Preliminary study on miRNA expression spectrum related to chemotherapy resistance in colon cancer

AIM: To screen the chemotherapy resistance-related microRNAs (miRNAs) of colon cancer using gene chip technique, and to explore the mechanism of miRNAs regulating chemotherapy resistance. METHODS: Gene chip technique was used to analyze the expression of miRNAs in colon cancer cell line HCT8 and vincristine-resistant cell line HCT8/v, and screen the miRNAs with significantly different expression. The results were verified by RT-qPCR. The target genes of these miRNAs were predicted, and the Gene Ontology (GO) analysis and the signaling pathway analysis of the predicted genes were carried out. RESULTS: Altogether 342 miRNAs with significantly differential expression were selected, in which 190 were up-regulated, and 152 were down-regulated. The verification results of RT-qPCR showed that the expression of miR-125-5p, miR-181c-5p and miR-153-3 was consistent with the results of chip detection. The expression of miR-130a-3p and miR-149-3p was not consistent with the results of chip detection. The results of GO analysis showed that the main pathway of chemotherapy resistance-related genes was RNA polymerase II regulatory region sequence-specific DNA binding. The chemotherapy resistance-related genes played roles mainly through positive regulation and are mainly located in intracellular membrane-bound organelles. The results of KEGG analysis showed that the pathways associated with the most enriched chemotherapy resistance-related genes were axon guidance pathway, insulin signaling pathway, and phospholipase D signaling pathway.CONCLUSION: miRNAs are closely related to chemotherapy resistance in colon cancer. Through the researches on miRNAs, we can have a deeper understanding of the mechanism of chemotherapy resistance and provide new ideas for reversing chemotherapy resistance in colon cancer.     

Effect of miR-375 on viability,cell cycle and apoptosis of colon cancer HCT116 cells

AIM: To investigate the effect of microRNA-375 (miR-375) on the viability, cell cycle and apoptosis of HCT116 cells.METHODS: The expression of miR-375 in different colorectal cancer cell lines was detected by real-time PCR. The miR-375 mimics was transfected into HCT116 cells by Lipofectamine^TM 2000. The mRNA expression of miR-375 and AEG-1 was detected by real-time PCR. The HCT116 cell viability was detected by MTT assay. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry.RESULTS: Real-time PCR showed that miR-375 expression was the lowest in HCT116 among 4 colorectal cancer cell lines. The expression level of miR-375 significantly increased in miR-375 mimics group compared with that in the negative control group. The high expression level of miR-375 significantly inhibited the mRNA expression of AEG-1. After transfection with miR-375 mimics, the cell viability was inhibited, the apoptotic rate was increased, the proportion of G₁-stage cells was increased, and the proportion of S-stage cells was decreased.CONCLUSION: miR-375 inhibits the viability, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells. miR-375 may act as a tumor suppressor in colorectal cancer by inhibiting AEG-1.     

Effect of miR-7-5p targeting Itch gene on insulin resistance model of HepG2 cells

AIM:To preliminarily explore the relationship between microRNA-7-5p (miR-7-5p) and Itch gene and their relationship with insulin resistance by establishing insulin resistance model of HepG2 cells in vitro for detecting differential expression of miR-7-5p and its predicted target gene Itch in the state of insulin resistance. METHODS:The insulin resistance model of HepG2 cells was induced by suitable concentration of plamitic acid. The possible target genes and the associated signaling pathways of miR-7-5p were predicted based on bioinformatic analysis. The expression levels of miR-7-5p and Itch were detected by RT-qPCR and Western blot in the HepG2 cells with insulin resistance. RESULTS:The HepG2 cell model of insulin resistance was successfully induced by treatment with 0.25 mmol/L palmitic acid for 24 h. Compared with negative control group, the expression level of miR-7-5p detected by RT-qPCR in insulin resistance group was significantly decreased (P<0.01). Bioinformatic analysis showed that a considerable number of target genes of miR-7-5p were enriched in the ubiquitin proteasome system. Among them, E3 ubiquitin ligase Itch gene was the most relevant target gene to insulin resistance. The results of Western blot showed that the protein expression of Itch was up-regulated in the HepG2 cells under insulin resistance (P<0.01). CONCLUSION:miR-7-5p may be involved in the pathophysiological process of insulin resistance, which may directly or indirectly affect the normal transduction of insulin signaling pathway by targeting Itch gene.     

Up-regulation of microRNA-187* inhibits proliferation of human colon cancer cell line HCT116

AIM:To investigate the expression of microRNA-187* (miR-187*) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187* up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS:The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187* level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), the possible target of miR-187*, was also detected. Synthetic miR-187* mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187* and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS:miR-187* was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187* in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187* expression was remarkably increased after transfection with miR-187* mimics, and ectopic expression of miR-187* significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187* mimics group, and proliferating cell nuclear antigen (PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187* mimics group were significantly increased. CONCLUSION: miR-187* is down-regulated in human colon cancer cell lines. Up-regulation of miR-187* not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Effect of miR-24 on chemotherapy sensitivity in lung cancer A549 cells

AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.     

Expression of miRNA-198 and miRNA-296-5P in PB3A-treated colon cancer cell line and its function prediction

AIM:To explore the effect of pinobanksin-3-acetate (PB3A) on microRNA (miRNA) expression profile of human colon cancer cells for providing new methods of treatment of colon cancer and development of targeted drug.METHODS:The method of miRNA expression profiling was used to observe the miRNA differential expression in human colon cancer SW480 cells after treated with PB3A.The expression of miRNA-198 and miRNA-296-5p in the SW480 cells was detected by RT-qPCR.The network databases of miRWalk,MicroT,miRanda and so on were used to predict the target genes regulated by these miRNAs,and pathway significant enrichment analysis was performed.RESULTS:miRNA microarray analysis showed that after treated with propolis flavonoid PB3A for 24 h,267 miRNAs with differential expression twice or more in the SW480 cells were observed.Among them,there were 30 miRNAs with 10-fold or more differential expression,in which 28 were up-regulated and 2 were down-regulated.The results of RT-qPCR showed that the expression levels of miRNA-198 and miRNA-296-5p were consistent with the results of miRNA microarray analysis,and the difference was statistically significant (P<0.05).Bioinformatic analysis revealed that miRNA-198 has 859 target genes,and miRNA-296-5p has 906 target genes.The target genes of miRNA-198 were clustered in pathways in cancer,axon guidance,Wnt signaling pathway,regulation of actin cytoskeleton,insulin signaling pathway and MAPK signaling pathway,while the target genes of miRNA-296-5p were clustered in axon guidance,Wnt signaling pathway,MAPK signaling pathway,endocytosis,melanogenesis,insulin signaling pathway and calcium signaling pathway.CONCLUSION:Propolis flavonoid PB3A affects the expression of miRNA in colon cancer SW480 cells.The abnormal expression of miRNA-198 and miRNA-296-5p may be involved in the inhibitory effect of PB3A on colon cancer.     

Differential expressions of ameloblastoma related genes determined by microarray

AIM: This study aims to screen the differentially expressed genes related to the pathogenesis of ameloblastoma by cDNA microarray. METHODS: The total RNAs were isolated from ameloblastoma and tooth germ, respectively. The RNAs were purified by oligotex. Both the mRNAs from two kinds of tissues were reversely transcribed to cDNA with the incorporation of fluorescent-labelled dUTP to prepare the hybridization probes. The mixed probes were hybridized to cDNA microarray. Tumor- related genes were screened through the analysis of fluroescent intensity. RESULTS: 722 genes exhibited significant changes in expression levels in the ameloblastoma in comparison with tooth germs tissues. 240 genes were overexpressed more than doubled (92 genes were more than 3-fold), and 482 genes were underexpressed to below 0.5 of the control level. CONCLUSION: Microarray technique facilitates large scale and rapid identification of potential target genes of ameloblastoma.     

Role of miR-183 in breast cancer progression

AIM:To investigate the expression of miR-183 in breast cell lines and tissue specimens and its effects on the biological behaviors of breast cancer cells. METHODS:Human breast cell lines and clinical tissue specimens of breast diseases were used in the study. The expression of miR-183 was determined by real-time PCR. The characteristics of cell proliferation, invasion and migration were examined after miR-183 was transfected by lipofection. RESULTS:Altered expression of miR-183 was found in the highly invasive breast cancer cells as compared with weakly invasive breast cells. The expression of miR-183 was also significantly decreased in the breast cancer tissues as compared with that in the benign breast disease tissues. The capacities of breast cancer cell invasion and migration were significantly increased after transfection of miR-183 inhibitor and were decreased after transfection of miR-183 mimic. No significant change of cell proliferation was observed after miR-183 transfection. CONCLUSION:miR-183 may play an important role in breast cancer progression, especially in the cell invasion and migration.     

miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

MicroRNA-497 inhibits viability,invasion and migration of thyroid papillary cancer cells

AIM:To investigate the underlying mechanisms of mircoRNA-497 (miR-497) inhibiting the viability, migration and invasion of papillary thyroid cancer (PTC) cells. METHODS:TargetScan 6.0 was used to predict the potential targets of miR-497. The target gene was confirmed by luciferase reporter assay, RT-qPCR and Western blot. The expression levels of miR-497 and its target gene in the PTC tissues were detected by RT-qPCR. Gene transfection, MTT assay, and cell migration and invasion assays were used to investigate the effects of miR-497 and its target gene on PTC cell viability, migration and invasion. RESULTS:AKT3 was demonstrated to be the direct target gene of miR-497. In addition, AKT3 expression was higher in the PTC tissues than that in normal tissues (P<0.05) and negatively correlated with miR-497 expression (r=-0.573 7, P<0.01). Furthermore, down-regulation of AKT3 also suppressed cell viability, migration and invasion of PTC, which played similar roles of miR-497 over-expression in PTC cells. CONCLUSION:miR-497 inhibits the viability, migration and invasion of PTC cells by directly targeting AKT3.     

Effect of miRNAs on apoptotic leukemia cells induced by demethylcantharidin

AIM: To explore the role of miRNA expression in the process of antitumor drug demethylcantharidin (DMC)-induced leukemia cell apoptosis. METHODS: K562 cells were treated with DMC to induce apoptosis. Microarray assessment was performed to detect the changes of miRNAs. The expression of miRNAs was further detected by RT-PCR and real-time PCR. The miRNA-relative genes were analyzed by gene information softwares,and their expression was determined by ELISA analysis. RESULTS: The results of microarray showed that more than 290 miRNAs were differentially expressed after DMC treatment. The expression of miR-16, miR-34 and miR-125 increased at more than 1.5 folds in DMC treatment group determined by RT-PCR and real-time PCR analysis, while both miR-106 and miR-150 were down-expressed over 60％. Using microRNA TargetScan and miRanda analysis software, we found that the expression of oncogenes ( bcl-2, E2F1, E2F3 ) and tumor suppressor genes ( RB1, p53 ) may be regulated by the above miRNAs. The expression of RB1 and P53 proteins significantly increased, while Bcl-2, E2F1 and E2F3 proteins were obviously down-regulated after DMC treatment.CONCLUSION: DMC induces K562 cell apoptosis by regulating the expression of miRNAs and their relative genes.     

Expression of miR-625-3p in colorectal carcinoma tissues and cells

AIM: To investigate the expression of microRNA-625-3p (miR-625-3p) in colorectal carcinoma (CRC) and its underlying mechanism. METHODS: Quantitative real-time PCR was employed to detect the levels of miR-625-3p expression in different CRC cell lines, CRC tissues and pair-matched adjacent normal tissues. The relationships between the expression levels of miR-625-3p and the patients' clinicopathological parameters were estimated. The effects of miR-625-3p on the apoptosis and the cell mitotic cycle of CRC cells were analyzed with propidium iodide staining and flow cytometry. The effect of miR-625-3p on the apoptosis-related proteins was analyzed by Western blot. RESULTS: The expression level of miR-625-3p in the CRC tissues was higher than that in the pair-matched adjacent normal tissues (P<0.05). The expression of miR-625-3p in the CRC tumor tissues was significantly correlated with the tumor infiltrative depth, TNM stage and distant metastasis (P<0.05). The expression levels of miR-625-3p in CRC SW620 cells were higher than that in SW480 cells. The CRC cell mitotic cycle was significantly inhibited and cell apoptosis was significantly promoted when the expression of miR-625-3p was inhibited (P<0.05). The expression of Bax protein didn't change and the expression of Bcl-2 protein increased after miR-625-3p mimics were transfected into CRC SW620 cells(P<0.05). CONCLUSION: miR-625-3p may be a promising approach for the treatment of CRC by promoting cell proliferation and inhibiting apoptosis.