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Objective: To investigate the effects of miR-191-5p on cell migration, clone formation and proliferation of gastric cancer (GC) cells. Methods: The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 paired GC tissues and their adjacent normal tissues. miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells. The migration, clone formation and proliferation of GC cells were detected by the scratch wound assay, clone formation assay and cell counting kit-8 (CCK-8), respectively. Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor. The binding sites of cyclin-dependent kinase 6 (CDK6) and miR-191-5p were analyzed using TargetScan software, and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression. Western blot (WB) was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins. Results: miR-191-5p decreased in 53 cases (88%) of GC tissues compared to their controls. Furthermore, overexpression of miR-191-5p effectively inhibited the migration, clone formation and proliferation of GC cells ( P <0.05). Dual-fluorescence reporter confirmed that miR-191-5p bound to 3'UTR of CDK6. WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21. Conclusion: Down-regulation of miR-191-5p has a correlation with the progression of GC. Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.

Background: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells., Methods: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991., Results: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells., Conclusions: PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.

Background: FK506, also named tacrolimus, a new macrolide immunosuppressive agent, has been shown to possess anti-proliferation activities in some cancer cells. The aim of this study was to investigate the effect of FK506 on the cell proliferation and migration of lung cancer cell lines and its mechanism., Methods: A549 and H1299 cell lines were cultured in vitro. The effect of FK506 on cell viability and DNA synthesis ability of A549 and H1299 were measured by CCK-8 assay and EDU-labeling assay, respectively. Flow cytometry assay was used to detect the cell cycle. The in vitro migration of lung cancer cells was detected by Boyden chamber assay and wound-healing assay after the treatment of FK506. The expression of p27, RB1, CDK4, CDK6 and MMP9 were detected using Western blot., Results: FK506 inhibited cell growth and induced cell cycle arrest in G0/G1 phase in A549 and H1299 cells in a dose- and time-dependent manner. Compared to the control groups, the migration of A549 and H1299 cells treated with FK506 were decreased obviously. Moreover, FK506 increased the expression of P27 and RB1, and reduced the expression of CDK4, CDK6 and MMP9., Conclusions: FK506 inhibit the cell growth and migration of lung cancer cells in vitro. The inhibitive effects may be associated with the up-regulation of p27 expression and inhibition CDK4, CDK6 and MMP9 expression.
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Objective: To assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro., Methods: QRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors., Results: Compared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21., Conclusion: NPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.

Objective: To construct the eukaryotic expression vector of miR -145 and evaluate the inhibitory effect of miR-145 targeting cyclin-dependent kinase 6 (CDK6) gene on the proliferation of human cervical carcinoma cells., Methods: The gene sequence of miR-145 was synthesized and cloned into pcDNA(TM);6.2-GW to construct recombinant plasmid pcDNA(TM);6.2-GW-miR-145. HeLa cells were divided into miR-145 group (transfected with the recombinant plasmid), NC group (untransfected) and blank group (transfected with a blank plasmid). The transcription levels of miR-145 and CDK6 were detected using real-time quantitative PCR (qRT-PCR), and Western blotting was used to evaluate the CDK6 protein expression. In addition, the inhibitory effect of miR-145 targeting CDK6 on the proliferation of HeLa cells was measured by MTT assay., Results: Sequencing and reverse transcrip0tion PCR demonstrated that we constructed successfully the recombinant plasmid pcDNA(TM);6.2-GW-miR-145, and that the miR-145 expression level in miR-145 group was significantly higher than that in blank group. The qRT-PCR and Western blotting showed that the CDK6 expression level in miR-145 group was significantly lower than that in blank group. Furthermore, miR-145 significantly inhibited the proliferation of HeLa cells (P<0.05)., Conclusion: The eukaryotic expression vector carrying miR-145 was successfully constructed, and miR-145 over-expression in HeLa cells could reduce the expression of CDK6.

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.