NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells

AIM:To study the mechanisms of nicotine-induced expression of intercellular adhesion molecule-1(ICAM-1).METHODS:Related luciferase reporter gene plasmids were constructed with molecular cloning techniques;above plasmids and intracontrol plasmid pSV-β-gal were co-transfected into human umbilical vein endothelial cells(HUVECs) with eukaryotic gene transfection techniques; the relative luciferase activities were detected in the transfected HUVECs.RESULTS:Series of luciferase reporter gene containing different sequences of human ICAM-1 promotor and site-directed mutants of NF-κB and Sp-1 in promotor were successfully constructed; Nicotine could increase the expression of luciferase reporter gene plasmid containing-579 bp(pGL3E-579/+36),-230 bp(pGL3E-230/+36) and mutated Sp-1 version(pGL3E-Sp-1-MU)(P＜0.05 vs control) of ICAM-1 promotor in the transfected HUVECs, whereas deletion derivative (pGL3E-134/+36) and mutation (pGL3E- NF-κB -MU) of downstream NF-κB site of ICAM-1 promotor prevent nicotine-induced increase in expression of luciferase reporter gene plasmid.CONCLUSION:NF-κB site of promotor mediates nicotine-induced ICAM-1 expression in human umbilical vein endothelial cells.     

MicroRNA-98 suppresses viability and invasion ability of colorectal can-cer cells by targeting EZH2

AIM: To study the target relationship between microRNA-98 (miR-98) and enhancer of Zeste homolog 2 (EZH2), and the effect of miR-98 on the viability and invasion ability of colorectal cancer cells.METHODS: The target relationship between EZH2 and miR-98 was predicted by TargetScan software and confirmed by dual-luciferase reporter assay. The miR-98 mimic and miR-98 inhibitor were transfected into human colorectal cancer SW480 cells and SW620 cells. The protein expression level of EZH2 was determined by Western blot. The cell viability was measured by MTT assay, and the invasion ability was detected by Transwell assay. EZH2 over-expression vector was transfected into the colorectal cancer cells, and the cell viability and invasion ability were measured.RESULTS: miR-98 targeted EZH2 and down-regulated EZH2 protein expression in the SW480 cells and SW620 cells. miR-98 over-expression significantly decreased, while miR-98 knockdown dramatically increased the viability and invasion ability of SW480 cells and SW620 cells. Additionally, EZH2 over-expression enhanced the viability and invasion ability of SW480 cells and SW620 cells.CONCLUSION: miR-98 inhibits the viability and invasion ability of SW480 cells and SW620 cells by targeting EZH2, which may provide new therapeutic target and method for colorectal cancer treatment.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

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.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

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.     

MicroRNA-3666 regulates expression of phosphatase and tensin homologue deleted on chromosome ten in leukemic cells

AIM: To explore the regulatory effect of microRNA-3666 (miR-3666) on the expression of its target gene phosphatase and tensin homologue deleted on chromosome ten (PTEN) in leukemic cells. METHODS: miR-3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR. miR-3666 targeting PTEN 3-untranslated region (3UTR) was predicted by TargetScan software. 3UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK2. The reporter activity was evaluated by the Dual-Luciferase Reporter Assay System after the luciferase promoter vector and miRNA were co-transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor (anti-miR-3666) or a synthetic control miRNA (anti-miR-C). The expression of PTEN protein in the above transfected K562 cells was determined by Western blotting. RESULTS: miR-3666 was up-regulated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells. The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666. Inhibition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K562 cells. CONCLUSION: miR-3666 is over-expressed in leukemic cells. The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN.     

Effects of rs5418 site variation in SLC2A4 promoter region on gene expression

AIM: To investigate the effect of G/A mutation in rs5418 site of solute carrier family 2,facilitated glucose transporter,member 4 protein(SLC2A4) promoter region on gene expression. METHODS: The core promoter region of SLC2A4 gene was amplified by PCR. Mutant and wild-type recombinant expression vectors containing promoter of SLC2A4 gene were constructed by recombinant gene technique and the strategy of site-directed mutagenesis. Recombinant vectors were transfected into HEK293T cells by lipofectamine and the expression activity of the reporter gene in the recombinant expression vectors with different alleles was detected by a dual-luciferase reporter assay system. RESULTS: The 716-bp SLC2A4 promoter was amplified and the recombinant expression vectors pGL3-SLC2A4-prom(A) and pGL3-SLC2A4-prom(G) were successfully constructed. The luciferase reporter vector containing SLC2A4 promoter with rs5418-A alleles produced significantly higher relative luciferase activity (19.49±4.41) than that with rs5418-G allele (13.04±4.45; P<0.05). CONCLUSION: The G→A variation of rs5418 site in SLC2A4 promoter region increases the expression of SLC2A4 gene,thereby affecting the gene function.     

Cholesterol-sensitive action sites in promoter of prohibitin gene

AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200　bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer.     

Metastasis-related function of osteopontin between two different colorectal cancer cell lines

AIM:To analyze the metastasis-related function of osteopontin(OPN) in colorectal cancer cell lines. METHODS:The sense- and antisense-osteopontin eukaryotic expression plasmids were transfected into Colo205 and SW480 cell lines. The metastatic function was detected by flow cytometry, immunohistochemistry, homogeneous and heterogeneous adhesion in different cell lines. RESULTS:High expression of OPN reduced E-cadherin expression and enhanced CD44v6 expression in colorectal cancer cells. Homogeneous adhesion was weakened, but heterogeneous adhesion was enhanced among these cells. CD44v6 expression was intensified to accelerate colorectal cancer cell adhering with ECM and invading into blood vessels and liver. CONCLUSION:OPN is one of potential and important factors in the process of colorectal cancer liver metastasis.     

miR-335 regulates proliferation of human osteosarcoma MG-63 cells by targeting Sp1

AIM：To investigate the regulatory role of miR-335 on the proliferation of human osteosarcoma cell line MG-63. METHODS：Luciferase reporter gene assay was used to detect the specific binding ability of miR-335 to Sp1 3’-untranslated region (UTR). Real-time PCR and Western blotting were used to evaluate the expression of Sp1 mRNA and protein, respectively. MTT assay was used to analyze the proliferation of MG-63 cells. RESULTS：The luciferase reporter gene assay showed that miR-335 targeted Sp1 3’-UTR. Western blotting and real-time PCR showed that miR-335 inhibited the protein expression of Sp1, but had no effect on the mRNA expression of Sp1. Transfection of Sp1 expression plasmid increased the protein and mRNA expression of Sp1. MTT assay showed the viability of MG-63 cells transfected with miR-335 was significantly decreased compared with negative control. Transfection of Sp1 expression plasmid partly reversed the inhibitory effect of miR-335 on the proliferation of MG-63 cells. CONCLUSION: miR-335 inhibits the proliferation of human osteosarcoma MG-63 cells, which may be related to its targeting on Sp1 3’-UTR and the subsequent down-regulation of Sp1 expression.     

Inhibitory effect of anti-miR-196a on invasion and migration of human pancreatic cancer PANC-1 cells

AIM:To investigate the expression of miR-196a in different pancreatic cancer cell lines and to observe the effect of anti-miR-196a on the biological behaviors of human pancreatic cancer PANC-1 cells. METHODS:The expression of miR-196a in the pancreatic cancer cells was examined by real-time quantitative PCR. Anti-miR-196a was chemically synthesized and transfected into PANC-1 cells by Lipofectamine 2000. The cell proliferation was measured by CCK-8 assay. The apoptosis was determined by flow cytometry. Matrigel invasion assay was performed to examine the migration and invasion of the tumor cells. The wild-type and mutant-type NF-κB inhibitor α(NFKBIA) 3'UTR luciferase reporter vectors were constructed. The relative activity of Renilla luciferase was detected to confirm the binding site of miR-196a on NFKBIA mRNA. RESULTS:The expression of miR-196a in human pancreatic cancer cell lines was significantly higher than that in human pancreatic ductal epithelial H6c7 cells. The expression of anti-miR-196a in miR-196a group was down-regulated. After transfected with miR-196a, no change of cell proliferation and apoptosis was observed, but the abilities of invasion and migration were reduced(P<0.01). Compared with negative control, wild-type NFKBIA3'UTR or mutant-type NFKBIA 3'UTR, cotransfection of anti-miR-196a and wild-type NFKBIA 3'UTR significantly increased the relative activity of Renilla luciferase. CONCLUSION:miR-196a is one of the oncomiRs and may be a target microRNA of human pancreatic cancer for gene therapy.     

Influence of human mutant p27 gene on the biological behaviors of colorectal cancer cells

AIM: To observe the influence of human mutant p27 gene (p27mt) on the growth and so as to investigate the function and mechanism of p27mt in gene therapy for colorectal cancer.METHODS: Colorectal cancer cell line SW480 was infected with recombinant replication defective adenovirus Ad-p27mt,and expression of p27mt protein was detected by Western blotting.The inhibitory effect of p27mt on SW480 and cell cycle were determined by flow cytometry,and DNA fragment was analyzed to identify the occurrence of apoptosis.RESULTS: After transfected with Ad-p27mt,p27 protein was highly expressed in SW480 cells.77.96% colorectal cancer cells were blocked in phase G₀/G₁,while in Ad-LacZ group and blank control group,27.57% and 25.29% cells were blocked in the same phase,respectively.Growth curve showed Ad-p27mt had an obviously inhibitory effect on the growth of SW480 cells.DNA fragment assay demonstrated that p27mt was able to induce the apoptosis of colorectal cancer cells.CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle,and most cells are blocked in phase G₀/G₁.This blockage is related with the growth inhibition and apoptosis induced by p27mt.     

miR-221 changes resistance of lung cancer cells to gefitinib by regulating PTEN

AIM: To explore the effect of microRNA-221 (miR-221) on resistance of lung cancer cells to gefitinib, and to investigate its related mechanism. METHODS: RT-qPCR was used to detect the levels of miR-221 expression between gefitinib-sensitive cell line PC9 and gefitinib-resistant cell line PC9/GR. The PC9/GR cells were transfected with miR-221 inhibitor by Lipofectamine 2000. The drug sensitivity of these cells to gefitinib was determined by CCK-8 assay. The protein expression level of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was determined by Western blot. The 3'-UTR of PTEN was cloned into luciferase reporter vector and its luciferase activity was detected to verify whether miR-221 targets PTEN. RESULTS: The expression level of miR-221 in the PC9/GR cells was significantly higher than that in the PC9 cells (P<0.05). The protein expression level of PTEN in the PC9/GR cells was lower than that in the PC9 cells (P<0.05). The IC₅₀ of gefitinib was significantly reduced in the PC9/GR cells after transfection with miR-221 inhibitor (P<0.05). The protein expression level of PTEN in the cells transfected with miR-221 inhibitor was increased as compared with control group and blank group (P<0.05). Inhibition of miR-221 expression enhanced the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: miR-221 enhances the resistance of lung cancer cells to gefitinib by down-regulating the protein expression of PTEN.