Background:Glioblastoma remains a highly invasive primary brain malignancy with an undesirable prognosis.Growing evidence has shed light on the importance of microRNAs(miRs),as small non-coding RNAs,in tumor developme...Background:Glioblastoma remains a highly invasive primary brain malignancy with an undesirable prognosis.Growing evidence has shed light on the importance of microRNAs(miRs),as small non-coding RNAs,in tumor development and progression.The present study leverages the in-silico and in-vitro techniques to investigate the significance of hsa-miR-181a-5p and the underlying hsa-miR-181a-5p-meidated signaling pathway in glioblastoma development.Methods:Bioinformatic studies were performed on GSE158284,GSE108474(REMBRANDT study),TCGA-GTEx,CCLE,GeneMANIA,Reactome,WikiPathways,KEGG,miRDB,and microT-CDS to identify the significance of hsa-miR-181a-5p and its underlying target.Afterward,the U373 cell line was selected and transfected with hsa-miR-181a-5p mimics,and the cell viability,clonogenicity,migration,mRNA expression,apoptosis,and cell cycle were studied using the MTT assay,colony formation test,migration assay,qRT-PCR,andflow cytometry respectively.Results:hsa-miR-181a-5p expression is decreased in glioblastoma samples.The in-silico results have shown that hsa-miR-181a-5p could regulate the MAPK pathway by targeting AKT3.The experimental assays have shown that hsa-miR-181a-5p decreases the migration of glioblastoma cells,arrests the cell cycle,and increases the apoptosis rate.Besides downregulating MMP9 and upregulating BAX,hsa-miR-181a-5p downregulates MET,MAP2K1,MAPK1,MAPK3,and AKT3 expression in U373 cells.The in-vitro results were consistent with in-silico results regarding the regulatory effect of hsa-miR-181a-5p on the MAPK pathway,leading to tumor suppression in glioblastoma.Conclusions:hsa-miR-181a-5p inhibits glioblastoma development partially by regulating the signaling factors of the MAPK pathway.展开更多
To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell line...To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTSmiRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSIONOur data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.展开更多
Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during rad...Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during radiation-induced Epithelium-to-mesenchymal transition(EMT).Methods TGF-β1 or IR was used to induce EMT.After mi R-663 a transfection,cell migration and cell morphological changes were detected and the expression levels of mi R-663 a,TGF-β1,and EMT-related factors were quantified.Results Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-β1 or radiation were suppressed by mi R-663 a.Furthermore,both X-ray and carbon ion irradiation resulted in the upregulation of TGF-β1 and downregulation of mi R-663 a,while the silencing of TGF-β1 by mi R-663 a reversed the EMT process after radiation.Conclusion Our findings demonstrate an EMT-suppressing effect by mi R-663 a via TGF-β1 in radiationinduced EMT.展开更多
OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligo...OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.展开更多
Background Lung cancers are classified as squamous cell carcinoma （SQ）,adenocarcinoma （AC） and small cell lung carcinoma （SCLC）.SQ is the major subtype of lung cancer.Currently,there are no targeted therapies fo...Background Lung cancers are classified as squamous cell carcinoma （SQ）,adenocarcinoma （AC） and small cell lung carcinoma （SCLC）.SQ is the major subtype of lung cancer.Currently,there are no targeted therapies for SQ due to lack of understanding its driving oncogenes.In this study,we validated an SQ specific biomarker hsa-miR-205 in Chinese patients with lung cancer and screened its candidate target genes for further functional studies to enrich knowledge in SQ target therapies.Methods Quantitative reverse-transcription PCR （quantitative RT-PCR）was performed on 197 macro-dissected （cancerous cells ＞75％） surgical lung tissues （45 SQ,44 AC,54 SCLC and 54 adjacent normal tissues） to validate the expression profiles of miR-205.Furthermore,the targets of this microRNA were predicted through the gateway miRecords and mapped to lung cancer-associated pathways using the KEGG （Kyoto Encyclopedia of Genes and Genomes） database.Then quantitative RT-PCR was performed on an independent cohort of 44 snap-frozen surgical lung tissues to concurrently assess the expression profiles of miR-205 and its 52 putative targeted genes.Results MicroRNA-205 yielded high diagnostic accuracy in discriminating SQ from AC with an area under the curve （AUC） of 0.985,and discriminating SQ from SCLC with an AUC of 0.978 in formalin-fixed paraffin-embedded （FFPE）surgical lung tissues.Predicted targets of miR-205 were associated with 52 key members of lung cancer signaling pathways.Ten target genes （ACSL1,AXIN2,CACNA2D2,FOXO3,PPP1R3A,PRKAG3,RUNX1,SMAD4,STK3 and TBL1XR1） were significantly down-regulated in SQ and had a strong negative correlation with miR-205,while one target gene （CDH3） was up-regulated in SQ and exhibited a strong positive correlation with miR-205.Conclusions We confirmed the high diagnostic accuracy of miR-205 in discriminating SQ from AC and SCLC in Chinese patients.Moreover,we identified 11 significant target genes of miR-205 which could be used for further functional studies as the basis for the development of SQ targeted therapies.展开更多
文摘Background:Glioblastoma remains a highly invasive primary brain malignancy with an undesirable prognosis.Growing evidence has shed light on the importance of microRNAs(miRs),as small non-coding RNAs,in tumor development and progression.The present study leverages the in-silico and in-vitro techniques to investigate the significance of hsa-miR-181a-5p and the underlying hsa-miR-181a-5p-meidated signaling pathway in glioblastoma development.Methods:Bioinformatic studies were performed on GSE158284,GSE108474(REMBRANDT study),TCGA-GTEx,CCLE,GeneMANIA,Reactome,WikiPathways,KEGG,miRDB,and microT-CDS to identify the significance of hsa-miR-181a-5p and its underlying target.Afterward,the U373 cell line was selected and transfected with hsa-miR-181a-5p mimics,and the cell viability,clonogenicity,migration,mRNA expression,apoptosis,and cell cycle were studied using the MTT assay,colony formation test,migration assay,qRT-PCR,andflow cytometry respectively.Results:hsa-miR-181a-5p expression is decreased in glioblastoma samples.The in-silico results have shown that hsa-miR-181a-5p could regulate the MAPK pathway by targeting AKT3.The experimental assays have shown that hsa-miR-181a-5p decreases the migration of glioblastoma cells,arrests the cell cycle,and increases the apoptosis rate.Besides downregulating MMP9 and upregulating BAX,hsa-miR-181a-5p downregulates MET,MAP2K1,MAPK1,MAPK3,and AKT3 expression in U373 cells.The in-vitro results were consistent with in-silico results regarding the regulatory effect of hsa-miR-181a-5p on the MAPK pathway,leading to tumor suppression in glioblastoma.Conclusions:hsa-miR-181a-5p inhibits glioblastoma development partially by regulating the signaling factors of the MAPK pathway.
基金Supported by the Deutsche Forschungsgemeinschaft,No.(DFG) SFB-TRR77 “Liver cancer”(Project B1)
文摘To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTSmiRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSIONOur data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.
基金funded by the National Natural Science Foundation of China[no.U1932208,YANG Kai]the Science and Technology Research Project of Gansu Province[no.17JR5RA307 and 145RTSA012,WANG Ju Fang]the Science and Technology Research Project of Gansu Province[no.21JR7RA108,DING Nan]
文摘Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during radiation-induced Epithelium-to-mesenchymal transition(EMT).Methods TGF-β1 or IR was used to induce EMT.After mi R-663 a transfection,cell migration and cell morphological changes were detected and the expression levels of mi R-663 a,TGF-β1,and EMT-related factors were quantified.Results Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-β1 or radiation were suppressed by mi R-663 a.Furthermore,both X-ray and carbon ion irradiation resulted in the upregulation of TGF-β1 and downregulation of mi R-663 a,while the silencing of TGF-β1 by mi R-663 a reversed the EMT process after radiation.Conclusion Our findings demonstrate an EMT-suppressing effect by mi R-663 a via TGF-β1 in radiationinduced EMT.
文摘OBJECTIVE To investigate the effects of microRNA-15a （miR- 15a） on the growth of the lymphoma Raji cell line. METHODS A eukaryotic expression vector of precursor mLR-15a （pre-miR-15a） was constructed. Paired oligonucleotides for pre- miR-15a expression were designed and synthesized chemically. Annealed oligonucleotides were inserted into a pGCSIL- GFP vector under a U6 promoter to construct a pre-miR-15a expression plasmid. Oligonucleotides with a scrambled sequence which were used as a negative control were also constructed into the pGCSIL-GFP vector. A recombinant expression vector was identified through PCR and sequencing. A pre-miR-15a expression plasmid and a negative control plasmid were all transferred into Raji cells with lipofectamine 2000. Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was employed to explore expression of miR-15a. The expression levels of the protein were assayed via immunofluorescence. The growth effect of Raji cells was measured by CCK8 assay. Apoptosis was determined using Hoechst dyeing and flow cytometry. The growth-inhibitory effect on Raji cells was measured using a CCK8 assay. RESULTS The results showed that the insertion sequence was correct. The expression level of miR-15a was obviously higher in Raji cells transferred with pre-miR-15a plasmid than in the blank group and in the negative control group. After Raji cells were transferred with the pre-miR-15a expression plasmid for 48 h, Bcl-2 protein expression levels were obviously decreased, which indicated that there was a significant difference as compared with the negative control group and blank group （P 〈 0.05）. The CCK8 assay showed that transfection of pre-miR-15a expression plasmid decreased the cell growth at 24, 48 and 72 h post-transfection. After Raji cells were transferred with pre-miR-15a expression plasmid, apoptotic cells can be seen with Hoechst dyeing, and the cell apoptotic rate was obviously higher than that in blank group and negative control group. CONCLUSION Pre-miR-15a can induce apoptosis and inhibit the growth of Raji cells.
基金This research was supported by the grants from the National Natural Science Foundation of China （No. 81071918） and the Science and Technology Commission of Shanghai Municipality （No. 10411950300 and No. 9410705300）.Acknowledgments： We thank Zhu Zhidong at Shanghai Biot~chnology Corporation for assistance with the statistics.
文摘Background Lung cancers are classified as squamous cell carcinoma （SQ）,adenocarcinoma （AC） and small cell lung carcinoma （SCLC）.SQ is the major subtype of lung cancer.Currently,there are no targeted therapies for SQ due to lack of understanding its driving oncogenes.In this study,we validated an SQ specific biomarker hsa-miR-205 in Chinese patients with lung cancer and screened its candidate target genes for further functional studies to enrich knowledge in SQ target therapies.Methods Quantitative reverse-transcription PCR （quantitative RT-PCR）was performed on 197 macro-dissected （cancerous cells ＞75％） surgical lung tissues （45 SQ,44 AC,54 SCLC and 54 adjacent normal tissues） to validate the expression profiles of miR-205.Furthermore,the targets of this microRNA were predicted through the gateway miRecords and mapped to lung cancer-associated pathways using the KEGG （Kyoto Encyclopedia of Genes and Genomes） database.Then quantitative RT-PCR was performed on an independent cohort of 44 snap-frozen surgical lung tissues to concurrently assess the expression profiles of miR-205 and its 52 putative targeted genes.Results MicroRNA-205 yielded high diagnostic accuracy in discriminating SQ from AC with an area under the curve （AUC） of 0.985,and discriminating SQ from SCLC with an AUC of 0.978 in formalin-fixed paraffin-embedded （FFPE）surgical lung tissues.Predicted targets of miR-205 were associated with 52 key members of lung cancer signaling pathways.Ten target genes （ACSL1,AXIN2,CACNA2D2,FOXO3,PPP1R3A,PRKAG3,RUNX1,SMAD4,STK3 and TBL1XR1） were significantly down-regulated in SQ and had a strong negative correlation with miR-205,while one target gene （CDH3） was up-regulated in SQ and exhibited a strong positive correlation with miR-205.Conclusions We confirmed the high diagnostic accuracy of miR-205 in discriminating SQ from AC and SCLC in Chinese patients.Moreover,we identified 11 significant target genes of miR-205 which could be used for further functional studies as the basis for the development of SQ targeted therapies.
