miR-458b-5p regulates ovarian granulosa cells proliferation through Wnt/β‐catenin signaling pathway by targeting catenin beta-1

Wang Wenwen,Teng Jun,Han Xu,Zhang Shen,Zhang Qin,Tang Hui 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.6

Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development. Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear.Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (<i><i>CTNNB1</i></i>), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels.Results: We demonstrated that the expression of miR-458b-5p and <i>CTNNB1</i> showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that <i>CTNNB1</i> is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of <i>CTNNB1</i> in Wnt/β-Catenin pathway and play functional roles in cell proliferation.Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting <i>CTNNB1</i>, suggesting that miR-458b-5p and its target gene <i>CTNNB1</i> may potentially play a role in chicken ovarian follicular development.

성장생물학 : 콜라비가 돼지 지방전구세포와 3T3-L1 cell의 증식과 분화에 미치는 영향

송미연 ( Mi Yeon Song ),이재준 ( Jae Joon Lee ),차선숙 ( Seon Sook Cha ),정정수 ( Chung Soo Chung ) 한국동물자원과학회(구 한국축산학회) 2013 한국축산학회지 Vol.55 No.1

본 연구는 콜라비가 돼지 지방전구세포와 3T3-L1 세포의 증식과 분화에 미치는 영향을 구명하기 위해 수행하였다. 돼지 지방전구세포는 신생자돈의 등지방에서 분리했다. 세포를 접종한 1일 후에 세척했고(day 0), 세포증식에 미치는 영향을 구명하기 위해서 2일 동안(day 0∼day 2) 25ng/ml과 100ng/ml의 콜라비 알코올 추출 물(과피와 과육)를 처리했다. 세포분화를 구명하기 위해서는 DMEM/F- 12 배지에 6일 동안(day 0∼day 6) 배양하고 배양초기 2일 동안(day 0~day 2) 콜라비를 처리하고 day 6에 세포 분화를 측정했다. 콜라비 과피 25ng/ml와 100ng/ml은 돼지 지방전구세포의 증식을 각각 4.59%, 17.7% 억제했고, 콜라비 과육은 각각 11.4%, 19.2% 억제했다. 반면 돼지 지방전구세포의 분화는 억제하지 않았다. 콜라비가 3T3-L1 cell의 증식과 분화에 미치는 작용을 구명하기 위해, 돼지 지방전구세포처럼, 세포 배양초기 2일간 콜라비를 처리했는데 콜라비 과피와 과육 둘 다 세포의 증식과 분화에 영향을 미치지 않았다. 본 연구의 결과를 요약하면, 콜라비는 돼지 지방전구세포의 증식을 억제했으나 분화는 억제하지 않았고, 한편 3T3-L1 cell의 증식과 분화 모두 영향을 미치지 않았다. The current study was carried out to determine the effects of Kohlrabi(Brassica oleracea var. gongylodes) on proliferation and differentiation of pig preadipocytes and 3T3-L1 cells. Pig preadipocytes were isolated from the backfat of the new-born pigs. Twenty-four hours after seeding, the cells were washed with DMEM/F-12(designated day 0). To measure the cell proliferation, the cells were treated with 25ng/ml and 100 ng/ml ethanol extracts of Kohlrabi(peel and flesh) for two days(day 0~2). To measure differentiation, the cells were treated with Kohlrabi for two days(day 0~2) and cell differentiation was measured on day 6. Twenty-five ng/ml and 100ng/ml of Kohlrabi peel decreased proliferation of pig preadipocytes by 4.59% and 17.7%, respectively, compared with the control and Kohlrabi flesh by 11.4% and 19.2%, respectively. However, Kohlrabi did not inhibit cell differentiation. To measure the effects of Kohlrabi on proliferation and differentiation of 3T3-L1 cells, the cells were treated with Kohlrabi for two days in culture, like pig preadipocytes. Kohlrabi(both peel and flesh) did not show any effects on cell proliferation and differentiation. In summary, the results of the current study showed that Kohlrabi decreased proliferation of pig preadipocytes, but no inhibitory effects on differentiation of the cells. Kohlrabi had no effects on proliferation and differentiation of 3T3-L1 cells.

Reactive Oxygen Species Co-Operated with Sex Hormones Inhibit Proliferation of Hepal-6 Cells

Wang Ai-Guo,Kim Nam-Soon,Lee Dong-Seok The Korean Society for Biomedical Laboratory Scien 2005 Journal of biomedical laboratory sciences Vol.11 No.3

Reactive oxygen species (ROS) and sex hormones affect the proliferation of cells and are believed to play important roles in tumorigenesis. However, little is known regarding how these two factors interact to affect cell proliferation. In this study, hepal-6 cells were treated with ROS and sex hormones (testosterone and steroidal) either separately or in combination. The sex hormones had no significant influence the cell proliferation up to a concentration of $1{\mu}M$. However, cell proliferation was inhibited when the cells were treated simultaneously with $H_2O_2$, which alone was found to promote cell proliferation at the concentrations of $15{\mu}M$. In conclusion, this study indicates that instead of promoting the cell proliferation, ROS interact with sex hormones to inhibit the Hepa 1-6 cell proliferation.

Proliferated Leydig Cells for Engineered Testis-like Tissue Regeneration with Testosterone-Secreting Ability

Hongda Bi,Xiaoyun Wang,Wei Liu,Yilin Cao,Guangdong Zhou,Xin Xing 한국조직공학과 재생의학회 2014 조직공학과 재생의학 Vol.11 No.5

Tissue engineering approach provides a hopeful strategy for reconstructing testis testosterone-secreting functions. However, limited source and low proliferative activity in vitro of Leydig cells (LCs, the main testosteroneproducing cells) makes testis-like tissue regeneration difficult to be achieved. This study explored the feasibility of in vitro expanding LCs and their potential application in testis-like tissue regeneration. LC lineage cells were isolated from Sprague-Dawley (SD) rats by differential adhesion method and cell composition was identified by expressions of 3β-HSD, LHR, LIFR, and c-kit. A modified expansion medium (EM) system was used to test the feasibility of in vitro expanding LC lineage. The results showed that the attached cells reached a high purification of LC lineage (>90%, indicated by positive expression of 3β-HSD) and that EM significantly enhanced proliferation of LC lineage compared to regular medium, which was testified to be related to the presence of stem LCs that was implied by positive expressions of LIFR and c-kit as well as the transition of 3β-HSD expression from negative to positive in partial cells. Importantly, the proliferated LCs showed relatively sustained testosterone-secreting ability in vitro and these cells combined with biodegradable scaffolds successfully regenerated testis-like tissue with sustained testosteronesecreting function in vivo, which was supported by the enhanced serum testosterone level in castrated rats. All these results indicated that the differential adhesion method could efficiently isolate and purify LC lineage and that EM system could efficiently promote proliferation and functional maintenance of LC lineage, providing a good cell source for testes-like tissue regeneration. Tissue engineering approach provides a hopeful strategy for reconstructing testis testosterone-secreting functions. However, limited source and low proliferative activity in vitro of Leydig cells (LCs, the main testosteroneproducing cells) makes testis-like tissue regeneration difficult to be achieved. This study explored the feasibility of in vitro expanding LCs and their potential application in testis-like tissue regeneration. LC lineage cells were isolated from Sprague-Dawley (SD) rats by differential adhesion method and cell composition was identified by expressions of 3β-HSD, LHR, LIFR, and c-kit. A modified expansion medium (EM) system was used to test the feasibility of in vitro expanding LC lineage. The results showed that the attached cells reached a high purification of LC lineage (>90%, indicated by positive expression of 3β-HSD) and that EM significantly enhanced proliferation of LC lineage compared to regular medium, which was testified to be related to the presence of stem LCs that was implied by positive expressions of LIFR and c-kit as well as the transition of 3β-HSD expression from negative to positive in partial cells. Importantly, the proliferated LCs showed relatively sustained testosterone-secreting ability in vitro and thesecells combined with biodegradable scaffolds successfully regenerated testis-like tissue with sustained testosteronesecreting function in vivo, which was supported by the enhanced serum testosterone level in castrated rats. All these results indicated that the differential adhesion method could efficiently isolate and purify LC lineage and that EM system could efficiently promote proliferation and functional maintenance of LC lineage, providing a good cell source for testes-like tissue regeneration.

Suppression of CDK2 expression by siRNA induces cell cycle arrest and cell proliferation inhibition in human cancer cells

( Xiang E Long ),( Zhao Hui Gong ),( Lin Pan ),( Zhi Wei Zhong ),( Yan Ping Le ),( Qiong Liu ),( Jun Ming Guo ),( Jiu Chang Zhong ) 한국생화학분자생물학회 (구 한국생화학회) 2010 BMB Reports Vol.43 No.4

Cyclin-dependent kinase 2 (CDK2) is a member of serine/threonine protein kinases, which initiates the principal transitions of the eukaryotic cell cycle and is a promising target for cancer therapy. The present study was designed to inhibit cdk2 gene expression to induce cell cycle arrest and cell proliferation suppression. Here, we constructed a series of RNA interference (RNAi) plasmids which can successfully express small interference RNA (siRNA) in the transfected human cells. The results showed that the RNAi plasmids containing the coding sequences for siRNAs down-regulated the cdk2 gene expression in human cancer cells at the mRNA and the protein levels. Furthermore, we found that the cell cycle was arrested at G0G1 phases and the cell proliferation was inhibited by different siRNAs. These results demonstrate that suppression of CDK2 activity by RNAi may be an effective strategy for gene therapy in human cancers. [BMB reports 2010; 43(4): 291-296]

Interleukin-21 induces proliferation and modulates receptor expression and effector function in canine natural killer cells

Shin, D.J.,Lee, S.H.,Park, J.Y.,Kim, J.S.,Lee, J.J.,Suh, G.H.,Lee, Y.K.,Cho, D.,Kim, S.K. Elsevier 2015 Veterinary immunology and immunopathology Vol. No.

<P>Interleukin (IL)-21 is an important modulator of natural killer (NK) cell function. However, little is known about IL-21 function in canine NK cells because the phenotype of these cells remains undefined. In this study, we selectively expanded non-B and non-T large granular NK lymphocytes (CD3(-)CD21(-)CD5(-)CD4(-)TCR alpha beta-TCR gamma delta(-)) ex vivo from the peripheral blood mononuclear cells (PBMCs) of healthy dogs using a combination of IL-2, IL-15, and IL-21 in the presence of 100 Gy-irradiated K562 cells. We investigated the effects of varying the duration and timing of IL-21 treatment on stimulation of proliferation, expression of NK-related receptors, anti-tumor activity and production of interferon (IFN)-gamma. The expanded NK cells in each treatment group became enlarged and highly granular after 21 days in culture. NK cells proliferated rapidly in response to activation by IL-21 for 3 weeks, and IL-21 was able to induce changes in the mRNA expression of NK cell-related receptors and enhance the effector function of NK cells in perforin- and granzyme-B-dependent manners. The duration, frequency and timing of IL-21 stimulation during culture affected the rate of proliferation, patterns of receptor expression, cytokine production, and anti-tumor activity. The optimal conditions for maximizing the IL-21-induced proliferation and effector function of NK cells in the presence of IL-2 and IL-15 were seen in cells treated with IL-21 for the first 7 days of culture but without any further IL-21 stimulation other than an additional 2-day treatment prior to harvesting on day 21. The results of this study suggest that synergistic interactions of IL-21 with IL-2 and IL-15 play an important role in the proliferation, receptor expression, and effector function of canine NK cells. (C) 2015 Elsevier B.V. All rights reserved.</P>

Novel Pathway for Hypoxia-Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF-1α, FASN, and mTORC1.

Lee, Hyun Jik,Ryu, Jung Min,Jung, Young Hyun,Oh, Sang Yub,Lee, Sei-Jung,Han, Ho Jae AlphaMed Press 2015 Stem Cells Vol.33 No.7

<P>The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCB-hMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCB-hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. Stem Cells 2015;33:2182-2195</P>

Molecular insights and immune responses of big belly seahorse syndecan-2 (CD362): Involvement of ectodomain in regulating cell survival, proliferation, and wound healing

D.S. Liyanage,W.K.M. Omeka,M.D. Neranjan Tharuka,Sumi Jung,Sukkyoung Lee,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

핵의학적 세포증식 영상

여정석 대한핵의학회 2004 핵의학 분자영상 Vol.38 No.2

Tumor cell proliferation is considered to be a useful prognostic indicator of tumor aggressiveness and tumor response to therapy, but in vitro measurement of individual proliferation is complex and tedious work. PET imaging provides a noninvasive approach to measure tumor growth rate in situ. Early approaches have used ^(18)F-FDG or methionine to monitor proliferation status. These 2 tracers detect changes in glucose and amino acid metabolism, respectively, and therefore provide only an indirect measure of proliferation status. More recent studies have focused on DNA synthesis itself as a marker of cell proliferation. Cell lines and tissues with a high proliferation rate require high rates of DNA synthesis. [^(11)C]Thymidine was the first radiotracer for noninvasive imaging of tumor proliferation. The short half-life of ^(11)C and rapid metabolism of [^(11)C]thymidine in vivo make the radiotracer less suitable for routine use. Halogenated thymidine analogs such as 5-iodo-2-deoxyuridine (IUdR) can be successfully used as cell proliferation markers for in vitro studies because these compounds are rapidly incorporated into newly synthesized DNA. IUdR has been evaluated as a potential in vivo tracer in nuclear medicine, but the image quality and the calculation of proliferation rates are impaired by its rapid in vivo degradation. Hence, the thymidine analog 3'-deoxy-3'-^(18)F-fluorothymidine (FLT) was recently introduced as a stable proliferation marker with a suitable nuclide half-life and stable in vivo. [^(18)F]FLT is phosphorylated to 3-fluorothymidine monophosphate by thymidine kinase 1 and reflects thymidine kinase 1 activity in proliferating cell. [^(18)F]FLT PET is feasible in clincal use and well correlates with cellular proliferation. Choline is a precursor for the biosynthesis of phospholipids (in particular, phosphatidylcholine), which is the essential component of all eukaryotic cell membranes and [^(11)C]choline, which is a new marker for cellular proliferation. (Korean J Nucl Med 38(2):198-204, 2004)

Functional characterization of human oncoprotein gankyrin in Zebrafish

So Yeon Kim,Wonhee Hur,최정은,Daniel Kim,Jin Sang Wang,Hye-Yeon Yoon,Lian-Shu Piao,윤승규 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.1

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma(HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2- hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.