MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell / 中国医学科学院学报

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

Upregulated Long Noncoding RNA LINC01234 Predicts Unfavorable Prognosis for Colorectal Cancer and Negatively Correlates With KLF6 Expression

BACKGROUND: LINC01234, a long noncoding RNA (lncRNA), is overexpressed in several cancers, including colorectal cancer (CRC). We investigated the role of LINC01234 in CRC development and confirmed its correlation with Krüppel-like factor 6 (KLF6), a tumor suppressor gene that is dysregulated in CRC. METHODS: We tested mRNA levels using quantitative reverse transcription PCR (qRT-PCR). Tissue samples from patients with CRC, inflammatory bowel disease (IBD), hyperplastic polyp, and adenoma were included. Correlations between clinicopathological parameters, overall survival (OS) rate, and LINC01234 were analyzed using Kruskal-Wallis H test. Additionally, cell proliferation, apoptosis, and tumor formation in nude mice were tested to investigate the mechanism of LINC01234. Western blotting was used to determine protein levels. RESULTS: LINC01234 expression was significantly upregulated in CRC tissues and CRC cell lines than in non-tumor tissues and normal epithelial cells, respectively. LINC01234 was associated with high tumor stage, larger tumor size, and metastasis. Patients with higher LINC01234 expression showed reduced OS. Cell proliferation was inhibited by LINC01234 knockdown, whereas apoptosis was enhanced. Mice injected with SW480 cells with LINC01234 knockdown displayed decreased tumor volume, weight, and Ki-67 levels compared with those injected with control cells. KLF6 was negatively regulated by LINC01234. Overexpression of KLF6 showed effects similar to those observed following LINC01234 knockdown on cell proliferation and apoptosis. CONCLUSIONS: LINC01234 could be a prognostic biomarker in CRC patients. Upregulation of LINC01234 in CRC promotes tumor development through negative regulation of KLF6.

Regulation of Krüppel-like factor 6 via activating transcription factor 4 pathway to apoptosis of human lens epithelial cells / 中华实验眼科杂志

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.

Effect of high glucose on the expression of KLF6 in human lens epithelial cell / 中华实验眼科杂志

Background Epithelial-mesenchymaltransition (EMT)isamajorcontributortothe pathogenesis of posterior capsular opacification(PCO).Kruppel-like factor 6 (KLF6) is a zinc finger protein,which can be stimulated by high glucose in proximal tubule cells and involved in transforming growth factor beta (TGF-β)induced EMT of diabetic nephropathy.ObjectiveThis study was designed to investigate the effect of high glucose on the expression of KLF6 and its target genes( TGFB1,TGFBR1,COLIA1,HSP47) in human lens epithelial cells (LECs).MethodsHuman LECs(SRA01/04) were cultured and exposed to different concentration of glucose.The expressions of KLF6 mRNA and protein were analyzed by real time polymerase chain reaction( real time PCR) and western blot after treatment with high glucose.The expressions of KLF6 target genes were analyzed by real time PCR to evaluate the EMT of SRA01/04 cells.ResultsCompared with the control group(5.5 mmol/L),the relative mRNA levels of t-KLF6 and wt-KLF6 in SRA01/04 treated with high glucose(22.2,44.4,66.6 mmol/L) increased obviously (F =72.53,42.02,P＜0.01 ).Then,the concentration of 22.2 mmol/L was used in the next experiments.The relative mRNA levels of t-KLF6 and wt-KLF6 increased to the peaks after treatment with high glucose for 12 h,and began to decrease after 24 h until lower levels after 48 h ( F =100.12,125.52,P ＜ 0.01 ).Western blot showed that the expression of KLF6 protein was also upregulated by high glucose treatment.With the promotion of the expression of KLF6 gene,the relative mRNA levels of TGFB1,TGFBR1,COLlAl and HSP47 of treated cells also respectively increased after treatment for 12 h,and began to decrease after 24 h until nearly at the levels of the control groups after 48 h( F=6.73,162.35,64.39,12.05,P＜0.05 ).ConclusionsIt was concluded that high glucose induced the expression of KLF6 in human LECs,and KLF6 transiently stimulated the expression of target genes TGFB1,TGFBRl,COLlAl and HSP47 which were mainly involved in the mechanism of EMT.

Loss of expression of wtKLF6 in hepatocellular carcinoma and its effect on hepatocellular carcinoma cell proliferation / 中华肝胆外科杂志

Objective To investigate the expression and genetic alterations of KLF6 in hepatocellular carcinoma (HCC) and explore their functional mechanisms in the oncogenesis and development of HCC. Methods Real-time quantitative-PCR, direct sequencing and LOH approaches were used to detect KLF6 genetic abnormalities in HCC. The experiment had 2 groups, an experimental group and a control group. In the experimental group, the transfected plasmid pcDNA3.0 was recombined with KLF6 and tranfected into HCC HepG2 cells. MTT, flow cytometry and Western blotting were used to observe the effect of anti-oncogene wild type KLF6 on HepG2 cells by transgenic method for 48 h.Results Expression levels of KLF6 were significantly downregulated in HCCs(P＜0. 01), as detected by qRT-PCR. LOH occurred in 11 (52%) of the 21 tumors, and all the samples with LOH showed KLF6 down-regulation. The mutational frequency was 29%, and sequence changes located in activation domain of KLF6. Meanwhile, MTT assay showed a significant antiproliferative effect of the transfected wtKLF6 on HepG2 cells(42.7%, P＜0.05). Fluorescence-activated cell sorting analysis revealed that KLF6 induced apoptosis. Conclusion The deregulation of KLF6 together with genetic abnormalities of allelic imbalance and mutations may play an important role in HCC pathogenesis.

Mechanism and expression of KLF6 tumor suppressor gene in related tumors / 国际肿瘤学杂志

It has been revealed that the major mechanism of KLF6 is to upregulate p21 in an p53-independent manner leading to inhibition of cell proliferation. Lacking of KLF6 activity or its abnormal expression may be related to multiple tumors'development and prognosis such as prostate cancer, liver cancer, gastric cancer and other tumors. Furthermore, KLF6 may become a novel candidate for molecular target therapy.

Frequent Down-regulation and Deletion of KLF6 in Primary Hepatocellular Carcinoma / 华中科技大学学报（医学）（英德文版）

Kruppel-like factor 6 (KLF6) was reported as tumor suppressor in multiple cancers.However,loss of chromosomal locus spanning KLF6 is relatively infrequent in previous published studies.To explore the role of KLF6 in hepatocellular carcinoma (HCC),we examined the gene for expression change,loss of heterozygosity (LOH) and mutation in 26 HCC samples.The expression levels of KLF6 were significantly down-regulated in HCCs,as detected by qRT-PCR.LOH occurred in 11 (52%) of 21 tumors,and all the samples with LOH showed KLF6 down-regulation.The mutational frequency was 24%,and sequence changes located in activation domain of KLF6.Furthermore,MTT assay showed a significant antiproliferative effect of the wt KLF6 transfected in HepG2 hepatoblastoma cells.Fluorescence-activated cell sorting analysis revealed that KLF6 could induce apoptosis.These findings indicate that deregulation of KLF6,together with genetic abnormalities of allelic imbalance and mutations,may play a role in HCC pathogenesis.