ObjectiveTo investigate the role of Peptidase domain containing associated with muscle regeneration 1 (PAMR1) in the proliferation, migration, and prognosis of hepatocellular carcinoma (HCC) through cellular experiments and clinical sample validation. Methods① Bioinformatics analysis was performed on datasets from the GEO public database to identify and screen for key genes, ultimately selecting PAMR1 for further study. Findings were validated using data from the TCGA database and six primary HCC surgical specimens prospectively obtained from the Army Characteristic Medical Center of Army Medical University from February 2024 to June 2024. ② PAMR1-overexpressing cell lines were established using HCC cell lines Huh7 and Lm3. Cells were transfected with either the recombinant plasmid pcDNA3.1(+)-PAMR1 as overexpression group or the empty vector pcDNA3.1(+) as negative control group. The effects of PAMR1 on HCC cell proliferation and migration were assessed using the Cell Counting Kit-8 (CCK-8) assay and wound healing assay, respectively. ③ Pathological specimens and clinical data were retrospectively collected from 61 patients with primary HCC who underwent surgical resection at the Army Characteristic Medical Center of Army Medical University between May 2019 and April 2020. The impact of PAMR1 expression on disease-free survival was evaluated. Results① PAMR1 was identified as a candidate gene through GEO database screening and was found to be downregulated in HCC tissues based on both TCGA data and the six HCC surgical specimens. ② The CCK-8 assay revealed that cell proliferation was significantly inhibited in the PAMR1 overexpression group compared to the negative control group (P<0.05). Similarly, the wound healing assay demonstrated reduced migratory capability in PAMR1 overexpression group (P<0.05). ③ Multivariate cox proportional hazards regression analysis of patient data indicated that high PAMR1 expression serves as an independent protective factor for disease-free survival in HCC (HR= 0.335, P=0.026). ConclusionPAMR1 serves as a crucial gene in HCC, high PAMR1 expression can significantly suppressing tumor cell proliferation and migration and indicates a favorable prognosis.
This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.
ObjectiveTo summarize the progress of study on the relationship between endoplasmic reticulum stress and cell proliferation and provide evidence with reliable evidence-based data to the experiment on the field of tissue damage repair, organ proliferation, and regeneration.MethodThe relevant literatures about the progress of multiple signaling pathways related to the endoplasmic reticulum stress in the cell proliferation and injury repair in recent years were reviewed.ResultsThe endoplasmic reticulum stress participated in the process of proliferation and regeneration in the intestinal epithelial cells, skeletal muscle cells, islet cells, and hepatocytes through different pathways, which involved the three pathways of unfolded protein reaction that interacted with interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, Wnt, etc.ConclusionsAlthough endoplasmic reticulum stress has been widely debated in the field of determining cell fate, after we reviewed recent studies on endoplasmic reticulum stress in maintaining cell survival and promoting cell proliferation, the complexity, diversity, and importance of the endoplasmic reticulum stress in promoting cell proliferation have been presented in front of us. It not only promotes cell proliferation through the classical signaling pathway with Wnt protein, but also acts to repair tissue and promote proliferation by interacting with Musashi protein independently of the Notch pathway. The complex reaction pathway interacts with different stimulating factors in different cells, providing research directions and exploration possibilities for cell proliferation, injury repair, and organ regeneration, reveales the critical role of endoplasmic reticulum stress in cell proliferation.
Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.
Objective To investigate the effects of cediranib on hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway and proliferation, migration and invasion of liver cancer cells. Methods The hypoxia microenvironment was simulated in vitro, and different doses of cediranib were used to intervene the human hepatoma cell HepG2, MTT assay was used to detect the proliferation of human hepatoma cell HepG2, Transwell chamber assay was used to detect the invasion and migration of human hepatoma cell HepG2, tumor formation in nude mice was used to detect the growth of human hepatoma cell HepG2 in vivo, the angiogenesis of tumor tissue and expression level of HIF-1α/VEGF pathway protein were detected by immunohistochemistry. Results Compared with the control group, the proliferation rate, invasion and migration abilities, and the expression of HIF-1α/VEGF pathway proteins of human hepatoma cell HepG2 were significantly decreased in the different concentration of cediranib treatment group (P<0.05), the tumor volume and microvessel formation of tumor tissues in nude mice were significantly reduced (P<0.05). Conclusion Cediranib may inhibit the proliferation, migration and invasion of liver cancer cells by inhibiting HIF-1α/VEGF signaling pathway.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
The aim of this article is to study the regulatory feedback loop between β-catenin and IQ motif containing GTPase activating protein 1 (IQGAP1), as well as the effect of this regulation loop in colon cancer cell proliferation. Western blot was used to detect the expression of IQGAP1 and β-catenin after changing their expression respectively by transfection in SW1116 cells. CCK-8 cell proliferation assay was used to detect the effect of IQGAP1 involved in the proliferation of SW1116 cells promoted by β-catenin. The results of Western blot indicated that β-catenin could positively regulate IQGAP1, while IQGAP1 silencing could up-regulate β-catenin, forming a negative feedback loop. The results of CCK-8 showed that IQGAP1 silencing inhibited β-catenin-mediated proliferation in SW1116 cells. In conclusion, our research reveals a negative regulatory feedback loop between β-catenin and IQGAP1 which has a remarkable effect on the proliferation ability of colon cancer cells.
Objective To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs). Methods The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg (control group), 75 mm Hg (low negative pressure group), and 150 mm Hg (high negative pressure group) (1 mm Hg=0.133 kPa), and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase (ALP) activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I (COL-I) and osteocalcin (OC) in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot. Results The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group (P<0.05), but there was no significant difference between the low negative pressure group and the control group (P>0.05); at 5-7 days, the cell number showed significant difference between 3 groups (P<0.05). The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction (P>0.05); the ALP activity showed significant difference (P<0.05) between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction (P<0.05). The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group (P<0.05), and in the high negative pressure group than the low negative pressure group (P<0.05). Conclusion With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.
Objective To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. Methods Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. ResultsThere were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group<uDBM group<hDBM group<tDBM group<fDBM group. After co-culture for 14 days, the expressions of ALP, calcified nodules, and collagen fibers in each group were consistent with the distribution of BMP-2 concentration in DBM. The order of ALP content from low to high was cDBM group<uDBM group<hDBM group<tDBM group<fDBM group, and the differences between the groups were significant (P<0.05). Linear regression analysis showed that \begin{document}$\hat y $\end{document}=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. Conclusion The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.