Effect of PD-L1 on proliferation,migration,and invasion of human oral squamous carcinoma cells / 吉林大学学报(医学版)

Objective:To discuss the expression of programmed cell death-ligand 1(PD-L1)in the oral squamous cell carcinoma(OSCC)cells and its effect on biological behavior of the OSCC CAL27 cells,and to clarify the possible mechanism.Methods:Western blotting method was used to detect the expression levels of PD-L1 protein in the oral epithelial HOK cells and OSCC CAL27,TCA8113,and SCC15 cells;immunofluorescence staining method was used to detect the expression and localization of PD-L1 protein in the CAL27 cells.The CAL27 cells were divided into control group(transfected with si-NC)and si-PD-L1 group(transfected with si-PD-L1).Western blotting method was used to detect the interference efficiency of the cells in two groups;CCK-8 assay was used to detect the proliferative activities of the cells in two groups at different time points;plate clone formation assay was used to detect the numbers of clone formation of the cells in two groups;cell scratch healing assay was used to detect the scratch healing rates of the cells in two groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in two groups.Results:The expression level of PD-L1 protein in the OSCC cells was higher than that in the HOK cells(P<0.05 or P<0.01);PD-L1 expressed in the cytoplasm and nucleus of the CAL27 cells.The CCK-8 assay and plate clone formation assay results showed that compared with control group,the proliferative activities of the CAL27 cells in si-PD-L1 group at different time points were significantly decreased(P<0.05 or P<0.01),and the numbers of clone formation were significantly decreased(P<0.01).The cell scratch healing assay results showed that compared with control group,the scratch healing rates of the cells in si-PD-L1 group were significantly decreased(P<0.05 or P<0.01).The Transwell chamber assay results showed that compared with control group,the numbers of migration and invasion cells in si-PD-L1 group were significantly decreased(P<0.01).Conclusion:The expression of PD-L1 in the OSCC cells is higher than that in normal oral epithelial cells,and knocking down PD-L1 expression can inhibit the proliferation,clone formation,migration and invasion capabilities of the OSCC cells.

Effect of chelerythrine on migration,invasion,and epithelial-mesenchymal transition of human ovarian cancer SKOV3 cells / 吉林大学学报(医学版)

Objective:To discuss the inhibitory effect of chelerythrine(CHE)on the migration,invasion,and epithelial-mesenchymal transition(EMT)of the human ovarian cancer SKOV3 cells,and to clarify the associated mechanism.Methods:The SKOV3 cells were cultured in vitro and divided into control group and 2.5,5.0,10.0,20.0,and 40.0 μmol·L-1 CHE groups.Methylthiazolydiphenyl-tetrazolium(MTT)assay was used to detect the inhibitory rates of proliferation of the cells in various groups.The SKOV3 cells were cultured in vitro and divided into control group,transforming growth factor-β1(TGF-β1)group,TGF-β1+5 μmol·L-1 CHE group,and TGF-β1+10 μmol·L-1 CHE group.Cell scratch assay was used to detect the migration rates of the cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups;Western blotting method was used to detect the expression levels of E-cadherin,N-cadherin,and Vimentin proteins in the cells in various groups;immunofluorescence staining method was used to detect the fluorescence intensities of E-cadherin and N-cadherin in the cells in various groups.Results:The MTT assay results showed that compared with control group,the inhibitory rates of proliferation of the cells in 5.0,10.0,20.0,and 40.0 μmol·L-1 CHE groups were significantly increased(P<0.05 or P<0.01).The cell scratch assay results showed that compared with control group,the migration rate of the cells in TGF-β1 group was increased(P<0.01);compared with TGF-β1 group,the migration rates of the cells in TGF-β1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly decreased(P<0.01).The Transwell chamber assay results showed that compared with control group,the numbers of migration and invasion cells in TGF-β1 group were significantly increased(P<0.05);compared with TGF-β1 group,the numbers of migration and invasion cells in TGF-β1+5 μmo·l L-1 CHE group and TGF-β1+10 μmo·l L-1 CHE group were significantly decreased(P<0.01).The Western blotting results showed that compared with control group,the expression level of E-cadherin protein in the cells in TGF-β1 group was significantly decreased(P<0.01),while the expression levels of N-cadherin and Vimentin proteins were increased(P<0.05 or P<0.01);compared with TGF-β1 group,the expression levels of E-cadherin protein in the cells in TGF-β1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly increased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins were significantly decreased(P<0.01).The immunofluorescence staining results showed that compared with control group,the fluorescence intensity of E-cadherin in the cells in TGF-β1 group was decreased,and the fluorescence intensity of N-cadherin was increased;compared with TGF-β1 group,the fluorescence intensities of E-cadherin in the cells in TGF-β 1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly increased,and the fluorescence intensities of N-cadherin were decreased.Conclusion:CHE can inhibit the proliferation,migration,invasion,and EMT of the human ovarian cancer SKOV3 cells.

Inhibitory effect of berberine on migration and invasion of human glioma T98G cells and its mechanism / 吉林大学学报(医学版)

Objective:To discuss the regulatory effect of berberine(BBR)on fatty acids in the human glioma T98G cells and its effect on the cell proliferation,migration,and invasion,and to clarify its potential mechanism.Methods:The T98G cells at logarithmic growth phase were divided into control group and different concentrations(25,50,and 100 mg·L-1)of BBR groups.Cell wound healing assay was used to detect the migration rates of the cells in various groups;Transwell chamber assay was used to detect the invasion rates of the cells in various groups.The T98G cells at logarithmic growth phase were divided into control group and 100 mg·L-1 BBR group,and Mass spectrometry was used to detect the fatty acid contents in the cells in two groups.The T98G cells at logarithmic growth phase were divided into control group and different concentrations(50,100,and 150 mg·L-1)of BBR groups;Western blotting method was used to detect the expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(AKT),phosphorylated AKT(p-AKT),sterol regulatory element-binding protein 1(SREBP-1),and fatty acid synthase(FASN)in the cells in various groups.The expression of FASN was suppressed by gene silencing technology,and the T98G cells at logarithmic growth phase were divided into control group,shFASN1 group,and shFASN2 group.Western blotting method was used to detect the expression levels of FASN protein in the cells in various groups;clone formation assay was used to detect the clone formation of the cells in various groups;cell wound healing assay was used to detect the migration rates of the cells in various groups.Results:Compared with control group,the migration rates and invasion rates of the cells in different concentrations of BBR groups were decreased in a concentration-dependent manner(P<0.01),and the fatty acid content in the cells in 100 mg·L-1 BBR group was significantly decreased(P<0.01).Compared with control group,the expression levels of p-PI3K,p-AKT,SREBP-1,and FASN proteins in the cells in 150 mg·L-1 BBR group were significantly decreased(P<0.05 or P<0.01),and the expression level of SREBP-1 protein in the cells in 100 and 150 mg·L-1 BBR groups were significantly decreased(P<0.01).After suppression of FASN expression,compared with control group,the expression levels of FASN protein in the cells in shFASN1 and shFASN2 groups were significantly decreased(P<0.01),and the expression level of FASN protein in the cells in shFASN2 group was lower than that in shFASN1 group(P<0.05);compared with control group,the numbers of clone formation and migration rates of the cells in shFASN1 and shFASN2 groups were significantly decreased(P<0.01),and the migration rate of the cells in shFASN2 group was significantly lower than that in shFASN1 group(P<0.05).Conclusion:BBR interferes with fatty acid synthesis in the glioma T98G cells by reducing the expression of the PI3K/AKT/SREBP-1/FASN pathway related proteins,and decrease their migration and invasion capabilities.

Inhibitory effect of downregulating HMGB2 expression on epithelial-mesenchymal transition of liver cancer LM3 cells and its AKT/m TOR signaling pathway mechanism / 吉林大学学报(医学版)

Objective:To discuss the effect of downregulating of high mobility group box protein 2(HMGB2)expression on the biological behavior of the liver cancer cells and the epithelial-mesenchymal transition(EMT)process,and to clarify its mechanism.Methods:The human liver cancer LM3 cells at logarithmic growth phase were divided into negative control group and HMGB2 RNA interference group(HMGB2 siRNA group);the cells in two groups were transfected with RNA oligonucleotides(RNA oligos)with irrelevant sequences and RNA oligos designed to knock down HMGB2,and the Lipofectamine 2000 was regarded as the vector.The expression levels of HMGB2 mRNA and protein in the cells in two groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods;cell scratch assay and Transwell chamber assay were used to detect the migration and invasion abilities of the cells in two groups;the expression levels of E-cadherin,N-cadherin,and Vimentin proteins and protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway related proteins in the cells in two groups were detected by Western blotting method.Results:Compared with negative control group,the expression levels of HMGB2 mRNA and protein in the cells in HMGB2 siRNA group were significantly decreased(P<0.05),the cell scratch healing rate was significantly decreased(P<0.01),the number of invasion cells was significantly decreased(P<0.01),and the expression level of E-cadherin protein in the cells was significantly increased(P<0.01),while the expression levels of N-cadherin,Vimentin,mTOR,AKT,and phosphorylated AKT(p-AKT)proteins in the cells were significantly decreased(P<0.05 or P<0.01).Conclusion:Downregulating the expression of HMGB2 can reduce the migration and invasion abilities of the liver cancer LM3 cells and inhibit the EMT,and its mechanism may be related to regulating the expression of the AKT/mTOR pathway related proteins.

Effect of CIP2A knockdown on proliferation and migration of hypopharyngeal cancer cells / 中国耳鼻咽喉头颈外科

OBJECTIVE The expression of cancerous inhibitor of protein phosphatase 2A(CIP2A)in hypopharyngeal carcinoma FaDu cells(FaDu cells)was reduced by shRNA to understand its role in the occurrence and development of hypopharyngeal carcinoma.METHODS Specific shRNA sequence was designed,lentivirus was packaged and transfected into hypopharyngeal carcinoma FaDu cells,and CIP2A expression was specifically knocked down.The expression of CIP2A was detected by RT-PCR and Western blot.RESULTS 1.After shRNA knocked down CIP2A in FaDu cells,the CIP2A mRNA expression in the experimental group(CIP2A knocked down group)was significantly lower than that in the blank group,and the CIP2A protein expression in the experimental group was also significantly lower than that in the blank group.2.Cell cloning and CCK8 experiments showed that the cell proliferation ability of the experimental group was significantly decreased compared with that of the blank group(t=50.86,P<0.01;t=12.406,P<0.001);The results of cell scratch test showed that the transverse migration ability of the experimental group was significantly decreased compared with the blank group,and the longitudinal migration ability of the experimental group was significantly decreased compared with the blank group by Transwell test(t=40.038,P<0.01;t=12.247,P<0.001).CONCLUSION After knockdown CIP2A expression in hypopharyngeal carcinoma FaDu cells,the proliferation and migration ability of hypopharyngeal cancer cells decreased,suggesting that CIP2A is involved in regulating the biological behavior of hypopharyngeal cancer cells and can be used as a potential anticancer target.

Effects of Jianpi Bushen Jiedu Prescription regulating JAK2/STAT3 pathway on proliferation and migration of hepatocellular carcinoma cells / 国际中医中药杂志

Objective:To explore the effects of Jianpi Bushen Jiedu Prescription on the proliferation and migration of hepatocellular carcinoma cells; To discuss its possible mechanism.Methods:Using human highly metastatic liver cancer cell line (HCCLM3) as the research object, they were randomly divided into control group and TCM group (100, 200, 400, 800, 1 600, 3 200 μg/ml Jianpi Bushen Jiedu Prescription) and Western medicine group (2.5, 5, 10, 20, 40 μmol/L sorafenib) using a random number table method. Cell viability was detected using cell counting reagent (CCK-8) method; HCCLM3 cells were divided into control group and TCM (Jianpi Bushen Jiedu Prescription 800 μg/ml) group and combined group (Jianpi Bushen Jiedu Prescription 800 μg/ml +sorafenib 20 μmol/L). Western blot method was used to detect the protein expressions of kinase/signaling transducer and transcriptional activator (JAK2/STAT3) pathway related proteins (p-JAK2, JAK2, p-STAT3, STAT3) in each group.Results:Compared with the control group, viability and mobility of HCCLM cell in TCM group and Western medicine group decreased ( P<0.01 or P<0.05); compared with the control group, the protein expressions of P-JAK2, JAK2, P-STAT3 and STAT3 in the TCM group and the combined group decreased ( P<0.05), and the JAK2 protein expression in the combined group was lower than that in the TCM group ( P<0.05). Conclusion:Jianpi Bushen Jiedu Prescription can inhibit the proliferation and migration of HCC cells by regulating JAK2/STAT3 pathway.

lncRNA VIM-AS5 expression and its effect on proliferation and migration of human breast cancer cell lines / 基础医学与临床

Objective To explore the clinical significance of long non-coding RNA(lncRNA)VIM-AS5 expres-sion in human breast cancer tissues and its regulatory mechanism involved in cancer cell proliferation and mi-gration.Methods The Lnc2Cancer 3.0 database was used to analyze the expression of VIM-AS5 in breast cancer tissues and its correlation with the clinical stage and survival time of breast cancer patients.RT-qPCR was used to detect the expression of VIM-AS5 in breast cancer cell lines BT-549,MDA-MB-435,MDA-MB-231 and CAL-51.Plasmid with VIM-AS5 overexpression and negative control were all transfected into CAL-51 cells through liposome recorded as VIM-AS5 group and NC group,respectively.The proliferation and migration of CAL-51 cells were detected by colony formation assay and scratch healing method,respectively.Dual-lucif-erase reporter gene experiment verified the targeting relationship between VIM-AS5 and miR-500a.RT-qPCR was used to detect the expression of miR-500a in CAL-51 cells.Western blot was used to detect the expression of JAK/STAT3 pathway in CAL-51 cells.Results The expression of VIM-AS5 in breast cancer tissues was significantly lower than that in adjacent tissues(P＜0.01).VIM-AS5 expression was negatively correlated with the clinical stage of breast cancer patients(P＜0.01).The survival time of breast cancer patients with low VIM-AS5 expression was significantly shorter than that of breast cancer patients with high VIM-AS5 ex-pression(P＜0.01).Compared with mammary epithelial cell line MCF-10 A cells,VIM-AS5 expression was significantly reduced in breast cancer cells(P＜0.01).The counting number of colony formed in the VIM-AS5 group was significantly lower than that in the NC group(P＜0.01).The cell migration rate in the VIM-AS5 group was significantly lower than that in the NC group(P＜0.01).Dual-luciferase reporter gene experiment confirmed that miR-500a was the target gene of VIM-AS5(P＜0.01).VIM-AS5 can negatively regulate the expression of miR-500a(P＜0.01).Compared with the NC group,the expression of JAK/STAT3 pathway proteins JAK,p-STAT3,c-Myc,Bcl-2,and CDK3 in CAL-51 cells of the VIM-AS5 group were significantly decreased.Conclusions VIM-AS5 is low-expressed in breast cancer cells,and up-regulation of VIM-AS5 may inhibit the proliferation and migration of breast cancer cells CAL-51 by targeting at miR-500a/JAK/STAT3 pathway.

The biological function and mechanism of IDH1 gene in intrahepatic cholangiocarcinoma cell HuCCT1 / 解放军医学杂志

Objective To explore the role and possible molecular mechanism of Isocitrate dehydrogenase 1(IDH1)gene in proliferation and migration of intrahepatic cholangiocarcinoma(iCCA)cell HuCCT1.Methods HuCCT1 cells with IDH1 gene knockout(HuCCT1IDH1-/-)were constructed by CRISPR/Cas9 gene editing technology.To investigate the capacities of proliferation,migration and invasion of HuCCT1WT(HuCCT1 cells with wild-type IDH1 gene)and HuCCT1IDH1-/-cells,assays of CCK-8,clone formation,scratch and transwell were performed.Western blotting was used to detect the expression levels of epithelial-mesenchymal transition(EMT)associated proteins E-cadherin,N-cadherin,Vimentin,MMP-9,Wnt3a and β-catenin in two groups of cells.The transcriptome sequencing data of HuCCT1WT and HuCCT1IDH1-/-cells were analyzed by bioinformatics methods,Western blotting was used to verify the expression of signaling pathway-related proteins.Results Compared with HuCCT1WT cells,HuCCT1IDH1-/-cells showed the number of proliferation and clone formation significantly reduced(P<0.05),the proportion of cells blocked in G2/M phase was significantly increased(P<0.01),the rate of scratch healing was significantly decreased(P<0.01),and the number of migrated cells(P<0.001)and invaded cells(P<0.05)was significantly reduced.qRT-PCR assay showed that the expression levels of IDH1,Vimentin,MMP-9 and genes related to the regulation of G2/M cycle proliferation,Cyclin A2,Cyclin B1 and CDK1 mRNA were down-regulated in HuCCT1IDH1-/-cells(P<0.05),and the expression of CDH1 mRNA encoding E-cadherin was up-regulated(P<0.01);Western blotting assay showed that the expression level of E-cadherin in HuCCT1IDH1-/-cells was significantly increased(P<0.05),and the expression level of N-cadherin,Vimentin and MMP-9 protein was significantly decreased(P<0.05)than that in HuCCT1WT cells.Data of transcriptome sequencing revealed 1476 differentially expressed genes(DEGs)between two groups of HuCCT1 cells.Go enrichment analysis showed the DEGs were significantly enriched in cell biological processes associated with inflammatory response,cell signaling and cell metabolism.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis suggested that the DEGs may be involved in some signaling pathways such as Wnt,MAPK,Rap1,Hippo and TNF,which are closely related to the regulation of proliferation and invasion of tumor cells.Western blotting verification results showed that compared with HuCCT1WT cells,the relative expression of Wnt3a and β-catenin proteins of HuCCT1IDH1-/-cells was significantly decreased(P<0.05).Conclusions IDH1 gene may participate in the control of biological functions of HuCCT1 cells,including cell proliferation,migration,invasion and epithelial mesenchymal transition.The mechanism may be related to the activation of the Wnt/β-catenin signaling pathway.

Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6 / 上海交通大学学报（医学版）

Objective·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6.Methods·The HN6 cell line was selected,cultivated,and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis:control group,3.125 μg/mL group,6.25 μg/mL group,12.5 μg/mL group,25 μg/mL group,50 μg/mL group,and 100 μg/mL group.After 24 and 48 h of cultivation,CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity.Subsequent experiments were divided into control group,25 μg/mL group and 50 μg/mL group.Flow cytometry was used to examine the effects of gingipain extract on cell cycle.Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability.Real-time PCR(RT-PCR)and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells.Results·Stimulated with gingipain extract for 24 h,the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL(P=0.025),50 μg/mL(P=0.000),and 100 μg/mL(P=0.049)groups compared to the control group.After 48 h,proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024),12.5 μg/mL(P=0.006),25 μg/mL(P=0.000),50 μg/mL(P=0.000),and 100 μg/mL(P=0.000)groups compared to the control group.Cell cycle analysis revealed that,after 24 h of gingipain stimulation,the proportion of HN6 cells in the G1 phase decreased,while the proportion in the S+G2 phase significantly increased compared to the control group(25 μg/mL group:P=0.024;50 μg/mL group:P=0.001).Compared to the control group,the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased(P=0.001).Compared to the control group,the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased.RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased,the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased,while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group.Conclusion·Gingipain extract could promote proliferation,migration,and invasion of oral squamous cell carcinoma HN6 cells.

HNRNPF promotes the proliferation,migration and invasion of prostate cancer / 复旦学报（医学版）

Objective To investigate the expression and clinical relevance of heterogeneous nuclear ribonucleoproteins F(HNRNPF)in prostate cancer and its effect on the proliferation,migration and invasion of prostate cancer cells.Methods The expression and immune infiltration characteristics of HNRNPF in prostate cancer and its correlation with the clinicopathological characteristics of prostate cancer patients were analyzed using TCGA database and GEO database.The HNRNPF gene was silenced by RNA interference in prostate cancer cell PC-3 and DU145,then the changes in cell proliferation ability was detected by CCK-8,EdU and colony formation assays,and the changes in cell migration and invasion abilities were detected by Transwell and wound-healing assays.Results The expression of HNRNPF was significantly increased in prostate cancer compared with normal prostate tissue and significantly associated with T stage,Gleason score,prostate specific antigen and the infiltration level of multiple immune cells of prostate cancer patients.The prostate cancer patients with high expression of HNRNPF had shorter overall survival and disease-specific survival.HNRNPF silencing decreased the proliferation,migration,and invasion abilities of prostate cancer cells PC-3 and DU145.Conclusion HNRNPF is a gene that is highly expressed in prostate cancer,has significant clinical relevance,and can promote the proliferation,migration,and invasion of prostate cancer cells.

Lamin B2 promotes migration of retroperitoneal liposarcoma cells by regulating AKT signaling pathway / 中华普通外科杂志

Objective:To investigate the effect of Lamins B2 (LMNB2) on the migration of human retroperitoneal liposarcoma (RPLS) cells SW872.Methods:Immunohistochemistry was used to analyze the the differential expression levels of LMNB2 in 33 RPLS tissue samples . The correlation between LMNB2 expression and clinical prognosis and clinicopathological features was analyzed. siRNA was used to lower the expression level of LMNB2 in tumor cells, and the effect of LMNB2 on the scratch healing ability and migration ability of SW872 cells was examined by using wound-healing assay and transwell migration assay. The expression levels of p-AKT and AKT in each group cells were detected by Western blot.Results:Patients with high LMNB2 expression had a lower recurrence-free survival and overall survival compared to those with low LMNB2 expression, and were more likely to experience recurrence, ( χ2=4.872, P=0.027; χ2=4.180, P=0.041; χ2=7.127, P=0.008). The migration ability of cells was significantly reduced following the silencing of LMNB2 expression ( t=11.240, P<0.01; t=7.445, P<0.01). The expression level of p-AKT in the silencing group was significantly lower than that in the control group, while there was no significant difference in the expression level of AKT between the two groups ( t=9.784, P<0.01). Conclusion:LMNB2 may promote the migration of human retroperitoneal liposarcoma cells SW872 by regulating AKT signaling pathway.

Application of microfluidics in anti-infectious immunity / 中华微生物学和免疫学杂志

The immune response against infection is a multifaceted process encompassing the activation and migration of diverse immune cells, as well as the clearance of pathogens. The behaviors of immune cells and the identification of pathogens play pivotal roles as indicators for disease diagnosis and prediction. In recent years, the utilization of microfluidic chip technology has gained substantial attention within the areas of biology, pharmacology, and clinical research and diagnosis. This is primarily attributed to the numerous advantages it offers, including miniaturization, enhanced throughput, heightened sensitivity, expedited analysis, and reduced sample consumption. As a result, microfluidic technology has facilitated the development and utilization of immune cell behavioral assays, bacterial growth studies, and drug-screening assays. This paper is to review the application of microfluidic technology in the field of anti-infection immunity research, focusing on the analysis of migratory behavior of innate immune cells, deformation of their nuclei, and rapid identification of pathogenic bacteria and viruses. The primary objective of this review is to advance the application of microfluidic technology in research on both anti-infection immunity and clinical diagnosis.

Impact of intracellular calcium reduction on neutrophil migration in sepsis patients / 中华微生物学和免疫学杂志

Objective:To investigate the relationship between intracellular calcium level and neutrophil migration dysfunction in patients with sepsis.Methods:This study retrospectively collected 21 blood samples of patients with sepsis in the First Hospital of Jilin University from December 2017 to September 2018, and 20 healthy people were included as the control group. Peripheral blood neutrophils were isolated from the healthy controls and patients with sepsis using magnetic-activated cell sorting. Multichannel microfluidic microarray technology was used to detect the chemotactic migration of neutrophils. The levels of calcium in neutrophils from healthy controls and sepsis patients as well as in neutrophils from healthy controls that were pretreated with calcium chelators BAPTA-AM and EDTA were detected by flow cytometry using the calcium indicator Fluo-4.Results:The intracellular calcium levels were lower in neutrophils from sepsis patients than in those from healthy controls ( P<0.01). BAPTA-AM and EDTA could reduce the calcium level in neutrophils of healthy controls ( P<0.01). Microfluidics revealed that the migration speed, distance and gap-passing rate of neutrophils in microfluidics were significantly reduced after the decrease of intracellular calcium ( P<0.01). Conclusions:Reduced calcium levels in neutrophils of patients with sepsis may be closely related to the decreased cell migration. This study suggests that the migration impairment of neutrophils can be improved by regulating intracellular calcium levels, which provides a new idea for further research.

Silencing Nodal inhibits the biological behavior of retinal vascular endothelial cells under high glucose conditions / 中华眼底病杂志

Objective:To observe the effect of Nodal on the biological behavior of retinal vascular endothelial cells (RF/6A cells) in monkeys with high glucose.Methods:RF/6A cells were divided into normal group, mannitol group, high glucose group, high glucose combined with non-specific small interfering RNA treatment group (HG+NC group), high glucose combined with small interfering Nodal treatment group (HG+siNodal group). The transfection efficiency of siNodal was observed by real-time fluorescence quantitative PCR and western blot protein immunoblotting. The effect of Nodal on the proliferation of RF/6A cells was detected by thiazole blue colorimetry. The effect of Nodal on migration ability of RF/6A cells was detected by cell scratch assay. The effect of Nodal on the formation of RF/6A cell lumen was measured by Matrigel three-dimensional in vitro. The expression of extracellular signal phosphorylated regulated kinase 1/2 (pERK1/2) in RF/6A cells was detected by western blot protein immunoblotting. One-way analysis of variance was used to compare groups.Results:Compared with HG+NC group, Nodal protein ( F=33.469) and mRNA relative expression levels ( F=38.191) in HG+siNodal group were significantly decreased, cell proliferation was significantly decreased ( F=28.548), and cell migration ability was significantly decreased ( F=24.182). The number of cell lumen formation was significantly decreased ( F=52.643), and the differences were statistically significant ( P<0.05). Compared with HG+NC group, the relative expression of pERK1/2 protein in HG+siNodal group was significantly decreased, and the difference was statistically significant ( F=44.462, P<0.01). Conclusions:Silencing Nodal expression can inhibit proliferation, migration and tube formation of RF/6A cells induced by high glucose. It may act by inhibiting pERK1/2 expression.

Effect of estrogen-related receptor α-mediated lipophagy on proliferation and migration abilities of nasopharyngeal cancer cells / 肿瘤研究与临床

Objective:To investigate the effect of estrogen-related receptor α (ESRRA)-mediated lipophagy on the proliferation and migration abilities of nasopharyngeal carcinoma cells.Methods:A total of 16 clinical samples diagnosed by pathology in the Affiliated Hospital of Nantong University from 2021 to 2023 were selected, including 8 normal nasopharyngeal mucosa tissues and 8 nasopharyngeal carcinoma tissues. Immortalized normal nasopharyngeal epithelial cell line NP69 and nasopharyngeal carcinoma cell lines C666-1, CNE2, TW03-EBV and TW03 were selected. The cell lines C666-1 and CNE2 were divided into the siR-NC group (transfected with small interfering RNA negative control sequence) and siR-ESRRA group (transfected with small interfering RNA against ESRRA gene). The relative expression levels of ESRRA were detected by Western blotting and immunohistochemical assay. EdU assay was used to detect the proliferation ability of C666-1 and CNE2 cells, and Transwell assay was used to detect the migration ability. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of ESRRA and perilipin 3 (PLIN3) mRNA. The formation of lipophagy in C666-1 and CNE2 cells was observed by transmission electron microscopy. The co-localization of LC3, PLIN3 and LAMP2 with lipid droplets labeling with Bodipy was detected by immunofluorescence assay. Dual-luciferase reporter gene assay was used to verify the targeting relationship between ESRRA and PLIN3.Results:The relative expression level of ESRRA in nasopharyngeal carcinoma tissues was higher than that in normal nasopharyngeal mucosa tissues(1.15±0.75 vs. 0.32±0.21, t = 3.02, P = 0.009). The relative expression level of ESRRA in nasopharyngeal carcinoma cell lines C666-1 (1.539±0.044), CNE2 (1.420±0.030), TW03-EBV (2.867±0.044), and TW03 (1.323±0.022) were higher than that in normal nasopharyngeal epithelial cell line NP69 (0.094±0.002), and the difference was statistically significant ( F = 34.08, P < 0.001).The results of EdU assay showed that the proportions of EdU labeled positive cells in CNE2 cells of siR-NC group and siR-ESRRA group were (70.44±4.06)% and (51.51±0.92)% ( t = 7.88, P = 0.001), and the proportions in C666-1 cells were (62.25±3.89)% and (54.91±0.27)% ( t = 3.26, P = 0.031). The results of Transwell assay showed that the number of migrating cells in CNE2 and C666-1 cells was less than that in siR-NC group [CNE2 cells: (181±7) cells vs. (261±21) cells; C666-1 cells: (201±16) cells vs. (256±7) cells], and the differences were statistically significant ( t = 6.30, P = 0.003; t = 5.43, P = 0.006). According to qRT-PCR results, the relative expression level of PLIN3 mRNA in the siR-ESRRA group was higher than that in the siR-NC group (CNE2 cells: 1.58±0.16 vs. 0.83±0.17, t = 5.59, P = 0.005; C666-1 cells: 1.37±0.12 vs. 1.06±0.06, t = 3.86, P = 0.018). The dual-luciferase reporter gene assay results indicated a targeted binding interaction between PLIN3 and ESRRA. Transmission electron microscopy observation showed that the lipid droplets in nasopharyngeal carcinoma cells increased and the binding to autophagosomes decreased after knockdown of ESRRA. The results of immunofluorescence assay demonstrated that, in contrast to the siR-NC group, there was a decrease in the co-localization of LC3 and LAMP2 and an increase in the co-localization of lipid droplets with PLIN3. Conclusions:ESRRA is highly expressed in nasopharyngeal carcinoma tissues and cells. As a transcription repressor, ESRRA may work to prevent PLIN3 from being transcribed, decrease lipid droplet stability, mediate lipophagy, and promote proliferation and migration of nasopharyngeal carcinoma cells.

Expression of lncRNA HOXA-AS2 in Gastric Cancer and Its Effect on the Biological Behavior of Gastric Cells / 医学研究杂志

Objective To investigate the expression of LncRNA HOXA-AS2 in gastric cancer tissues and its effect on the malig-nant biology of gastric cancer.Methods The expression levels of lncRNA HOXA-AS2 in gastric cancer tissues and gastric cancer cell lines were detected by qPCR;the effect of lncRNA HOXA-AS2 on the prognosis of gastric cancer patients was analyzed by Kaplan-Meier Plotter,an online website for bioinformatics analysis;the correlation between the expression levels of lncRNA HOXA-AS2 and the clinical and pathological characteristics of gastric cancer patients;cell lines interfering with the expression of lncRNA HOXA-AS2 were constructed,and the effects of down-regulation of lncRNA HOXA-AS2 on the proliferation ability,migration ability and invasion ability of gastric cancer cells were analyzed using CCK8,clone formation assay,scratch assay and Transwell assay.Results The expression lev-el of lncRNA HOXA-AS2 was significantly upregulated in gastric cancer tissues compared with paraneoplastic tissues;the expression lev-el of lncRNA HOXA-AS2 was significantly higher in gastric cancer cell lines compared with human normal gastric mucosal cells GES(P＜0.05);survival analysis showed that high expression of lncRNA HOXA-AS2 was associated with poor prognosis of gastric cancer patients;lncRNA HOXA-AS2 expression level correlated with gastric cancer stage,lymph node metastasis and differentiation(P＜0.05);the expression level of lncRNA HOXA-AS2 was significantly decreased in gastric cancer cells transfected with SiRNA(P＜0.05),and their cell proliferation,migration,and invasion ability were also significantly decreased(P＜0.05).Conclusion lncRNA HOXA-AS2 plays an oncogene role in gastric cancer and is associated with prognosis.Down-regulation of lncRNA HOXA-AS2 ex-pression can inhibit the proliferation,migration,and invasion ability of gastric cancer cells.

Effect of RhoC silencing on migration and invasion of oral squamous cell carcinoma / 中国病理生理杂志

AIM:To explore the expression of RhoC in oral squamous cell carcinoma(OSCC)and its effects on the malignant biological behavior of OSCC cells.METHODS:The UALCAN and K-M plotter databases,alongside tis-sue sample analyses,facilitated understanding RhoC expression in cancer and its links to clinicopathological traits.Two small interfering RNAs(RhoC-siRNA)were constructed according to the RhoC gene sequence.The mRNA and protein ex-pression levels of RhoC in OSCC cells were determined.The protein levels of FAK,p-FAK,MAPK,p-MAPK,matrix me-talloproteinase-2(MMP-2)and MMP-9 were also examined by Western blot.Furthermore,the invasion and migration of OSCC cells were analyzed by Transwell assay and scratch test.Finally,the pulmonary metastasis model of nude mice was established.RESULTS:The results of the databases showed that RhoC was highly expressed in OSCC tissues,which was closely related to pathological stage,pathological grade and lymph node metastasis,but not significantly related to the sur-vival rate of patients.Furthermore,compared with paracancer tissues,the mRNA and protein expression levels of RhoC were increased in OSCC tissues(P<0.01).Silencing of RhoC prominently reduced the migration and invasion of OSCC cells as well as the protein levels of p-FAK,p-MAPK,MMP2 and MMP9(P<0.05).The protein levels of MAPK and FAK were unchanged(P>0.05).The fluorescence intensity of the experimental group was significantly lower than that of the control group,and the results of HE staining showed that the number of lung nodules in the experimental group was sig-nificantly reduced(P<0.05).CONCLUSION:RhoC can effectively influence the migration and invasion of OSCC cells,and its potential mechanism may be related to FAK/MAPK/MMPs signaling pathway.

Expression of centromere protein-H in adrenocortical carcinoma and its impact on viability and migration of adrenocortical carcinoma cells / 中国病理生理杂志

AIM:To investigate the expression of centromere protein-H(CENP-H)in adrenocortical carcino-ma(ACC)and its relationship with disease progression and prognosis,and to explore the impact of CENP-H gene knock-down on the viability and migration of ACC cells.METHODS:The mRNA expression level of CENP-H in 76 ACC pa-tients and 128 healthy controls,and its correlations with tumor stages and prognosis were analyzed by GEPIA2 database.The mRNA expression of CENP-H in different stages of ACC and its correlation with disease prognosis were further ana-lyzed by ULCAN database.The protein expression of CENP-H was examined by immunohistochemical staining of paraffin-embedded ACC and normal adrenal gland specimens.Knockdown of CENP-H by siRNA(siCENP-H)was performed in human ACC cell line H295R.The viabilty of H295R cells transfected with siCENP-H or siNC was measured by CCK-8 as-say,the cell migration was detected by wound-healing assay,and the protein levels of CENP-H,p-ERK1/2,t-ERK1/2,p-P38,t-P38,p-JNK1/2 and t-JNK1/2 were detected by Western blot.RESULTS:The mRNA level of CENP-H was signifi-cantly higher in ACC than that in normal controls,and was correlated with tumor stages and prognosis.The protein level of CENP-H was significantly higher in ACC specimens than that in normal adrenal gland.Knockdown of CENP-H in H295R cells resulted in decreased cell viability and migration.The protein levels of p-P38 and p-JNK1/2 were decreased in si-CENP-H group.CONCLUSION:CENP-H is highly expressed in ACC,and is correlated with tumor stages and poor prognosis.Knockdown of CENP-H can inhibit the viability and migration of ACC cells,and its mechanism may related to inactivation of P38 and JNK signaling pathways.

Osteopontin activates the PI3K/AKT pathway by upregulating LGALS3BP in promotion of hepatoma cell migration / 中国比较医学杂志

Objective To investigate the effect and mechanism of osteopontin(OPN)in hepatoma cell migration through galectin-3 binding protein(LGALS3BP).Methods Human hepatoma cell lines SMMC-7721,SMMC-P(stably transfected with empty eukaryotic expression vectors),and SMMC-OPN(stably transfected with the OPN gene)were cultured.mRNA expression levels of OPN and LGALS3BP were measured by RT-qPCR.Western blot assays were used to analyze the relative protein expression of OPN and LGALS3BP and PI3K/AKT pathway.Wound healing assays were performed to explore the cell migration ability.After transfection with LGALS3BP-targeting small interfering RNA(si-LGALS3BP)or negative control small RNA(si-NC)into SMMC-OPN cells,cell migration and relative expression of PI3K/AKT pathway-related proteins were assessed.Results Compared with SMMC-7721 and SMMC-P,the migratory ability of SMMC-OPN cells was significantly reinforced,and expression of LGALS3BP was obviously upregulated at both mRNA and protein levels.Moreover,relative expression of p-PI3K/PI3K and p-AKT/AKT proteins was significantly increased.Wound healing assays showed that the si-LGALS3BP obviously suppressed the migratory ability of SMMC-OPN cells.Furthermore,relative expression of p-PI3K/PI3K and p-AKT/AKT proteins in SMMC-OPN cells was significantly decreased after transfection of si-LGALS3BP.Conclusions OPN activates the PI3K/AKT pathway by upregulating LGALS3BP expression to promote hepatoma cell migration.

PIEZO1 Channel is Involved in Electric Field Guided Cell Migration / 生物化学与生物物理进展

ObjectiveDisruption of epithelial layer may instantaneously induce the generation of endogenous electric fields, which was proved to play an important role in guiding the cell migration and promoting wound healing. PIEZO1 is a kind of mechanic sensitive channel, may be regulated by voltage, is proved to involve in chemotactic migration of cells and play an important role in the process of wound healing. In this paper, the role of PIEZO1 and its downstream proteins FAK and integrin β1 in the electric field guided cell migration were investigated by HaCaT cells (human immortalized keratinocyte). MethodsCell migration was tracked by Living Cell Imaging System in directed current (DC) electric field (EF). Inhibitors and RNAi techniques were applied to study the function of PIEZO1 and other related proteins in electric fields. Western blot was used to detect the expression and phosphorylation levels of integrin β1 and FAK in electric field guided migration under EF stimulation. ResultsPiezo1 RNAi as well as Ruthenium red and GsMTx4 treatment all significantly inhibited the electrotaxis of HaCaT cells. Electric field stimulation with GsMTx4 treatment alone increased FAK phosphorylation level and the expression of integrin β1. Electric field promoted the expression level of integrin β1 and the phosphorylation level of FAK. Inhibiting the expression of PIEZO1 by RNAi significantly attenuated the phosphorylation level of FAK under EF stimulation. Inhibition of integrin β1 and FAK by inhibitor significantly decrease the electric field guided cell migration. ConclusionPIEZO1 as well as integrin β1 and FAK are involved in the electric field guided cell migration of HaCaT cells. Electric field signals regulate the expression of integrin β1 and the activation of FAK through PIEZO1-mediated signal pathway to orchestrate cell migration.