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The pathogenesis of hepatocellular carcinoma is complex and has not been fully clarified. KLF4, also known as gut-enriched Kruppel-like factor (GKLF), plays the dual regulatory role of tumor suppressor and tumor promoter. KLF4 plays an anti-cancer role in liver cancer. This article introduces the biological function and regulatory mechanism of KLF4 in the progression of liver cancer, and research on the mechanism of action of KLF4 in liver cancer is expected to provide new ideas for targeted therapy and prognosis monitoring of liver cancer.
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The pathogenesis of hepatocellular carcinoma is complex and has not been fully clarified. KLF4, also known as gut-enriched Kruppel-like factor (GKLF), plays the dual regulatory role of tumor suppressor and tumor promoter. KLF4 plays an anti-cancer role in liver cancer. This article introduces the biological function and regulatory mechanism of KLF4 in the progression of liver cancer, and research on the mechanism of action of KLF4 in liver cancer is expected to provide new ideas for targeted therapy and prognosis monitoring of liver cancer.
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Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.
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Objective To construct and identify a lentiviral vector carrying mouse Krüppel-like factor 4 (KLF4) gene, and establish RAW264.7 cell line of peritoneal macrophages that over-expressed KLF4. Methods KLF4 gene was cloned using the measure of polymerase chain reaction (PCR). Then the recombinant transfer vector pLVX-KLF4 (pLVX-KLF4-mCMV-ZsGreen-PGK-Puro) was constructed. The pLVX-KLF4 was confirmed through PCR, restriction enzyme digestion and sequencing. The correct recombinant transfer vector together with its two helper virus vectors (psPAX2 and pMD2.G) were cotransfected into the 293T cells by Lipofectamine? 3000. The supernatant of 293T was harvested to infect RAW264.7 cells. Flow cytometry (FCM) was used to test the viral titer of the expression level of green fluorescent protein. The expression of KLF4 mRNA in RAW264.7 cells was measured by real-time PCR. Results The restriction enzyme digestion, PCR and sequencing confirmed that the transfer lentiviral vector pLVX-KLF4 was constructed successfully. KLF4 mRNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells (P<0.05). In transfected RAW264.7 cells, KLF4 mRNA was over-expressed (P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was 2.05×108 TU/mL measured by FCM.The flow cytometry results showed that the S phase fraction was prolonged and G0/G1 was arrested in the over-expressed KLF4 of RAW264.7 cells. The EdU showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells. Conclusion The recombinant lentiviral vector, which can effectively express KLF4 mRNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.
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Objective To study the expression and targeted relationship of miR-103/KLF4 (Krüppel like factor) in A549 and resistant cell lines of lung cancer.Methods To culture the A549 cell lines and A549/DDP (cisplatin) resistant cell lines in vitro and detect survival rates by methyl thiazolyl tetrazolium (MTT) method.Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to test the expression of miR-103/KLF4 of these cell lines.Dual-luciferase reporter gene experiment to detect the targeted relationship.Results A higher expression of miR-103 and a obvious lower expression of KLF4 were observed in A549/DDP resistant cell lines.MiR-103 target KLF4-3'UTR (3'untranslated regions) directly in lung cancer cells.Conclusions In A549/DDP resistant cells,miR-103 can regulate KLF4 at target.
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Objective To observe the morphologic changes of rat peritoneum tissue in different age groups, so as to explore the age-related molecular and pathology characteristics. Methods Eighteen male SD rats were evenly randomized into three groups:1-month group (the juvenile group); 8-month group (the youth group); and 18-month group (the elderly group). The morphological changes of parietal peritoneum were observed by Hematoxylin-Eosin and β-gal staining. The peritoneal thickness, peritoneal mesothelial cells and blood vessel count under unit area were analyzed semi-quantitatively. The expressions of transforming growth factor-beta 1(TGF-β1) and krüppel like factor 15(KLF15) mRNA or protein in the rat mesenteric tissues were evaluated by RT-PCR and Western blotting analysis. Results The peritoneum gradually thickened with the increase of age, accompanied by reduced peritoneal mesothelial cells and increased interstitial vascular proliferation (P<0.05). The expression of TGF-β1 was gradually increased (P<0.05) and that of KLF15 was decreased (P<0.05) at mRNA and protein levels, and the expression of TGF-β1 was negatively associated with KLF15. Conclusion It is speculated that TGF-β1 may play a role in promoting the peritoneal aging process, while the KLF15 factor may inhibit the peritoneal senescence.
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Objective To observe the inhibitory effect of atorvastatin on bleomycin-induced pulmonary fibrosis in SD rats and study their possible mechanism.Methods 30 male SD mice under SPF condition with average body weight of 250g were randomly allocated to three groups (n =10,each) of saline control group (control group),bleomycin-induced pulmonary fibrosis group (pulmonary fibrosis group) and atorvastatin treatment group (treatment group).Bleomycin (5mg/kg) (versus 0.2 ml saline in control group) were endotracheally instilled in pulmonary fibrosis group and the treatment group in order to establish the model of pulmonary fibrosis.Subsequently,the rats in the treatment group received daily atorvastatin (10 mg/kg) orally.5 rats in each group were sacrificed on 7th and 28th day after intratracheal instillation.Their lung tissues were taken and tested.The histological changes in the lungs were evaluated by hematoxylin-eosin and masson stain.The tumor necrosis factor (TNF-α) level and hydroxyproline content in lung tissues were measured by enzymelinked immunosorbent assay (ELISA).The expressions of Kruppel-like factor 2 (KLF2) protein and mRNA in lung tissues were measured by Western blotting and Real-Time PCR.Results The lung tissue in model group had significant bleeding and oozing inflammatory response on the 7th day and pulmonary fibrosis on the 28th day.Bleeding and oozing inflammatory response and pulmonary fibrosis were subdued in treatment group on the same days as compared to the model group.Hydroxyproline and TNF-α contents in lung tissue were significantly higher in model group than in control group (both P＜0.05).KLF2 protein and KLF2-mRNA expressions in lung tissues were significantly lower in model group than in control group (both P＜0.05).The above changes were partially reversed in treatment group.Compared to model group,treatment group showed that hydroxyproline and TNF-α contents in lung tissues were significantly reduced (both P＜0.05) and KLF2 protein and KLF2 mRNA expressions in lung tissues were significantly increased (both P＜ 0.05).Conclusions Atorvastatin can reduce the secretion of TNF-α and alleviate bleomycin-induced pulmonary fibrosis.The mechanism inhibiting fibrosis might be associated with up-regulation of KLF2-mRNA expression.
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Objective: To investigate the regulating effect of Krüppel-like transcription factor 8 (KLF8) on the expression of vascular endothelial growth factor A (VEGFA) in hepatocellular carcinoma (HCC) and its potential mechanism. Methods: The expressions of KLF8 and VEGFA proteins and their correlation in paired HCC and adjacent paracancerous tissues were detected by immunohistochemistry staining. The KLF8 expression plasmid pcDNA3.1-KLF8 and the control plasmid were transfected into HCC SMMC7721 cells, respectively. The expressions of VEGFA and phosphoinositide-3-kinase (PI3K)/Akt pathway-related molecules including phospho-Akt (p-Akt), phospho-phosphoinositide-dependent kinase 1 (p-PDK1) and phospho-phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN) in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting. The effect of PI3K/Akt pathway inhibitor LY294002 on the expression of VEGFA protein in SMMC7721 cells with KLF8 overexpression was detected by Western blotting. Chicken chorioallantoic membrane (CAM) model was used to detect the inducing effect of SMMC7721 cells with up-regulation of KLF8 expression on angiogenesis in chicken embryos. Results: The expression levels of KLF8 and VEGFA proteins were much higher in HCC tissues than those in the adjacent paracancerous tissues (P < 0.01). KLF8 protein expression was positively correlated with VEGFA protein expression in HCC tissues (r = 0.622, P < 0.01). The expression levels of KLF8, VEGFA, p-Akt, p-PDK1 and p-PTEN proteins were up-regulated in SMMC7721 cells transfected with pcDNA3.1-KLF8 plasmids (P < 0.05). LY294002 inhibited VEGFA expression in SMMC7721 cells with KLF8 overexpression (P < 0.05). The SMMC7721 cells with KLF8 overexpression had a higher potential in inducing angiogenesis in chick embryos (P < 0.01). Conclusion: KLF8 gene induces VEGFA expression and angiogenesis in HCC via PI3K/Akt signal pathway.
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Objective To investigate the correlation between Kruppel-like transcription factor 6 (KLF6) and gastric cancer.Methods By using immunohistochemistry (IHC) and reverse transcription PCR,the expression of KLF6 protein and mRNA in human gastric carcinoma specimens and adjacent tissues from 73 patients were examined.Results The KLF6 protein expression rates were 39.7 ％ (29/73) and 90.41％(66/73) in 73 cases of gastric cancer and adjacent tissues,The KLF6 protein expression was significantly lower in human gastric carcinomas than the adjacent tissues by chi-square test (P ＜ 0.01).The KLF6 expression rates were 25.0 ％ (10/40),57.6 ％ (19/33) in poorly and well differentiated cancer.The difference between the two groups was significant (P ＜ 0.01).The optical density ratio of KLF6 mRNA were 0.1586±0.1071 and 0.8899±0.0638 in the gastric cancer and cancer adjacent tissue respectively,the difference between the two groups was significant (P ＜ 0.01).Conclusion KLF6 expression is low in gastric carcinoma,and moreover,KLF6 expression is significantly lower in poorly differentiated cancers than the well differentiated gastric cancers,there are negative correlation between KLF6 and gastric cancer,therefore KLF6 could be a useful marker for gastric cancer.
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Objective To investigate the antitumour effects of transfected gut-enriched Krüppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice.
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Objective To study the mRNA expression of two imprinted genes,paternally expressed gene 1(PEG1) and paternally expressed gene 3(PEG3),and their promoter methylation in human placenta of abnormal birth weight(BW)neonates. Methods Placentas were obtained after full term delivery,without any maternal complications during pregnancy,and were divided into 3 groups according to BW:high BW group(n=22,BW≥4000 g),normal BW group(n=14,BW>2500 g and<4000 g)and low BW group(n=24,BW≤2500 g).The mRNA expression of PEG1 and PEG3 were determined by real-time quantitative polymerase chain reaction.Promoter methylation was measured by the bisulfite genomic sequencing method.Results among different groups were compared. Results (1)The mRNA expression of PEGl and PEG3 were 11.66±9.01 and 16.45±10.13 in the high BW group,0.84±0.49 and 0.85±0.67 in the low BW group and 1.10±0.77 and 1.11±0.60 in the normal BW group,respectively.Both genes were significantly up-regulated in the high BW group compared to the normal BW group(P<0.05),but no significant difference was found between the normal and low BW group (P>0.05). (2) The levels of PEG1 promoter methylation were (49. 7± 2. 3) %, (50. 2 ± 2. 1 )% and (50. 3 ± 1.9)% in the high, low and normal BW group (P>0.05), while the levels of PEG3 promoter methylation in the high BW was significantly lower than in the normal BW group [(13.1± 2. 7) % vs (16.2 ±1.8)%, P<0. 05], but no difference was shown between the low BW group (16.7± 3.5)% and the normal BW group (P>0.05). (3)Negative correlation was detected between the expression of PEG:3 and DNA methylation level within the objective fragment of promoter region (r= -0. 963, P<0. 01). Conclusions The up-regulation of PEG1 and PEG3 may be associated with high BW. The reduction of methylation pattern in the promoter region of PEG3 might be involved in the up-regulation of PEG3 and contribute to the mechanism of high BW fetus in turn.