Terpinen-4-ol inhibits proliferation of VSMCs exposed to high glucose via regulating KLF4/NF-κB signaling pathway / 中国中药杂志

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

Inhibitory Effect of Liuwei Dihuangwan on Triple-negative Breast Cancer Through MAPKKK1 and KLF4 / 中国实验方剂学杂志

ObjectiveTo study the underlying mechanism of Liuwei Dihuangwan in inhibiting triple-negative breast cancer through mitogen-activated protein kinase kinase kinase 1 （MAPKKK1） and Krüppel-like factor 4 （KLF4）. MethodFour hundreds SPF female Kunming mice aged 11.5 months were palpated once every 3 days. The model mice of spontaneous tumors were randomly divided into a model group （normal saline）， a paclitaxel group （0.025 g·kg-1·d-1， ip， 21 days）， and high-， medium- and low-dose Liuwei Dihuangwan groups （7.2， 3.6， 1.8 g·kg-1·d-1， ig）. Tumor tissues were separated until the moribund stage. The tumor volume and weight were measured， and the tumor inhibition rate and the survival time of the tumor mice were calculated （after 6 months， tumor-free mice were assigned into the normal group）. SPF SD rats were selected to prepare serum samples containing Liuwei Dihuangwan of different concentrations for cell culture， and MAPKKK1 in MDA-MB-231 cells was silenced. The protein expression of MAPKKK1 and KLF4 was detected by immunofluorescence and Western blot. ResultThe in vivo experimental results showed that compared with the conditions of the normal group， the protein expression of MAPKKK1 and KLF4 in tumor tissues of the model group dropped （P<0.01）. Compared with the model group， all medication groups showed reduced tumor volume and weight （P<0.05， P<0.01）， increased tumor inhibition rate， prolonged survival time of tumor mice （P<0.05）， and increased protein expression of MAPKKK1 （P<0.01）. Additionally， the paclitaxel group and the high-dose Liuwei Dihuangwan group exhibited increased protein expression of KLF4 （P<0.01）. The in vitro experiments showed that compared with the conditions of the normal group， the fluorescence intensities of MAPKKK1 and KLF4 in MDA-MB-231 cells in all medication groups were potentiated， and the protein expression of MAPKKK1 in the paclitaxel group and the high- and medium-dose Liuwei Dihuangwan groups， and the protein expression of KLF4 in the paclitaxel group and high-dose Liuwei Dihuangwan group increased （P<0.01）. After silencing of MAPKKK1， compared with the conditions of the negative plasmid group （unsilenced MAPKKK1）， the fluorescence intensities of MAPKKK1 and KLF4 and the protein expression decreased in the RNAi-27 positive plasmid group （silenced MAPKKK1） （P<0.05， P<0.01）. Compared with the RNAi-27 positive plasmid group， all medication groups had enhanced fluorescence intensities of MAPKKK1 and KLF4 and protein expression to different degrees （P<0.05， P<0.01）. ConclusionLiuwei Dihuangwan can inhibit the growth of triple-negative breast cancer， and the underlying molecular mechanism is related to the up-regulation of MAPKKK1 and activation of KLF4 expression.

The role of KLF4 in LPS induced cardiomyocyte injury / 中华急诊医学杂志

Objective:To investigate the role of KLF4 in lipopolysaccharide induced cardiomyocyte injury.Methods:Primary rat cardiomyocytes were isolated and cultured, and randomly divided into 5 groups: control group, negative control (NC), LPS group, KLF4 overexpression group, KLF4 overexpression+LPS group. MTT method was used to detect cell activity, ROS, SOD 2, GPX and MDA were detected by kit, TNFa, IL-1 β and IL-6 were detected by ELISA. TUNEL staining was used to detect apoptosis. The protein levels of TLR4 and Nrf2 were detected by Western blot.Results:The expression of KLF4 in cardiomyocytes was significantly higher than that in the NC group ( P<0.001). The cell activity of LPS group was significantly lower than that of NC group ( P < 0.001), and that of KLF4 overexpression +LPS group was higher than that of LPS group ( P<0.001). The levels of TNFa, IL-1 β and IL-6 in LPS group were significantly higher than those in the NC group ( P<0.0001), and the levels of TNFa, IL-1 β and IL-6 in KLF4 overexpression +LPS group were lower than those in LPS group ( P<0.0001). The levels of ROS and MDA in LPS group were significantly higher than those in the control group, while the activities of SOD2 and GPX were lower than those in the NC group ( P<0.0001); the levels of ROS and MDA in KLF4 overexpression +LPS group were lower than those in LPS group, while the activities of SOD2 and GPX were higher than those in LPS group ( P<0.0001). The number of apoptosis in LPS group was significantly higher than that in the NC group, and that in KLF4 overexpression +LPS group was lower than that in LPS group ( P< 0.001). The level of TLR4 wan higher and Nrf2 protein in the nucleus of LPS group was lower than that of the NC group. The level of TLR4 was lower and Nrf2 protein in the nucleus of KLF4 overexpression+LPS group was significantly higher than that of LPS group ( P < 0.001). Conclusions:KLF4 can alleviate LPS induced cardiomyocyte injury by regulating TLR4 and NRF2 signals.

Role and Possible Mechanism of LncRNA-MEG3 and KLF4 in Carcinogenesis and Progression of Nasopharyngeal Carcinoma / 肿瘤防治研究

Objective To investigate the role of LncRNAs-MEG3 in the carcinogenesis and progression of nasopharyngeal carcinoma (NPC) and the possible molecular mechanism. Methods qRT-PCR was used to detect the content of MEG3 and miR-543 in NPC cells. Luciferase reporter method was used to study the relation between MEG3 and miR-543, and the changes of cell proliferation and apoptosis induced by MEG3 or KLF4 were analyzed. Western blot was used to detect the expression of KLF4, Bcl-2 and Bax proteins. Results Compared with the control group, the expression of miR-543 in NPC cell line was significantly increased (P < 0.05), while the expression of MEG3 was decreased (P < 0.05). Luciferase report and Western blot showed that MEG3 could regulate the expression of KLF4 by adsorbing miR-543 to inhibit cell proliferation, promote cell apoptosis and affect the expression levels of Bcl-2 and Bax proteins. Conclusion LncRNA-MEG3 could regulate the expression of KLF4 by adsorbing miR-543 and then plays a role in inhibiting the occurrence and development of NPC. It may be a new biomarker for NPC targeted therapy.

Effect and mechanism of cyclanoline on nitrosamine-induced bladder cancer in rats / 中草药

Objective: To investigate the effect and mechanism of cyclanoline on nitrosamine (BBN)-induced bladder cancer in rats. Methods: Male SD rats were randomly divided into control group, model group, Cyc-low dose (20 mg/kg) group, Cyc-high dose (40 mg/kg) group and cisplatin (5 mg/kg) group. Rats were intragastrically administered with 0.5 mL BBN (0.4 kg/L, twice a week for 8 weeks) to establish bladder cancer model. In addition to control and model group given with normal saline, the other groups were intraperitoneally injected with drugs (once a day for 8 weeks). The bladder tissue was collected after experiment. HE staining was used to investigate the histopathological changes of the bladder in rats. The expressions of MMP9 and Ki67 were observed by immunohistochemistry method, and the expressions of KLF4, p21, CyclinD1, E-cadherin, N-cadherin, Vimentin, Wnt, β-catenin in the bladder tissues were detected by Western bloting. Results: Compared with the model group, cyclanoline effectively inhibited the infiltration of cystitis cells, promoted the degeneration of cancer cells, and reduced the proportion of cytoplasm. Cyclanoline significantly decreased the expressions of MMP9 and Ki67 (P < 0.05, 0.01), up-regulated the expressions of KLF4, p21 and E-cadherin (P < 0.05, 0.01), down-regulated the expressions of CyclinD1, Wnt, β-catenin, N-cadherin and Vimentin in bladder cancer tissues (P < 0.05, 0.01). Conclusion: Cyclanoline promotes the expression of KLF4, inhibits Wnt/β-catenin signaling transduction and the epithelial cell-mesenchymal transition of bladder cancer cells, thereby inhibiting the migration and invasion of bladder cancer cells. Meanwhile, cyclanoline regulates the expressions of p21 and CyclinD1 by up-regulating KLF4, affects the proliferation of bladder cancer cells, and thereby delays the pathological process of BBN-induced bladder cancer in rats.

Over-expression of KLF4 regulates EMT and migration of bladder cancer cells via Wnt/β-catenin signaling pathway / 中国肿瘤生物治疗杂志

@# Objective: To investigate the role and mechanism of Krüppel-like factor 4 (KLF4) in regulating epithelial-mesenchymal transition (EMT) and migration of bladder cancer cells. Methods: Bladder cancer 5637 and T24 cell lines that stably over-expressing KLF4 (LV-KLF4, experiment group) were constructed, and the negative control group (LV-NC) was also established; the mRNA and protein expressions of KLF4 were verified by qPCR and WB, respectively. Transwell chamber assay was used to detect the migration ability of cells in LV-KLF4 and LV-NC groups. WB was performed to detect the expression levels of EMT-related markers (E-cadherin, N-cadherin, Vimentin) and Wnt signaling pathway-related proteins. Immunofluorescence technique was used to detect the distribution of β-catenin in cells after over-expression of KLF4. Results: The 5637 and T24 cell lines over-expressing KLF4 gene were successfully constructed. Compared with the LV-NC group, the mRNA and protein expressions of KLF4 increased in LV-KLF4 groups (all P<0.01); the expression of E-cadherin increased (P<0.01), while the expressions of N-cadherin, vimentin, and the expression levels of total β -catenin, nuclear β -catenin, MMP 9 and c-Myc decreased (all P<0.01); moreover, the migration ability of cells decreased significantly (P<0.01); the fluorescence expression of β-catenin in cells also decreased significantly in LV-KLF4 group as compared to LV-NC group. Conclusion: Over-expression of KLF4 gene in bladder cancer cells may inhibit EMT process by regulating Wnt/β-catenin signaling pathway, and further inhibit the migration of bladder cancer 5637 and T24 cells.

Prognostic effects of the expression of Kruppel-like factor 4 and CD44 in patients with esophageal squamous carcinoma / 中华消化杂志

Objective To analyze the expression of Kruppel-like factor 4 (KLF4 ) and CD44 in esophageal squamous cell carcinoma (ESCC) tissues and adjacent non-cancerous tissues ,and to investigate their effects on the prognosis .Methods From June 2012 to September 2016 ,in The Second People′s Hospital of Nanyang ,a total of 100 patients with ESCC who receiving operation were selected .The ESCC tissues and the adjacent non-cancerous tissues (control) of the patients were collected .The expression levels of KLF4 and CD44 protein were detected by immunohistochemistry .The expressions of KLF4 and CD44 at mRNA and protein level of 50 paired fresh tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting ,respectively . T-test ,chi-square ,Kaplan-Meier method and Pearson correlation analysis were performed for statistical analysis .Results The positive expression rate of KLF4 protein in ESCC tissues was 55％ (55/100) ,which was lower than that in adjacent non-cancerous tissues (77％ ,77/100) ,and the difference was statistically significant (χ2 =10 .778 ,P=0 .001) .The positive expression rate of CD44 protein in ESCC tissues was 81％ (81/100) ,which was higher than that in adjacent non-cancerous tissues (11％ ,11/100) ,and the difference was statistically significant (χ2=112 .600 ,P<0 .01) .The expression level of KLF4 mRNA in 43 cases was lower than that in adjacent non-cancerous tissues ,the expression level of CD44 mRNA in 46 cases was much higher than that of adjacent non-cancerous tissues ,and the differences were statistically significant (χ2 =51 .837 and 70 .563 ,both P< 0 .01) .There were statistically significant differences in positive expression rates of KLF4 in cancer tissues between different gender , differentiation degree , invasion depth ,TNM stage and lymph node metastasis (all P<0 .05) .Similarly there were statistically significant differences in positive expression rates of CD 44 in cancer tissues between different differentiation degree ,invasion depth ,TNM stage and lymph node metastasis (all P< 0 .05) .The expression of KLF4 was negatively correlated with CD44 expression ,either at protein level or mRNA level (r= -0 .284、-0 .518 ,both P< 0 .01) .The median survival time of patients with positive KLF4 expression in cancer tissues was 33 months ,which was longer than that of patients with negative KLF4 expression (20 months) ,and the difference was statistically significant (χ2 =4 .021 , P= 0 .019) .The median survival time of patients with positive CD44 expression in cancer tissues was 24 months ,which was shorter than that of patients with negative CD44 expression (37 months) , and the difference was statistically significant (χ2 = 4 .379 , P= 0 .016) .The results of univariate analysis showed that TNM stage ,KLF4 expression and CD44 expression were correlated with overall survival time (all P<0 .05) . The results of multivariate analysis indicated that TNM stage ,lower KLF4 expression and higher CD44 expression were the independent risk factors for survival (all P<0 .05) .Conclusion Lower expression of KLF4 and higher expression of CD44 in ESCC may be closely correlated with its occurrence ,development and prognosis .

MiR-212 Attenuates MPP+-Induced Neuronal Damage by Targeting KLF4 in SH-SY5Y Cells

PURPOSE: Parkinson's disease (PD) is a common age-dependent neurodegenerative disease. MiR-212 has been demonstrated to exert protective effects in several neurological disorders. The present study aimed to investigate the role and underlying molecular mechanism of miR-212 in PD. MATERIALS AND METHODS: 1-methyl-4-phenylpyridinium (MPP+)-induced SH-SY5Y cells were applied as a PD model in vitro. RTqPCR was used to measure the expression of miR-212 and Kruppel-like factor 4 (KLF4) mRNA. Western blot analysis was performed to detect the protein levels of KLF4, Notch1 and Jagged1. Cell viability and apoptosis were determined by the Cell Counting Kit-8 and flow cytometry, respectively. Quantitative analysis of caspase-3 activity, lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1β) was conducted with corresponding ELISA kits. Dual-luciferase reporter assay was employed to evaluate the relationship between miR-212 and KLF4. RESULTS: MiR-212 was downregulated in MPP+-induced SH-SY5Y cells. Also, miR-212 alleviated MPP+-induced SH-SY5Y cell damage, embodied by increased cell viability, decreased caspase-3 activity, LDH release, ROS production, TNF-α, and IL-1β expression, as well as elevated SOD levels. KLF4 was a direct target of miR-212, and miR-212 repressed KLF4 expression in a post-transcriptional manner. Moreover, miR-212-mediated protection effects were abated following KLF4 expression restoration in MPP+-induced SH-SY5Y cells, represented as lowered cell viability and enhanced apoptotic rate. Furthermore, Notch signaling was involved in the regulation of miR-212/KLF4 axis in MPP+-induced SH-SY5Y cells. CONCLUSION: miR-212 might attenuate MPP+-induced neuronal damage by regulating KLF4/Notch signaling pathway in SH-SY5Y cells, a promising target for PD therapy.

miR-375 promotes prostate cancer cell migration and invasion by targeting KLF4 / 重庆医学

Objective To explore the expression,role and mechanism of miR-375 in prostate cancer (PCa) cells.Methods PCa cells were cultured and transfected with plasmid,the migration and invasion of PCa were detected by Transwell;the expression of miR-375 and KLF4 mRNA were detected by qPCR;the expression of KLF4 were detect by Western blot;the potential target genes of miR-375 were analyzed by bioinformatics and verified by dual luciferase report.Results The expression of miR-375 were significantly up-regulated in the PCa;Inhibited the expression of miR-375 could significantly inhibit the migration and invasion of PCa cells.KLF4 was the potential target genes of miR-375,which verified through bioinformatics analysis and dual luciferase report.The expression of KLF4 were significantly down-regulated in the PCa.Inhibited the expression of miR-375 could significantly up-regulated the expression of KLF4.Inhibited the KLF4 expression was able to reverse the suppressive effect miR-375 has on the migration and invasion of PCa cells.Conclusion miR-375 promotes the migration and invasion of PCa via inhibiting the expression of KLF4 and play the oncogene role.MiR-375 can be used as therapeutic targets for PCa,and provide a new direction for the treatment of PCa.

Expression and prognostic significance of SP1, KLF4 and p21 in ovarian serous carcinoma / 临床与实验病理学杂志

Purpose To explore the difference of expression and prognostic significance of SP1,KLF4 and p21 in low grade ovarian serous carcinoma (LGSC) and high grade ovarian serous carcinoma (HGSC).Methods The expression of SP1,KLF4 and p21 protein was examined with immunohistochemistry EliVision method in cases with LGSC and HGSC.Kaplan-Meier analysis and Cox multivariate survival analysis were used to assess the impact of SP1,KLF4 and p21 expression on prognosis of LGSC and HGSC.Results SP1,KLF4 and p21 expression were detected respectively in 74.5％,17.0％ and 11.7％ HG-SC cases,and in 65.2％,34.8％ and 26.1％ LGSC cases.Compared to control group,the expression level of SP1 was significantly higher (P ＜ 0.05),but the expression level of KLF4 and p21 were significantly lower (P ＜0.05).There was no significant difference of SP1,KLF4 and p21 expression between HGSC and LGSC (P ＞ 0.05).The expression of SP1,KLF4 and p21 were associated with FIGO stage,meanwhile SP1 associated with residual tumor size in HGSC (P ＜ 0.05).There was a significant negative correlation between SP1 and KLF4,p21 proteins in HGSC (P ＜ 0.05).Kaplan-Meier analysis revealed that there were significantly poor overall survival (OS) of 5 years for patients with HGSC displaying high expression of SP1,or low expression of KLF4 and p21 (P ＜0.05),but no significantly improved OS for patients with LGSC (P ＞ 0.05).Cox analysis showed that SP1 overexpression is an independent prognosis factor for HGSC.Conclusion Overexpression of SP1 and low expresion of KLF4 and p21 contribute to carcinogenesis of HGSC and LGSC.They are associated with a poor prognosis of HGSC,but not LGSC,meanwhile SP1 is an independant prognosis factor for HGSC.

miR-25 promotes gastric cancer cells proliferation by directly targeting KLF4 / 实用医学杂志

ObjectiveTo investigate whether miR-25 targets KLF4 to regulate the growth of gastric cancer cells and lucidate the mechanism of miR-25 producing functional effects in gastric cancer cells. Methods The expression levels of miR-25 and KLF4 were detected by qRT-PCR in 35 pairs of gastric cancer tissues of patients. Target genes of miR-25 were predicted using Targetscan and verified though dual luciferase activity assay and west-ern blot. MTT assay and clone formation assay were detected after downregulated miR-25 and overexpressed KLF4 respectively. Results miR-25 was highly expressed in gastric cancer tissues with KLF4 downexpresstion. Downreg-ulated miR-25 and overexpressed KLF4 respectively can significantly reduce the proliferation of gastric cancer cells,the results were opposite when overexpression of KLF4. Conclusion miR-25 promotes gastric cancer cells proliferation by targeting KLF4.

Effect of tetramethylpyrazine combined with cisplatin on VEGF, KLF4 and ADAMTS1 in Lewis lung cancer mice

Objective To further explore the function of combine use of tetramethylpyrazine (TMP) and cisplatin (DDP) in lung carcinoma. Methods We used the combination drug to treat Lewis lung cancer mice, investigated the expression level of vascular endothelial growth factor (VEGF), Kruppel-like factor 4 (KLF4) and A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) and to further explore the inhibitory effects and potential mechanism of TMP combined with DDP on tumor angiogenesis. Results The tumor growth was suppressed in TMP group, DDP group and TMP combined with DDP group. Furthermore, the weights and volume of tumor, the expression level of VEGF, KLF4 and ADAMTS1 were found significantly changed between experiment group and control group. These findings suggest that TMP with DDP had additional or synergistic effects to inhibit the tumor growth effectively, might be achieved through reducing the expression of angiogenesis promoting factor VEGF and increasing expression of angiogenesis inhibitors KLF4 and ADAMTS1. Conclusion KLF4 and ADAMTS1 may be synergically involved in the angiogenesis in mouse Lewis lung cancer through the different signal ways.

KLF 4 knockdown inhibits epithelial-mesenchymal transition, cell migration and invasion of prostate cancer cells / 肿瘤

Objective: To investigate the effects of Krüppel-like factor 4 (KLF 4) knockdown on epithelialmesenchymal transition (EMT), cell migration and invasion of prostate cancer cells. Methods: The prostate cancer cells with stable knockdown of KLF 4 named as LNCaP-shKLF4 cells and the LNCaP-con cells as the control were constructed. The expressions of KLF4 mRNA and protein in LNCaP-con and LNCaP-shKLF4 cells, and the expressions of EMTassociated gene mRNA and protein in the prostate cancer PC3-shKLF4 cells with stable knockdown of KLF 4 and the PC3-con cells (as the control) as well as the LNCaP-shKLF4 and LNCaP-con cells were detected by real-time fluorescence quantitative-PCR and Western blotting, respectively. The abilities of migration and invasion of PC3-shKLF4 and PC3-con cells as well as LNCaP-shKLF4 and LNCaP-con cells were detected by Transwell chamber assay. Results: LNCaP-shKLF4 and LNCaP-con cells were successfully constructed. The expression levels of KLF4 mRNA and proteins in LNCaP-shKLF4 cells were lower than those in LNCaPcon cells (P<0.01, P<0.05). The expression levels of E-cadherin (E-cad) mRNA in PC3- shKLF4 and LNCaP-shKLF4 cells were higher than those in PC3-con and LNCaP-con cells, respectively (both P<0.01). The expression levels of N-cadherin (N-cad), Zinc finger E-box-binding homeobox 1 (Zeb1), Snail1, vimentin (Vim) and matrix metallopeptidase 1 (MMP1) mRNAs in PC3-shKLF4 and LNCaP-shKLF4 cells were lower than those in PC3-con and LNCaP-con cells, respectively (all P<0.05). The expression levels of E-cad protein in PC3-shKLF4 and LNCaP-shKLF4 cells were higher than those in PC3-con and LNCaP-con cells, respectively (both P<0.05). The expression levels of N-cad, Zeb1, Snail1, Vim and MMP1 mRNAs in PC3-shKLF4 and LNCaP-shKLF4 cells were lower than those in PC3-con and LNCaP-con cells, respectively (all P<0.05). The abilities of migration and invasion of PC3-shKLF4 and LNCaP-shKLF4 cells were weaker than those of PC3-con and LNCaP-con cells, respectively (P<0.01, P<0.05). Conclusion: KLF 4 knockdown in prostate cancer cells can activate the expression of epithelium-associated genes and inhibit the expressions of mesenchymal-associated genes, resulting in the inhibition of cell migration and invasion of prostate cancer cells in vitro.

Effect of tetramethylpyrazine combined with cisplatin on VEGF, KLF4 and ADAMTS1 in Lewis lung cancer mice

OBJECTIVE@#To further explore the function of combine use of tetramethylpyrazine (TMP) and cisplatin (DDP) in lung carcinoma.@*METHODS@#We used the combination drug to treat Lewis lung cancer mice, investigated the expression level of vascular endothelial growth factor (VEGF), Kruppel-like factor 4 (KLF4) and A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) and to further explore the inhibitory effects and potential mechanism of TMP combined with DDP on tumor angiogenesis.@*RESULTS@#The tumor growth was suppressed in TMP group, DDP group and TMP combined with DDP group. Furthermore, the weights and volume of tumor, the expression level of VEGF, KLF4 and ADAMTS1 were found significantly changed between experiment group and control group. These findings suggest that TMP with DDP had additional or synergistic effects to inhibit the tumor growth effectively, might be achieved through reducing the expression of angiogenesis promoting factor VEGF and increasing expression of angiogenesis inhibitors KLF4 and ADAMTS1.@*CONCLUSION@#KLF4 and ADAMTS1 may be synergically involved in the angiogenesis in mouse Lewis lung cancer through the different signal ways.

Over-expression of KLF4 inhibits the proliferation and migration of K562 cell line / 基础医学与临床

Objective To study the effect of over-expression of KLF 4 on the proliferation and migration ability of K562 cells.Methods The K562 cells with KLF4 over expression ( K562-KLF4 cells) were used as experimental group, and paralleled with the vector control ( K562-C1 cells) and blank K562 cell control.Real-time PCR was performed to detect the relative expression quantity of KLF4 mRNA of each group cells respectively;Western blot was performed to detect the level of KLF 4 protein of each group cells respectively .The cell proliferation was tested by MTS assay.The migration ability of K562 cells was detected by Transwell .Results Compared with K562-C1 cells and blank K562 cells, the relative expression of KLF4 mRNA of K562-KLF4 cells was significantly increased (P<0.05).The level of KLF4 protein of K562-KLF4 cells was also significantly increased, by 77.78%(P<0.05).The proliferation ability and migration ability of the K 562 cells with over-expressing KLF4 were inhibited sig-nificantly (P<0.05).Conclusions Over-expression of KLF4 could inhibit the proliferation and migration ability of K562 cells.

Stemness and Proliferation of Murine Skin-Derived Precursor Cells under Hypoxic Environment

Skin-derived precursors (SKPs) have potential to differentiate to various cell types including osteoblasts, adipocytes and neurons. SKPs are a candidate for cell-based therapy since they are easily accessible and have multipotency. Most mammalian cells are exposed to a low oxygen environment with 1 to 5% O2 concentration in vivo, while 21% O2 concentration is common in in vitro culture. The difference between in vitro and in vivo O2 concentration may affect to the behavior of cultured cells. In this report, we investigated the effect of hypoxic condition on stemness and proliferation of SKPs. The results indicated that SKPs exposed to hypoxic condition for 5 days showed no change in proliferation. In terms of mRNA expression, hypoxia maintained expression of stemness markers; whereas, oncogenes, such as Klf4 and c-Myc, were downregulated, and the expression of Nestin, related to cancer migration, was also downregulated. Thus, SKPs cultured in hypoxia may reduce the risk of cancer in SKP cell-based therapy.

Effect of Kruppel-like factor 4 on Notch pathway in hepatic stellate cells / 华中科技大学学报（医学）（英德文版）

The relationship between Kruppel-like factor 4 (KLF4) and the Notch pathway was determined to investigate the effect of KLF4 on the activation of hepatic stellate cells and underlying mechanisms. Fifty SPF BALB/c mice were randomly divided into two groups. A liver fibrosis model was established in 25 mice as the experimental group, and the remaining 25 mice served as controls. On the day 0, 7, 14, and 35, liver tissues were removed for immunofluorescent detection. The Notch pathway inhibitor DAPT was added to the primary original hepatic stellate cells, and KLF4 and Notch-associated factor expression was detected by qRT-PCR. Additionally, the hepatic stellate cell line LX-2 was used to establish control and experimental groups, and was cultured in vitro. LX-2 cells in the experimental groups were treated with DAPT and the Notch activator transforming growth factor-beta 1 separately, whereas those in the control group were given isotonic culture medium. After 48 h, KLF4 expression was examined by Western blotting. After transient transfection of LX-2 cells to increase KLF4, the expression of Notch factor was examined. Immunofluorescence analysis showed that, with the aggravation of liver fibrosis, the absorbance (A) values of KLF4 were decreased (day 0: 980.73±153.19; day 7: 1087.99±230.23; day 14: 390.95±93.56; day 35: 245.99±87.34). The expression of Notch pathway- related factors (Notch-1, Notch-2, and Jagged-1) in the hepatic stellate cell membrane was negatively correlated to KLF4 expression. With the increase of KLF4 expression, Notch-2 (0.73±0.13) and Jagged-1 (0.43±0.12) expression decreased, whereas Notch-1 level was not detectable. When the Notch pathway was inhibited, KLF4 levels generally increased (18.12±1.31). Our results indicate that KLF4 expression is negatively correlated to the Notch pathway in hepatic stellate cells, which may provide a reference for the treatment of hepatic fibrosis.

Effects of 5-Aza-CdR on growth of TPC-1 cell line and KLF4 expression / 中华内分泌外科杂志

Objective Methylation of anti-oncogene can be demethylated by related drugs which can help the inactivated gene to express again .This study aims to study the effects of the demethylating agent 5-Aza-2′-deoxycytidine on the growth of human thyroid papillary cancer cell line TPC-1 and mRNA and protein expres-sion of KLF4.Methods TPC-1 cells were treated with different concentration of 5-Aza-CdR.MTT was used to detect the influence of 5-Aza-CdR on cell proliferation .RT-PCR was used to detect mRNA and protein expression levels of KLF4.Results After being treated with 5-Aza-CdR for 24 hours, 48 hours, and 72 hours, the growth of TPC-1 cells was inhibited and the inhibition was in time and concentration depended manner .After treatment with 5-Aza-CdR, mRNA and protein expression levels of KLF 4 were increased, and the difference had statistical significance(P<0.05).Conclusion 5-Aza-CdR can inhibit the cell viability of TPC-1 cells through upregulat-ing KLF4 expression , which may provide experimental basis for 5-Aza-CdR in treating thyroid cancer .

The effect of KLF4 on invasion and migration by regulating MMP9 in hepatocellular carcinoma / 重庆医学

Objective To investigate the effects of Krüppel like factor 4 (KLF4)on matrix metalloproteinase 9 (MMP9)ex-pression in hepatocellular carcinoma(HCC).Methods A total of 50 primary hepatocellular carcinoma samples and their correspond-ing adjacent tissues specimens were collected.The expression of KLF4 and MMP9 were detected by IHC,Western blot and qRT-PCR.After KLF4 gene was transfected into hepatocellular carcinoma cell line (HepG2 cell line),the expressions of KLF4 and MMP9 were conformed by qRT-PCR and Western blot.Migration and invasion of HepG2 cell line transfected by KLF4 were detec-ted by wound-healing assay and invasion assay.Results Compared to corresponding adjacent tissues,The expression of KLF4 was significantly lower in HCCs(P <0.05),and MMP9 expression was remarkably higher in HCCs(P <0.05).KLF4 over-expression inhibited the expression of MMP9 on the protein and mRNA levels.Wound-healing assay and invasion assay confirmed that KLF4 regulated cell invasion and migration through regulating MMP9 expression.Conclusion KLF4 showed low expression in HCCs,and MMP9 was overexpressed.Up-regulation of KLF4 could decrease the expression of MMP9 in HepG2 cell line,which inhibited inva-sion and migration.

Study the roles of KLF2, KLF4 in prediction of acute lung injury in rats induced by endotoxin / 中华急诊医学杂志

Objective To investigate the expressions of KLF2 mRNA and KLF4 mRNA in the acute lung injury (ALl) rats induced by lipopolysaccharide (LPS),and to analyze the correlation between KLF2,KLF4 and ALI.Methods A total of 100 SD rats were randomly divided into 2 groups:normal control group and LPS treated group,then the latter group was randomly further divided into 3 subgroups as per the serum and lung tissue samples taken separately at 2,4 and 24h after modeling.The ALI model was made by injecting 5mg/kg LPS into tail vein.The pathological changes of lung tissue were observed in each group,and the expressions of KLF2,KLF4 mRNA in serum and lung tissue were detected by RT-PCR.The data of laboratory findings were analyzed with SPSS 17.0 software for statistical analysis.Results The histopathological changes showed the most obvious damage of lung tissue occurred at 4 hours after modeling.The expressions of KLF2 mRNA and KLF4 mRNA in the lung tissue and serum of control group were significantly higher compared to LPS treated subgroups (P ＜0.01).The expression of KLF2 mRNA in LPS treated subgroup at 2 hours was lower than that in LPS subgroups at 4 hours and 24 hours (P ＜ 0.01),while the expression of KLF4 mRNA in LPS treated subgroup at 4 hours was lower than that in LPS treated subgroups at 2 hours and 24 hours (P ＜ 0.01).Conclusions The expression of KLF2 mRNA was occurred earlier than the pathological changes in acute lung injury,while the expression of KLF4 was emerged synchronously,and both KLF2 and KLF4 could be used as candidates of predictive and diagnostics molecular markers of ALI.