LncRNA MCM3AP-AS1 serves as a competing endogenous RNA of miR-218 to upregulate GLUT1 in papillary thyroid carcinoma

ABSTRACT Objective: MCM3AP-AS1 has been characterized as an oncogenic long non-coding RNA (lncRNA) in several cancers including papillary thyroid cancer (PTC), but its role in PTC has not been fully elucidated. Considering the critical role of lncRNAs in cancer biology, further functional analysis of MCM3AP-AS1 in PTC may provide novel insights into PTC management. Subjects and methods: Paired tumor and non-tumor tissues were collected from 63 papillary thyroid carcinoma (PTC) patients. Expression levels of MCM3AP-AS1 , miR-218 and GLUT1 in tissue samples were analyzed by qRT-PCR. Cell transfection was performed to explore the interactions among MCM3AP-AS1 , miR-218 and GLUT1 . Cell proliferation assay was performed to evaluate the effects of MCM3AP-AS1 and miR-218 on cell proliferation. Results: MCM3AP-AS1 accumulated to high levels in PTC tissues and was affected by clinical stage. MCM3AP-AS1 showed a positive correlation with GLUT1 across PTC tissues. RNA interaction prediction showed that MCM3AP-AS1 could bind to miR-218 , which can directly target GLUT1 . MCM3AP-AS1 and miR-218 showed no regulatory role regulating the expression of each other, but overexpression of MCM3AP-AS1 upregulated GLUT1 and enhanced cell proliferation. In contrast, overexpression of miR-218 downregulated GLUT1 and attenuated cell proliferation. In addition, miR-218 suppressed the role of MCM3AP-AS1 in regulating the expression of GLUT1 and cell proliferation. Conclusions: MCM3AP-AS1 may serve as a competing endogenous RNA of miR-218 to upregulate GLUT1 in PTC, thereby promoting cell proliferation. The MCM3AP-AS1/miR-218/GLUT1 pathway characterized in the present study might serve as a potential target to treat PTC.

miR-218-5p Targeting TPX2 Regulates p53 Pathway and Inhibits Malignant Progression of Lung Adenocarcinoma / 中国肺癌杂志

BACKGROUND@#Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its treatment and diagnosis remain a hot research topic. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is highly expressed in a variety of cancer cells and may be associated with the progression of LUAD. This study aimed to investigate the effect of TPX2 on the malignant progression of LUAD cells and the regulatory mechanisms.@*METHODS@#The expression of gene TPX2 in LUAD tissues from The Cancer Genome Atlas (TCGA) database was analyzed by bioinformatics analysis techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TPX2 and miR-218-5p in human lung normal cell lines and human LUAD cell lines. Western blot was used to detect TPX2 protein expression in cell lines and its effect on the expression of key proteins in the p53 signaling pathway. The relationship between TPX2 and miR-218-5p was predicted using bioinformatics and verified by dual luciferase reporter gene assay. Cell counting kit-8 (CCK-8) assay, cell clone formation, cell scratching, Transwell assay, and flow cytometry were used to detect the effects of miR-218-5p and TPX2 on LUAD cell function.@*RESULTS@#TPX2 was significantly overexpressed in LUAD cells, and knockdown of TPX2 inhibited LUAD cell proliferation, migration, and invasion, promoted apoptosis and induced G2/M phase block, and promoted the expression of key proteins in the p53 signaling pathway. miR-218-5p, an upstream regulator of TPX2, could inhibit its expression. Overexpression of miR-218-5p eliminated the malignant development caused by high expression of TPX2, inhibited the malignant processes of LUAD cells such as proliferation and migration as well as promoted the p53 signaling pathway.@*CONCLUSIONS@#miR-218-5p targets and inhibits TPX2 expression and exerts an inhibitory effect on the malignant progression of LUAD cells via p53.

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized in vitro. After experimental screening of unique recombinant miRNAs produced in vivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

MiR-218-5p Promotes Rotenone-induced Apoptosis of Gastric Cancer Cells by Inhibiting TDP1 Expression / 肿瘤防治研究

Objective To investigate the effect of overexpression of miR-218-5p and inhibition of TDP1 expression on rotenone-induced apoptosis of gastric cancer cells, and to elucidate its possible mechanism. Methods The expression levels of miR-218-5p and TDP1 in human normal gastric epithelial cells and four gastric cancer cells were detected by RT-PCR, and their correlation was analyzed. The targeting regulation of miR-218-5p on TDP1 was verified by dual luciferase reporter gene assay. Gastric cancer cell injury model was induced by 1.0 μmol/L rotenone. Cell cycle and apoptotic rate were detected by flow cytometry. TDP1 level in mitochondria and the expression of Bax and Cyt-c protein were detected by Western blot. Results The expression of miR-218-5p was low in gastric cancer cells (P < 0.05), and TDP1 was high (P < 0.01). There was a negative correlation between the expression of miR-218-5p and TDP1 (R2=0.9580, P=0.0212). Compared with the control group, SGC-7901 cells in the injured group developed G1 phase arrest and the apoptotic rate increased (P < 0.01). After transfection of miR-218-5p-mimic, the cell arrest and apoptotic rate further increased (P < 0.01), the expression of Bax and Cyt-c increased (P < 0.01), while the level of TDP1 in mitochondria decreased (P < 0.01). The G1 phase arrest of cells in TDP1 overexpression group was relieved, the apoptotic rate was decreased (P < 0.01), the level of TDP1 in mitochondria was increased (P < 0.01), and Bax and Cyt-c expression were decreased (P < 0.01). Conclusion MiR-218-5p can target TDP1 expression and induce apoptosis of gastric cancer cells. Its mechanism may be related to inhibiting mitochondrial DNA damage repair and function maintenance and activating mitochondrial endogenous apoptosis pathway.

MiR-218-5p Suppresses the Killing Effect of Natural Killer Cell to Lung Adenocarcinoma by Targeting SHMT1

PURPOSE: Lung adenocarcinoma (LA) is one of the major types of lung cancer. MicroRNAs (miRNAs) play an essential role in regulating responses of natural killer (NK) cells to cancer malignancy. However, the mechanism of miR-218-5p involved in the killing effect of NK cells to LA cells remains poorly understood. MATERIALS AND METHODS: The expression of miR-218-5p was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Serine hydroxymethyl transferase 1 (SHMT1) level was detected by qRT-PCR or western blots. Cytokines production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The killing effect of NK cells to LA cells was investigated using lactate dehydrogenase cytotoxicity assay kit. The interaction of miR-218-5p and SHMT1 was probed by luciferase activity assay. Xenograft model was established to investigate the killing effect of NK cells in vivo. RESULTS: miR-218-5p was enhanced and SHMT1 was inhibited in NK cells of LA patients, whereas stimulation of interleukin-2 (IL-2) reversed their abundances. Addition of miR-218-5p reduced IL-2-induced cytokines expression and cytotoxicity in NK-92 against LA cells. Moreover, SHMT1 was negatively regulated by miR-218-5p and attenuated miR-218-5p-mediated effect on cytotoxicity, IFN-γ and TNF-α secretion in IL-2-activated NK cells. In addition, miR-218-5p exhaustion inhibited tumor growth by promoting killing effect of NK cells. CONCLUSION: miR-218-5p suppresses the killing effect of NK cells to LA cells by targeting SHMT1, providing a potential target for LA treatment by ameliorating NK cells function.

Effect of miR-218-2-3P on proliferation and apoptosis of NK/T-cell lymphoma by targeting SIN3A / 上海交通大学学报（医学版）

Objective • To investigate the expression of miR-218-2-3P in NK/T-cell lymphoma, and the effect of miR-218-2-3P on the proliferation, apoptosis and cycle of NK/T-cell lymphoma by targeting SIN3A. Methods • Quantitative real-time PCR (qPCR) was used to detect the expressions of miR-218-2-3P in normal NK cells and NK/T-cell lymphoma cells NK92MI and NKYS. Lipofectamine 3000 was used to transfect the inhibitor containing nonsense sequences (inhibitors NC), miR-218-2-3P inhibitor and the same dose of transfection reagent without any fragment into NK92MI cells, which were divided into three groups. qPCR and Western blotting were used to detect the expression levels of miR-218-2-3P and SIN3A protein in the inhibitor NC group, the miR-218-2-3P inhibitor group and the blank control group, respectively. The cell proliferation activities of the three group were measured by CCK8 method. The apoptosis rates and cell cycles of the three group were determined by flow cytometry. The double luciferase reporter gene assay was performed to detect whether SIN3A was a target gene of miR-218-2-3P. NK92MI cells were transfected with miR-218-2-3P inhibitor+SIN3A small interfering RNA (si-SIN3A) and miR-218-2-3P inhibitor+nonsense sequences small interfering RNA (si-NC), respectively, which were divided into two groups. The cell proliferation activities of the two groups were detected by CCK8 method. Results • Compared with the normal NK cells, the expressions of miR-218-2-3P in NK92MI and NKYS cells significantly increased (both P<0.05). Compared with the blank control group and the inhibitor NC group, the proliferation activity of NK92MI cells in the miR-218-2-3P inhibitor group decreased (both P<0.05), the apoptosis rate increased (both P<0.05) and cell cycle arrest occurred in G0/G1 phase (both P<0.05). The double luciferase reporter gene assay showed that SIN3A was the target gene of miR-218-2- 3P. Compared with the blank control group and the inhibitor NC group, the SIN3A protein expression of the miR-218-2-3P inhibitor group was increased (both P<0.05). The down-regulation of SIN3A expression could restore the proliferation activity of cells weakened by miR-218-2-3P inhibitor (both P<0.05). Conclusion • miR-218-2-3P is highly expressed in the NK92MI and NKYS cell lines. miR-218-2-3P may affect the proliferation, apoptosis and cycle of NK/T-cell lymphoma through targeted regulation of SIN3A.

Effects of miR-218-1-3p on Cell Proliferation, Cycle, and Apoptosis of Non-small Cell Lung Cancer / 中国医科大学学报

Objective To investigate the effect of miR-218-1-3p on the proliferation,cycle,and apoptosis of A549 cells in non-small-cell lung cancer.Methods miR-218-1-3p was transfected into non-small cell lung cancer A549 cells by LipofectamineTM 2000 Reagent,and the expression of miR-218-3p was detected by real-time PC R.Invasion and migration were assayed using the Transwell method.The effect of miR-218-1-3p on the proliferation of A549 cells was assayed by the MTS method.Changes in the cell cycle and apoptosis of A549 cells transfected with miR-218-1-3p was detected by flow cytometry.Changes in indicators related to cell proliferation,cycle,and apoptosis were detected by fluorescence quantitative PCR.Results Compared to the control group,the cell proliferation of A549 cells was significantly inhibited (P ＜ 0.05) and the proportion of cells in the S and G2-M phases was significantly decreased when miR-218-1-3p was up-regulated.In addition,compared with the control group,the early apoptotic rate was significantly increased by up-regulating miR-218-1-3p.We further detected indicators related to cell proliferation,cycle,and apoptosis and found that CYCLIN-D1 and BCL-2 were significantly downregulated.Conclusion miR-218-1-3p may inhibit proliferation,induce cell cycle arrest,and promote cell apoptosis of non-small cell lung cancer A549 cells by regulating CYCLIN-D 1 and BCL-2.

Effects of miR-218 on SNX4 protein on proliferation and invasion of breast cancer cells / 中国免疫学杂志

Objective:To investigate the effect of miR-218 on the proliferation and invasion of breast cancer cells.Methods: The expression of miR-218 in breast cancer tissues and breast cancer cell lines was detected by qPCR.The relationship between the expression of miR-218 and the clinicopathological parameters of breast cancer were analyzed.Double luciferase assay was used to detect the relationship between miR-218 and SNX4.MTT assay and invasion assay were used to detect the proliferation and invasion of breast cancer cells after overexpression of miR-218.MTX assay and invasion assay were used to detect the recovery level of SNX4 on the proliferation and invasion of breast cancer cells.The effect of miR-218 on the tumorigenesis of breast cancer cell lines was examined by tumorigenesis in nude mice.Results: The expression level of miR-218 in breast cancer tissue and MCF-7 cell line was higher.The expression of miR-218 was associated with pathological stage of breast cancer and lymph node metastasis.SNX4 may be the target of miR-218.Overexpression of miR-21 could inhibit the proliferation and invasion of breast cancer cells.Overexpression of SNX4 could reverse the inhibitory effect of miR-218 on breast cancer cells.Overexpression of miR-218 could inhibit the breast cancer cell line in nude mice tumorigenic ability.Conclusion: miR-218 can up-regulate the expression of miR-218 in breast cancer,and miR-218 can regulate the expression of SNX4 in breast cancer cell proliferation and invasion.

Inhibiting effects of gambogenic acid in combination withmiR-218 on cervival cancer HeLa cells and its mechanisms / 中国药理学通报

Aim To study the inhibitory effects of gambogenic acid in combination with miR-218 on cervical cancer HeLa cells and its mechanisms.Methods Eukaryotic expression vector of miR-218(pmi8-218) was transfected into HeLa cells.Transcript levels of miR-218 were quantified by real-time quantitative PCR.HeLa cells were incubated with different concentrations of gambogenic acid alone or in combination with pmiR-218.The cell growth inhibiting ratio of HeLa cells was assessed by MTT assay.Cell apoptosis was measured by fluorescence activated cell sorting.The expression levels of Bcl-2, Bax and E-cadherin were measured by Western blot and qRT-PCR.Results Levels of miR-218 transcript significantly increased in pmiR-218 transfected HeLa cells.Overexpression of miR-218 may enhance the sensitivity of HeLa cells to gambogenic acid.Over expression of miR-218 could enhance the effect of gambogenic acid on inhibition cell proliferation, promoting apoptosis of HeLa cells.pmiR-218 could enhance the regulation of Bax expression and decrease the expression of Bcl-2 in HeLa cells.Conclusions Over expression of miR-218 may enhance the sensitivity of HeLa cells to gambogenic acid;miR-218 can enhance the effect of gambogenic acid on inhibition cell proliferation and promote the apoptosis of HeLa cells, and the mechanism may be related to down-regulation of Bcl-2/Bax expression.

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1 / 中国病理生理杂志

AIM:To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism .METHODS:The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed . pmiR-218 was transfected into HeLa cells .The number of viable HeLa cells was counted by the method of Trypan blue ex-clusion.The inhibitory rate of cell activity was detected by WST-8 assay.The expression of LIM and SH3 protein 1 (LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot.The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay .RESULTS:The lentivirus expression vector pmiR-218 tar-geting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing .Over-expression of miR-218 inhibi-ted the activity of HeLa cells with the inhibitory rates of 15%, 26%and 65%at 24 h, 48 h and 72 h, respectively .The difference between transfection group and blank control /negative control group was statistically significant .The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3’ UTR plasmid.The relative expression of miR-218 was increased after transfection with pmiR-218.Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25%and 75%respectively.Compared with blank control group and negative control group , the difference was statistically significant (P<0.05).CONCLUSION:pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner.miR-218 targets to the 3’UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells .

Roles and regulation mechanism of microRNA-218 in acute lymphocytic leukemia cell CCRF-CEM / 中国免疫学杂志

Objective:To detect the expression of microRNA-218 (miR-218) in human acute lymphocyte leukemia (ALL) T lymphocytes ( CCRF-CEM) ,explore its effects on the biological features of CCRF-CEM cells and the expression of its target gene c-kit, so as to provide new insights for leukemia treatment.Methods: Using the quantitative real-time polymerase chain reaction ( qRT-PCR) ,we detected the expression of miR-218 in the normal peripheral blood T lymphocytes and CCRF-CEM cells.Forty-eight hours after the miR-218 mimic was transfected into the CCRF-CEM cells,the expression of miR-218 in the CCRF-CEM cells was detected by qRT-PCR.The effect of miR-218 on the CCRF-CEM cell viability was detected using MTT.The effect of miR-218 on the proliferation and apoptosis of CCRF-CEM cell was analyzed using flow cytometry.c-kit gene was identified to be a target gene of miR-218 by luciferase reporter enzyme system,and the effect of miR-218 on the expression of KIT protein in cells were determined using Western blot.Results:As shown by qRT-PCR,compared with that in the normal peripheral blood T lymphocytes,the expressions of miR-218 in ALL T lymphocytes cell lines were significantly decreased ( P<0.01 ) .Compared with the control group, the expression of miR-218 increase significantly in CCRF-CEM cells transfected with miR-218 mimic for 48 hours ( P<0.01).MTT showed that the cell viability decreased significantly after the over-expression of miR-218 in the CCRF-CEM cells ( P<0.05 ) .Flow cytometry showed that the S-phase fraction significantly declined after the over-expression of miR-218 ( P<0.01 ) , and meanwhile the apoptosis of cells also significantly increased (P<0.01).Luciferase reporter gene assay showed that,compared with the control group,the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P<0.01).Compared with the control group,the expression of KIT protein in the CCRF-CEM cells transfected with miR-218 mimic for 48 hours significantly decreased ( P<0.01).Conclusion:The expression of miR-218 decreases in ALL T lymphocytes cell lines.MiR-218 can negatively regulate the expression of KIT protein,inhibit the proliferation and increase the apoptosis of CCRF-CEM cells.Treatment based on the enhanced expression of miR-218 may be a promising strategy for leukemia.

miR-218 expression in osteosarcoma tissues and its effect on cell growth in osteosarcoma cells

Objective: To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells. Methods: miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis. Results: The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ

miR-218 expression in osteosarcoma tissues and its effect on cell growth in osteosarcoma cells

OBJECTIVE@#To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells.@*METHODS@#miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis.@*RESULTS@#The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ(2)=5.380, P=0.020), clinical stage (χ(2)=6.692, P=0.010) and distant metastasis (χ(2)=4.180, P=0.041). MiR-218 was obviously over-expressed by exogenous miR-218 plasmids (t=19.42, P<0.001), and miR-218 overexpression significantly reduced cell proliferation (t=9.045, P<0.001) and induced apoptosis (t=12.38, P<0.001) in Saos-2 cells.@*CONCLUSIONS@#The low-expression of miR-218 is correlated with the poor clinicopathological features in osteosarcoma. Moreover, miR-218 overexpression reduces cancer cell proliferation and induces apoptosis in Saos-2 cells, suggesting that miR-218 may play a key role in the progression of human osteosarcoma.

Effect of miR-218 on biological functions of renal cell carcinoma cell line / 肿瘤研究与临床

Objective To explore the effects of miR-218 on biological functions of renal cell carcinoma cell line through regulating the expression levels of miR-218.Methods The pcDNA3.1-miR-218 was transfected into renal carcinoma cell line A498 and 769-P and the expression of miR-218 was detected.The cell activity,invasion,apoptosis and proliferation of transfected renal carcinoma cell line were analyzed in vitro.Results After plasmid transfected A498/769-P renal carcinoma cell line,the expression level of miR-218 (1.99,1.64) was significantly higher than that of the control group (1.00) (t =60.82,10.89,P ＜ 0.000 1) The cell viability (0.90±0.10,0.68±0.06) was lower than that of the control group (1.39±0.14,1.24±0.08) by CCK8 experiment (t =15.02,31.69,P ＜ 0.000 1).The cell invasion was lower than that of control group by Transwell experiment (t =15.78,18.80,P ＜ 0.000 1).The apoptosis ratio was higher by AnnxinV-FITC experiment,the apoptosis ration of control group and transfected group were respectively 0.25 ％,45.77 ％ in A498 cell line and 0.11％,45.57 ％ in 769-P cell line.The proliferation was lower than that of the control group (P ＜ 0.000 1).Conclusion Up-regulation of miR-218 expression can inhibit the growth of renal cell carcinoma cell line in vitro.

Construction and application in renal cell carcinoma 769-P cells of miR-218 expression plasmids / 肿瘤研究与临床

Objective To construct a stable expression plasmids miR-218,to lay the experimental foundation for the expression and mechanism of miR-218 in human renal cell carcinoma.Methods The primers were designed by Has-miR-218 and the miR-218 fragment were amplified by PCR,and then which was connected with the carrier pcDNA3.1 (+) to build a stable expression plasmids.The recombinant plasmids were trasfected into renal cell carcinoma cell line 769-P,and the expression of miR-218 in these cells were detected.Results The plasmids were constructat successfully,which was determind by enzyme digestion and sequencing results.The constructed expression plasmids pcDNA3.1-miR-218 transfection of human renal cell carcinoma cell line 769-P,miR-218 cxpression level of cells was significantly increased after trasfected by recombinant plasmids carrying miR-218 gene (relative expression quantity was 1.64).Conclusion The miR-218 expression plasmids pcDNA3.1-miR-218 were successfully constructed,the plasmids can be applied to the study of miR-218 function and mechanism in renal cell carcinoma.