NC Inhibitor Group Research Articles

Construction of Dual-luciferase reporter gene vector of 3’UTR of the COL4A3 gene and validation of its targeting relationship with miR-299

Objective To construct a dual-luciferase reporter gene vector and validate the targeting relationship between miR-299 and the COL4A3 gene, laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene. Methods This study was made from March, 2018 to December, 2018. Firstly, the potential binding sites between miR-299 and COL4A3-3'UTR were predicted using bioinformatics. Then, the wild and mutant COL4A3-3’UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors. The vectors were validated by enzyme digestion and gene sequencing. Finally, the cells were resuscitated, amplified, transfected and divided into 4 groups: COL4A3-WT+miR-299/NC group, COL4A3-WT+miR-299-inhibitor/NC-inhibitor group, COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group. Each group contains 3 holes, respectively. Luciferase activity in each group was determined using a dual-luciferase assay kit. The statistical analysis was conducted and differences between groups were compared by t test. Probabilities lower than 5%(P<0.05) were considered statistically significant. Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully. Luciferase assay demonstrated that in wild COL4A3 gene, luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38%) compared with the NC group (The average R/F value was 100.00%), with a statistical significant difference (P 0.05. Conclusion The dual-luciferase reporter gene vector of the 3’UTR of the COL4A3 gene is constructed successfully. In addition, dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3’UTR of the COL4A3 gene. Key words: Collagen type IV alpha3 chain; MicroRNA; 3’Untranslated region; Dual-luciferase reporter genevector; Chondrogenic differentiation

Down-regulation of microRNA-98 promoted apoptosis of TNF-α stimulated human fibroblast-like synoviocytes via up-regulating IL-10

In this study, we constructed a tumor necrosis factor α (TNF-α)-induced synovial cell inflammatory model using human synoviocytes (HS) cell line to explore the function of miR-98 in rheumatoid arthritis (RA). miR-98 mimics or miR-98 inhibitor were transfected into HS cells to up-regulate or down-regulate the expression of miR-98. The proliferation and apoptosis of HS cells were determined using CCK8 assay and flow cytometry, respectively. TargetScan website was utilized to predict the targets of miR-98. Luciferase assay was carried out to verify that IL-10 is a target of miR-98. Western blot was performed to analyze the expression of IL-10, apoptosis-related and NF-κB signaling pathway-related proteins. Our results demonstrated that the expression of miR-98 was up-regulated in HS cells stimulated by TNF-α. Down-regulation of miR-98 by inhibitor in TNF-α-stimulated HS cells dramatically inhibited cell proliferation and promoted cell apoptosis compared with the miR-98 inhibitor NC group. The protein expression of Bcl-2 was declined while the levels of Bax and Bim were increased by miR-98 inhibitor in TNF-α-stimulated HS cells. IL-10 was predicted and verified as a target of miR-98. qRT-PCR and western blot results revealed that the level of IL-10 was negatively regulated by miR-98. Finally, we identified that down-regulation of miR-98 reduced the expression level of p-p65 and p-IκBα in TNF-α-stimulated HS cells. In summary, our present study demonstrated that down-regulation of miR-98 inhibited the proliferation and promoted the apoptosis of TNF-α-stimulated HS partly by targeting IL-10 and regulating NF-κB signaling pathway, insinuating miR-98 as a candidate biomarker in RA.

Let-7a-5p inhibits BMSCs osteogenesis in postmenopausal osteoporosis mice

PurposeThe aim of this study was to investigate the mechanism of let-7a-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis (PMOP) mice. MethodsA mouse model of PMOP was established and osteoporosis model was identified by micro-CT scan. BMSCs in the sham group and PMOP group were cultured and osteogenic differentiation was induced. The expression of let-7a-5p in BMSCs was detected by qRT-PCR, and BMSCs was induced by osteogenic differentiation in sham and PMOP group. The BMSCs treated by let-7a-5p mimics, let-7a-5p inhibitor and negative control were named as let-7a-5p mimics group, mimics NC group, let-7a-5p inhibitor group and inhibitor NC group, respectively. ALP staining and alizarin red staining were used to detect osteogenic differentiation ability, qRT-PCR and western blot were used to detect the expression of Runt-related transcription factor 2 (Runx2) and Osterix. The targeting relationship between let-7a-5p and TGFBR1 were verificated by target scan and luciferase reporter gene assay. ResultsThe PMOP mouse model was successfully established. The expression of let-7a-5p in BMSCs of PMOP group was significantly higher than that in the sham group (P < 0.05). Let-7a-5p reduced the expression of ALP and the formation of calcified nodules, while also inhibited the expression of Runx2 and Osterix. TGFBR1 is the target gene of let-7a-5p. ConclusionLet-7a-5p might inhibit the osteogenic differentiation of BMSCs in PMOP mice by regulating TGFBR1.

Role of miR-137 in Notch1 mediated autophagy in proliferation and migration of hepatocellular carcinoma cells

Objective To explore the role of miR-137 in the proliferation and migration of hepatocellular carcinoma (HCC) cells by regulating Notch1 and mediating autophagy. Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics, miR-137 inhibitor and Notch1 interfe-ring RNA (siRNA), and divided into normal control group (NC group), miR-137 mimics group, miR-137 inhibitor group, Notch1 siRNA group.The expression levels of miR-137 and Notch1 mRNA after the transfection were detected by RT-PCR in SMMC7721 cells. Transwell experiments were performed to analyze the effect of miR-137 and Notch1 on the migration and invasion of SMMC7721 cells. The expression levels of β-catenin and vimentin in SMMC7721 cells were detected by immunohistochemistry. The number of autophagosomes was detected by double labeled adenovirus. Western blot was utilized to detect the expression of Notch1, E-Cadherin, N-Cadherin, vimentin, P62, and LC3. Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group (5.71±0.45) was significantly higher than that in miR-137 mimics group (0.21±0.06) with statistical significance (P<0.05). The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group (66.00±4.55) and Notch1 siRNA group (88.00±6.78) than that in the miR-137 inhibitor group (515.00±35.12) (P<0.05). The expression levels of β-catenin in miR-137 mimics group and Notch1 siRNA group were significantly increased and the expression level of vimentin was decreased (P<0.05). The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR-137 mimics group (5.50±3.70) was significantly fewer than that in miR-137 inhibitor group (32.75±4.11), and the difference was statistically significant (P<0.05). The expression levels of Notch1, N-cadherin, vimentin, and LC3 protein in miR-137 mimics group were much lower than that in miR-137 inhibitor group and NC group, and the expression levels of E-Cadherin and P62 protein were greatly increased. The expression level of Notch1, N-cadherin, and LC3 protein in Notch1 siRNA group were significantly lower than that in NC group, and the expression levels of E-cadherin and P62 protein were much higher than that in NC group. Conclusion MiR-137 can inhibit the proliferation, migration and invasion of HCC cells by inhibiting the expression of Notch1 and autophagy, which may become a new target for the treatment of HCC. Key words: Hepatocellular carcinoma; MiR-137; Notch1; Invasion and metastasis; Autophagy

Role of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells

To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells. Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients' lung adenocarcinoma and adjacent tissue and lymph nodes. Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability. Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene. Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN. The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6±3.1 and 7.8±2.2, respectively. The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were (23.2±15.3)%, (75.4±20.2)% and (60.4±25.1)%, respectively. The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were (43.2±2.2)%, (21.3±4.2)%, (24.3±5.3)%, and (35.2±5.1)%, and that at 48 h were (75.2±4.5)%, (52.6±5.2)%, (39.4±4.2)%, and (51.5±4.3)%, respectively. Dual luciferase reporter gene assay showed that the value of the luciferase in the miR-155 mimics group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively. The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR-155 mimics control group, miR-155 mimics inhibitor group, and miR-155 inhibitor control group were 0.5±0.3, 1.0±0.1, 2.2±0.2 and 1.2±0.1, respectively. The Western blot assay detected that the relative expression of PTEN protein levels in the miR-155 mimics group, miR-155 mimics control group, miR-155 inhibitor group and miR-155 inhibitor control group were 0.4±0.1, 1.0±0.3, 2.8±0.2 and 1.4±0.1, respectively. The differences in PTEN mRNA and protein expressions of the four groups were statistically significant (P<0.05 for all). miR-155 may promote the invasion and metastasis of lung adenocarcinoma through reducing the target PTEN gene expression.

BASF with new class of enzymes for production of chiral intermediates

The aim of this study was to investigate the mechanism of let-7a-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis (PMOP) mice.A mouse model of PMOP was established and osteoporosis model was identified by micro-CT scan. BMSCs in the sham group and PMOP group were cultured and osteogenic differentiation was induced. The expression of let-7a-5p in BMSCs was detected by qRT-PCR, and BMSCs was induced by osteogenic differentiation in sham and PMOP group. The BMSCs treated by let-7a-5p mimics, let-7a-5p inhibitor and negative control were named as let-7a-5p mimics group, mimics NC group, let-7a-5p inhibitor group and inhibitor NC group, respectively. ALP staining and alizarin red staining were used to detect osteogenic differentiation ability, qRT-PCR and western blot were used to detect the expression of Runt-related transcription factor 2 (Runx2) and Osterix. The targeting relationship between let-7a-5p and TGFBR1 were verificated by target scan and luciferase reporter gene assay.The PMOP mouse model was successfully established. The expression of let-7a-5p in BMSCs of PMOP group was significantly higher than that in the sham group (P < 0.05). Let-7a-5p reduced the expression of ALP and the formation of calcified nodules, while also inhibited the expression of Runx2 and Osterix. TGFBR1 is the target gene of let-7a-5p.Let-7a-5p might inhibit the osteogenic differentiation of BMSCs in PMOP mice by regulating TGFBR1.