of human erythroleukemia cell line TF1, and to explore the underlying mechanism. METHODS: The abundance of CFTR in the bone marrow mononuclear cells of patients with AML was measured by real-time PCR. After TF1 cells were incubated with CFTR specific inhibitor CFTRinh-172, cell viability, cell cycle distribution and cell apoptosis were analyzed by CCK-8 assay and flow cytometry. The Wnt signaling pathway-related proteins were determined by Western blot. RESULTS: CFTR was highly expressed in both patients with AML and leukemia cell lines. After incubated with CFTRinh172, the viability of TF1 cells was decreased, the proportion of the cells in G₀/G₁ phase was increased, while that in S phase declined (P<0.05). Furthermore, the cells treated with CFTRinh-172 exhibited higher apoptotic rate, accompanied with lower protein expression of β-catenin, c-Myc and cyclin D1 (P<0.05). CONCLUSION: CFTR expression is dramatically increased in AML. Inhibition of CFTR restrains the growth and promotes the apoptosis of TF1 cells via classical Wnt signaling pathway.     

Significant anti-proliferation of human acute lymphoblastic leukemia cells by combined treatment with chloroquine and dexamethasone

AIM：To explore the promoting action of chloroquine on the anti-proliferation effect of dexamethasone on acute lymphoblastic leukemia cells. METHODS：CCK-8 assay was used to assess the viability of the dexamethasone-resistant human acute lymphoblastic leukemia CEM-C1 cell line treated with the combination of chloroquine and dexamethasone. Western blotting, quantitative real-time PCR and LysoTracker Red staining were utilized to examine the mechanism. RESULTS：Combination of chloroquine and dexamethasone significantly inhibited the proliferation of CEM-C1 cells compared with control group (P<0.01). The combination of chloroquine and dexamethasone increased the abundance of glucocorticoid receptor and inhibited lysosomal function, while lysosomal inhibitor bafilomycin A1 also increased glucocorticoid signaling. CONCLUSION：Dexamethasone combined with chloroquine triggers an anti-proliferation effect on CEM-C1 cells via a lysosome-mediated pathway.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Expression of miRNA-93 in acute lymphocytic leukemia

AIM: To investigate the expression of microRNA (miRNA)-93 in acute lymphocytic leukemia (ALL) and its effect on the proliferation of acute T-cell leukemia Jurkat cells.METHODS: The expression of miRNA-93 in the bone marrow samples of patients with ALL was measured by real-time PCR. After down-regulation of miRNA-93 by transfection with miRNA-93 inhibitor in the Jurkat cells, the cell viability, cell proliferation and cell cycle distribution were detected by CCK-8 assay, EdU assay and flow cytometry, respectively. Furthermore, the protein levels of cell cycle-related molecules such as cyclin D1, cyclin-dependent kinase 4 (CDK4), phosphorylation retinoblastoma (Rb) and P27 were measured by Western blot.RESULTS: miRNA-93 was highly expressed in the patients with ALL, and the expression level was highest in the high risk patients. Down-regulation of miRNA-93 inhibited Jurkat cell viability, arrested cell cycle in G₁/S transition. In addition, the protein levels of cyclin D1, CDK4 and p-Rb were significantly decreased, the protein expression of P27 was increased in Jurkat cells trasfected with miRNA-93 inhibitor.CONCLUSION: miRNA-93 expression is increased in ALL patients. Down-regulation of miRNA-93 restrains cell proliferation in the acute T cell leukemia cell line Jurkat via regulating cell cycle-related molecules.     

Long noncoding RNA ZFAS1/miR-150/ROCK1 axis regulates proliferation and migration of vascular smooth muscle cells

AIM: To investigate whether long noncoding RNA ZNFX1 (zinc finger NFX1-type containing 1) antisense RNA 1 (ZFAS1) promotes the proliferation and migration of vascular smooth muscle cells (VSMCs) by regulating microRNA-150 (miR-150)/ROCK1, and the involving mechanism of atherosclerosis. METHODS: Platelet-derived growth factor-BB (PDGF-BB) was used to induce proliferation and migration of VSMCs. Real-time PCR was used to detect the content of ZFAS1 in the VSMCs. After further down-regulating the expression of ZFAS1 by siRNA, the viability of VSMCs was detected by MTT assay, and the proliferation was measured by EdU staining. The migration ability of VSMCs was detected by Transwell method. The expression levels of miR-150 and ROCK1 were detected by RT-qPCR, and the protein level of ROCK1 was determined by Western blot. Luciferase reporter assay was used to confirm that ROCK1 was the target gene of miR-150. Finally, miR-150 expression was inhibited, and the proliferation and migration ability of VSMCs and expression of ROCK1 after down-regulation of ZFAS1 expression were examined. RESULTS: PDGF-BB up-regulated the expression of ZFAS1 in the VSMCs. After down-regulating the expression of ZFAS1, the proliferation and migration abilities of VSMCs were inhibited (P<0.05), the expression level of miR-150 was increased (P<0.05), and the expression level of ROCK1 was decreased (P<0.05). The results of luciferase reporter assay showed that miR-150 directly targeted ROCK1. Inhibition of miR-150 expression attenuated the inhibition of proliferation and migration of VSMCs by ZFAS1 expression knock-down (P<0.05) and up-regulated the expression level of ROCK1 (P<0.05). CONCLUSION: ZFAS1 promotes the proliferation and migration of VSMCs induced by PDGF-BB by regulating miR-150/ROCK1.     

Study on expression of long non-coding RNA in colon cancer tissues and adjacent tissues

AIM: To screen the differentially expressed long non-coding RNA (lncRNA) in colon cancer, and to explore its expression in colon cancer tissues and adjacent tissues. METHODS: The "Colon adenocarcinoma:Person neoplasm cancer status" which consisted of 36 cases of colon cancer tissues and 29 cases of normal colonic tissues was downloaded from the lncRNAtor database. The candidate genes were selected from these differentially expressed lncRNAs based on artificial criterion (P<0.01; fold change ≥ 2 or<0.5) and then validated by real-time PCR in 60 pairs of colon cancer tissues and adjacent tissues. RESULTS: A total of 50 lncRNAs were differentially expressed in colon cancer tissues, including 28 up-regulated and 22 down-regulated (P<0.01). The verifying results displayed that HNF1A-AS1 and ZDHHC8P1 were up-regulated (P<0.01), and SUZ12P expression was down-regulated (P<0.05), but the expression of AC069513.3 was not statistically significant between colon cancer tissues and adjacent tissues. The abilities of HNF1A-AS1, ZDHHC8P1, SUZ12P and AC069513.3 to discriminate the colon cancer from normal adjacent tissue by the ROC curve with an AUC of 0.729 (sensitivity 78%, specificity 67%), 0.617 (sensitivity 68%, specificity 55%), 0.689 (sensitivity 66%, specificity 55%) and 0.518 (sensitivity 52%, specificity 48%) were observed. CONCLUSION: Long non-coding RNA HNF1A-AS1 and ZDHHC8P1 are up-regulated and SUZ12P is down-regulated in colon cancer tissues, suggesting that they may be involved in the pathogenesis of colon cancer.     

Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway

AIM: To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue, normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR. Transwell assay was used to detect the invasion ability of ovarian cancer cells after PVT1 silencing. The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test. The interaction between PVT1 and microRNA (miR)-551 was analyzed by dual-luciferase reporter assay. The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test. The expression of Wnt signaling pathway-related proteins was determined by Western blot after PVT1 silencing. The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS: The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P<0.05). The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest. PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells. After PVT1 silencing, miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells. The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing (P<0.05). Compared with negative control group, the tumor volume and weight in PVT1-siRNA group were significantly decreased (P<0.05).CONCLUSION: PVT1 plays an important role in the development of ovarian cancer. PVT1 regulates the invasion and migration abilities of ovarian cancer cells through Wnt signaling pathway.     

lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

AIM:To investigate whether long non-coding RNA MALAT1 (lncRNA-MALAT1) targets and down-regulates microRNA-570-3p (miR-570-3p) expression to further promote the proliferation of gastric cancer cells. METHODS:Gastric cancer cell line SGC7901 was cultured in vitro and divided into 3 groups:blank control, si-MALAT1 and si-MALAT1 NC. The si-MALAT1 and si-MALAT1 NC groups were transfected with MALAT1 siRNA and its negative control, respectively. The cell proliferation was evaluated by MTS assay. The expression of miR-570-3p was detected at different time points in the pure SGC7901 gastric cancer cell line, and the expression of lncRNA-MALAT1 and miR-570-3p in different groups was detected by RT-qPCR. The potential complementary binding sites of lncRNA-MALAT1 and miR-570-3p were predicted by RegRNA. The MALAT1 gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2. Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1 or MALAT1-Mut were successfully constructed. miR-570-3p mimic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the 293T cells with the luciferase repor-ters containing MALAT1 or MALAT1-Mut. Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1 and miR-570-3p. RESULTS:Compared with blank control group and si-MALAT1 NC group, the A₄₉₀ value in si-MALAT1 group was significantly decreased (P<0.01). The expression of miR-570-3p presented an obvious declining trend over time. The expression of lncRNA-MALAT1 in si-MALAT1 group was remarkably decreased, whereas the expression of miR-570-3p was obviously increased. The dual-luciferase reporter assay indicated that the MALAT1 reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control (P<0.01), and the luciferase activity of MALAT1 reporter was obviously up-regulated in miR-570-3p inhibitor group compared with miR-570-3p mimic group (P<0.01). However, miR-570-3p mi-mic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control showed no effect on the luciferase activity of MALAT1-Mut reporter. CONCLUSION:lncRNA-MALAT1 targets and down-regulates miR-570-3p expression to further promote the proliferation of gastric cancer cells.     

Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

ARHI gene inhibits cell growth,induces G2/M phase arrest and apoptosis of acute myeloid leukemia cell line U937

AIM: To investigate the expression of aplasia rashomolog member I (ARHI) gene in acute myeloid leukemia cells (AML) and to study the effects of ARHI on the growth of AML cell line U937.METHODS: The mRNA expression of ARHI in AML cells, 293FT cells, AML primary cells and healthy volunteer blood cells were detected by RT-PCR. After transfection with the MSCV-IRES-GFP-ARHI plasmid to the U937 cells, the growth curve was analyzed by MTT assay. U937 cells were re-suspended by fresh medium and cultured for 24 h, then the cell cycle distribution and apoptotic rate were determined. RESULTS: The mRNA of ARHI was positively detectable in 293FT cells and healthy volunteer blood cells instead of AML cell line and AML primary cells. The growth curve showed that cell viability in U937 cells with high expression of ARHI (U937-ARHI) was lower than that in the control cells (U937-GFP) on 6th~8th day. The ratio of G₂/M phase and apoptotic rate in the U937-ARHI cells were increased compare with control group(P<0.05). CONCLUSION: The mRNA level of ARHI is low in AML cells. High expression of ARHI gene in U937 cells inhibits cell growth, arrests the cells at G₂/M phase and induces apoptosis.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.