miR-1299 suppresses cell proliferation of hepatocellular carcinoma (HCC) by targeting CDK6
Huaqiang ZhuGuangchuan WangXu ZhouXie SongHengjun GaoChaoqun MaHong ChangHongguang LiFangfeng LiuJun LuJinben Ma
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Cyclin-dependent kinase 6
MTT assay
Targeted therapies have been proven as promising in the treatment of breast cancer and have improved survival and quality of life in advanced breast cancer. We previously identified a novel peptide SA12 which showed significant activity in the inhibition of proliferation and induction of apoptosis in SKBr-3 cells.The present study investigated the potential antitumor role of SA12 in breast cancer cell lines MDA-MB-231 and MCF-7 through Cell Counting Kit-8 assay and colony formation assay, and examined the cell cycle distribution using flow cytometry analysis. Furthermore, the expression of cell cycle-related genes cyclin D1, CDK4, and tumor suppressor gene p16 were examined by real-time polymerase chain reaction and Western blot to explore the molecular mechanism.We determined that peptide SA12 suppressed the proliferation of MDA-MB-231 and MCF-7 cell lines through the G0/G1 phase cell cycle arrest. Moreover, the expressions of cell cycle-associated genes cyclin D1 and CDK4 were downregulated and the expression of tumor suppressor gene p16 was upregulated after treatment with SA12. MECP2 was required for the enhanced expression of p16 gene induced by SA12, which further inhibits CDK4/CDK6 activation and arrests the cell cycle progression from G0/G1 to S phase.We concluded that SA-12 inhibits the proliferation of MCF-7 and MDA-MB-231 cells through G0/G1 cell cycle arrest. Cell cycle related genes cyclin D1, CDK4, and p16 participate in the process, and MECP2 is essential for the enhanced expression of p16 gene induced by SA-12.
Cyclin-dependent kinase 6
G1 phase
MCF-7
Cyclin D
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It has been shown that a variety of cell cycle-related proteins play important roles in the process of carcinogenesis including hepatocarcinogenesis. In the present study, we evaluated mRNA and protein expression of G1 phase-related cell cycle molecules in the process of hepatocarcinogenesis, using Long-Evans Cinnamon (LEC) rats, an animal model of hepatocellular carcinoma (HCC). The expression of cyclin D1, cyclin-dependent kinase 4 (Cdk4) and Cdk6 was measured quantitatively by real-time polymerase chain reaction. Cyclin D1 mRNA expression was increased significantly in chronic hepatitis liver compared with normal liver, and then decreased in HCC and the surrounding precancerous liver of LEC rats. Levels of Cdk4 mRNA were increased significantly in HCC compared to precancerous and chronic hepatitis livers. In contrast, mRNA levels of Cdk6 did not change significantly during hepatocarcinogenesis. We also evaluated the protein levels of these G1 phase-related cell cycle molecules by Western blot analyses and confirmed similar results. Total amounts of retinoblastoma protein (pRb) in the liver did not change significantly in the process of hepatocarcinogenesis in LEC rats. However, levels of phosphorylated pRb were increased markedly in the process of hepatocarcinogenesis, and the highest in HCC compared to precancerous, chronic hepatitis and normal livers. These results indicate that cyclin D1 may be involved in the regeneration of hepatocytes rather than hepatocarcinogenesis, while Cdk4 but not Cdk6 may play an important role in the development of HCC.
Cyclin-dependent kinase 6
Retinoblastoma protein
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RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction.Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP.Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.
Knockdown resistance
RNA Silencing
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Fangchinoline (Fan) is a bioactive compound isolated from the Chinese herb Stephania tetrandra S. Moore (Fen Fang Ji). The aim of the present study was to investigate the effect of Fan on the proliferation of SPC-A-1 lung cancer cells, and to define the associated molecular mechanisms. Following treatment with Fan, Cell Counting Kit-8, phase contrast imaging and Giemsa staining assays were used to detect cell viability; flow cytometry was performed to analyze the cell cycle distribution; and reverse transcription-quantitative polymerase chain reaction and western blot assays were used to investigate changes in the expression levels of cell cycle-associated genes and proteins. In the present study, treatment with Fan markedly inhibited the proliferation of SPC-A-1 lung cancer cells and significantly increased the percentage of cells in the G0/G1 phase of the cell cycle in a dose-dependent manner (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), whereas the percentage of cells in the S and G2/M phases were significantly reduced following treatment (P<0.05 for 5 µm; P<0.01 for 10 µm). Mechanistically, Fan significantly reduced the mRNA expression levels of cyclin D1, cyclin-dependent kinase 4 (CDK4) and CDK6 (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), which are key genes in the regulation of the G0/G1 phase of the cell cycle. Furthermore, treatment with Fan also decreased the expression of phosphorylated retinoblastoma (Rb) and E2F transcription factor-1 (E2F-1) proteins (P<0.05 for 5 µm; P<0.01 for 10 µm). In summary, the present study demonstrated that Fan inhibited the proliferation of SPC-A-1 lung cancer cells and induced cell cycle arrest at the G0/G1 phase. These effects may be mediated by the downregulation of cellular CDK4, CDK6 and cyclin D1 levels, thus leading to hypophosphorylation of Rb and subsequent suppression of E2F-1 activity. Therefore, the present results suggest that Fan may be a potential drug candidate for the prevention of lung cancer.
Blocking (statistics)
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<p>Chemoresistance properties of MUC16 and effect of cisplatin on apoptosis of MUC16 knockdown cells. A & B, MUC16 knockdown (H1975-shMUC16 seq1 and 2) cells were highly sensitive to cisplatin (A) and gemcitabine (B). C, The percentage of apoptotic cells was significantly higher in MUC16 knockdown cells (H292-shMUC16) treated with 5μM cisplatin. In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown cells. D, We performed stable knockdown of Muc16 in K1418, the result shows that Muc16 is significantly decreased as compared to scramble cells. E, The p53 target gene p21 was significantly increased in MUC16 knockdown (H292-shMUC16) cells. *P<0.05, **P<0.01, ***P<0.001, and NS non-significant.</p>
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Objective To study the internal mechanism of inhibition of glioma cell cycle from G1 phase into S phase by miR-7.Methods After transfecting plasmid containing miR-7 into U251 glioma cell line,hybridization in situ and real-time PCR were used to detect the gene integration,and the cell cycle was detected by flow cytometry.The protein expression level of EGFR,CyclinD1,CyclinE,CDK2,CDK4,CDK6,P16,P21 and P27 were detected by Western blot.The corrclation among them was analyzed by software SPSS 13.0.Results The real-time PCR showed that miR-7 gene was successfully integrated into U251 glioma cells,and it was mainly localized in the nucleus and cytoplasm from in situ hybridization results.After miR-7 gene transfection,the cell proliferation rate slowed down,and most of them were inhibited in G1 phase in mitotic cells.As meanwhile,the percentage of S phase cells decreased significantly ( P ＜0.05).The expression level of miR-7 was negatively correlated with EGFR,CyclinD1,CDK4,and CDK6 ( P ＜ 0.01 ),and was negatively correlated with CyclinE and CDK2 (0.01 ＜ P ＜0.05),but was positively correlated with P16 ( P ＜0.01 ).There was no correlation with P21 and P27 ( P＞ 0.05 ).Conclusion Through effective silencing the expression of oncogene EGFR,miR-7 may inhibit the activity of protein complex CyclinD1/CDK4/6,which is an important cell cycle G1 phase regulatory.With the P16 synergies,they inhibit glioma cell cycle from G1 phase into S phase.So miR-7 is expected to become a new drug candidate for glioma treatment.
Key words:
Glioma; microRNA; Epidermal growth factor receptor
Cyclin-dependent kinase 6
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Objective To observe the effect of 1α,25( OH)2D3on gastric carcinoma cell proliferation and cell cycle progression and to investigate its possible mechanism. Methods Two gastric cancer cell lines BGC-823 and SGC-7901 were treated with 1α,25( OH)2D3in a dose dependent manner. Cell proliferation was determined by methyl thiazolyl tetrazolium MTT assay; the cell cycle changes were detected by using flow cytometry; the expression of genes of P21,cyclin D1,cyclin E1 and CDK6 related to the cell cycle was analyzed by RT-PCR. Results1α,25( OH)2D3significantly inhibited two gastric cancer cell lines proliferation in a dose dependent fashion,gastric cancer cells treated with 1α,25( OH)2D3caused significantly cell cycle arrest at G1phase; RT-qPCR showed that 1α,25( OH)2D3significantly increased P21 mRNA,and attenuated cyclin D1,cyclin E1,CDK6 mRNA expression in BGC-823,SGC-7901 cells( P 0. 01). Conclusions 1α,25( OH)2D3inhibites BGC-823,SGC-7901cell proliferation and induces cell cycle arresting in G1phase. The mechanism for the anti-proliferative and cell cycle arrest is probably related to overexpression of P21,down-regulated expression of cyclin D1,cyclin E1,CDK6.
Cyclin-dependent kinase 6
Cyclin B1
MTT assay
Cyclin D
Cyclin B
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The present study aimed to investigate the role of long noncoding RNA MACC1-AS1 in cervical squamous cell carcinoma (CSCC). In the present study MACC1-AS1 expression as analyzed using reverse transcription-quantitative PCR. The interactions between MACC1-AS1 and miR-34a was analyzed via overexpression experiments. Cell cycle and proliferation analyses were performed to analyze the roles of MACC1-AS1 in regulating cancer cell cycle progression and cell proliferation. It was observed that MACC1-AS1 was upregulated in CSCC, and its expression levels were elevated with the increase in clinical stage. Bioinformatics analysis revealed that MACC1-AS1 may be a sponge of miR-34a, which can target cyclin-dependent kinase 6 (CDK6). In CSCC cells, MACC1-AS1 overexpression led to upregulation of CDK6, while miR-34a overexpression had the opposite effect and reduced the effects of MACC1-AS1 overexpression in co-transfected cells. Cell cycle and proliferation analyses demonstrated that MACC1-AS1 and CDK6 promoted cell cycle progression and cell proliferation. By contrast, miR-34a had the opposite effect on cell cycle proliferation and cell proliferation, reducing the effects induced by MACC1-AS1 overexpression. Therefore, the lncRNA MACC1-AS1 may serve as a sponge of miR-34a to upregulate CDK6, thereby promoting cell cycle progression and cell proliferation.
Cyclin-dependent kinase 6
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We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.
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