Hippo pathway regulates somatic development and cell proliferation of silkworm.

Hippo signaling pathway (signaling pathway Hippo, hereinafter referred to as the Hippo pathway) was the earliest found in Drosophila (Schneck [1]), which can regulate the development of tissues and organs, even some components of the pathway were identified as tumor suppressor [2]. The pathway was more concerned in fruit flies and mice (Schneck [1]), but little attention has been given in silkworm, an important economic and lepidopteran model insect. In this manuscript, we identified major Hippo pathway related genes (Hippo, Salvador, Warts, Mats, Yorkie) in silkworm and named BmHpo, BmSav, BmWts, BmMats, BmYki. The domain organization of these genes was highly conserved in silkworm and other organisms suggesting that they could use similar protein-protein interactions to construct the Hippo kinase cascades. The expression profiles of these genes in silkworm during embryonic, larval, wandering, pupal and adult stages were analyzed, 14 tissues/organs of the day 3, 5th instar larvae (L5D3) as well. Experimental results showed that the expression of Hippo pathway had some influence on the development of silkworm. In order to find out the mechanism of Hippo pathway affecting silkworm development, BmHpo and BmYki were up-regulated and de-regulated in the cell line of Bombyx mori-BmN-SWU1(NS), and the changes of cell proliferation activity and cell cycle were detected. The distribution of BmYki was detected by immunofluorescence technique. This study provides insights into the genes of Hippo pathway which have a certain effect on somatic development and cell proliferation in silkworm.

HP-CagA+ Regulates the Expression of CDK4/CyclinD1 via reg3 to Change Cell Cycle and Promote Cell Proliferation.

Previous studies have shown that regeneration gene 3 (reg3) is significantly expressed in gastric mucosa tissues with Helicobacter pylori (HP) cytotoxin-associated gene A (CagA)-positive (HP-CagA+). CagA-positive HP increases the risk of gastric cancer. The purpose of this study was to investigate the correlation between reg3 and HP-CagA+ and explore the effects of reg3 on the proliferation of gastric cancer cells and the development of tissues and organs. We analyzed the expression of reg3 in human tissues and organs. The results showed that reg3 expression in gastric tissues was significantly higher than that in other tissues and organs. In addition, reg3 influenced the prognosis of gastric, lung, and ovarian cancers. Immunohistochemical analysis indicated that the expression of reg3 and CagA in cancerous tissues was higher than that in adjacent tissues. HP-CagA+ infection of gastric cancer cells promotes reg3 expression, suggesting that reg3 may be a target gene of CagA in gastric cancer, which together affects the formation and development of gastric cancer. reg3 and CagA promote cell proliferation, and then affect the development of mouse tissues and organs by regulating G1/S phase transition of the cell cycle via the formation of the cell cycle-dependent complex CDK4/CyclinD1. This is the first study that shows the influence of CagA on the cell cycle and induction of cell proliferation by promoting reg3 expression.

Ara-c induces cell cycle G1/S arrest by inducing upregulation of the INK4 family gene or directly inhibiting the formation of the cell cycle-dependent complex CDK4/cyclin D1.

Cytosine arabinoside (Ara-c) is a pyrimidine anti-metabolite that is capable of interfering with cellular proliferation by inhibiting DNA synthesis. Each inhibitor of cyclin-dependent kinase 4 (INK4) family member has the ability to bind to cyclin-dependent kinase 4 (CDK4) and inhibit the formation of the cell cycle-dependent CDK4/cyclin D1 complex, subsequently leading to cell cycle arrest in the G1/S phase. In this study, the expression of INK4 family genes in kidney cancer and the impact of these genes on patient prognosis were examined. Additionally, the effects of INK4 family genes and Ara-c on cell proliferation and tumor formation and development were examined. Finally, a potential association between Ara-c-induced cell cycle arrest and INK4-associated gene expression was evaluated. An upregulation of INK4 family genes was found to be positively correlated with the prognosis of patients with kidney cancer. Both the INK4 family genes and Ara-c were shown to induce cell cycle arrest and inhibit tumor formation and development. Moreover, Ara-c-induced cell cycle arrest was found to be associated with an Ara-c-induced upregulation of INK4 family gene expression, which ultimately inhibited the formation of the CDK4/cyclin D1 complex. These findings suggested that an upregulation of INK4 family genes has a positive effect on kidney cancer prognosis and can inhibit the formation and development of tumors. Moreover, Ara-c was shown to promote the upregulation of INK4 family genes, at the same time, Ara-c could directly regulate the cell cycle-dependent genes CDK4 and cyclin D1 (CCND1), independent of the INK4 family genes.

Effects of P27/Bmdacapo, in the CIP/KIP family, on cell proliferation, growth and development in the silkworm (Bombyx mori).

We investigated changes in expression of the CIP/KIP family-related genes and the cycle-dependent factors Pcna, Cdk4 and Cdk2 during the growth and development of mice, Drosophila and silkworms. When the organism was in a period of rapid development, the related genes of the CIP/KIP family had low expression level and the cell cycle-dependent genes were highly expressed. In mammals, the CIP/KIP family includes three genes, p21, p27/Dacapo and p57. However, only one gene, P27/Dacapo, exists in the CIP/KIP family in silkworm and the orthologous gene in the silkworm is named Bmdacapo. Down-regulation of Bmdacapo in silkworm embryos caused overdevelopment of the embryos and indicated that Bmdacapo can inhibit silkworm growth and development. Up-regulation of Bmdacapo in silkworm cells inhibited cell proliferation, whereas down-regulation of Bmdacapo promoted cell proliferation. In order to explore the mechanism of Bmdacapo regulated silkworm development and cell proliferation, the effect of Bmdacapo on cell cycle changes was examined. The results demonstrate that Bmdacapo was able to induce G1/S phase arrest in the cell cycle. In silkworm cells, Bmdacapo inhibits the expression of Pcna, CDK4 and CDK2, which affects the cell cycle and ultimately inhibits cell proliferation. This regulatory mechanism is particularly different from mammals.

p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation.

p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan-Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.