KLF8 Promotes Tumorigenesis, Invasion And Metastasis Of Colorectal Cancer Cells By Transcriptional Activation Of FHL2

Backgroud and ObjectionAs a novel-identigied oncogene, FHL2 (Four and a half LIM Protein 2) played important role in the development and progression of gastrointestinal (GI) cancers. Recently, it was known that FHL2 being able to modulate the function and expression of downstream genes, however, the regulation of FHL2 upstream genes had been seldomly reported. Combined with literature, we selected transcription factor KLF8 (Kruppel-like factor 8) as the potential regulatory factor of FHL2. One of Kruppel-like factors (KLFs) family members, KLF8, was a CACCC-box binding protein that associated with C-terminal binding protein (CtBP) to repress transcription. Recent studies reported that the expression of KLF8 in ovarian cancer, kidney cancer and breast cancer is higher. But, until now, no studies have reported a role of KLF8 in CRC (colorectal cancer) and the function of KLF8 in the development, invasion and metastasis. At the same time this issue was targeted to find the relationship between KLF8 and the upstream promoter sequences of FHL2 so that we can find the expression element of regulation FHL2, in the hope of being used in tumor biological target therapy.Methods and materialMaterialLovo, SW480, SW1116, Caco2, SW620,HT29 and DLD1 cells. Recombinant human TGF-β1, monoclonal anti-TGF-β1 antibody. Dual luciferase reporter gene detection kit, Chromatin Imumunopprecipitation (CHIP) Assay Kit. Quik Change Multi Site-Directed Mutagenesis Kit. Mouse anti-human FHL2 moloclonal antibody, Rabbit anti-human KLF8 polyclonal antibody. Rhodamine and phalloidin.Methods1. The impact of KLF8 on the EMT, invasion and metastasis of colorectal CRC1.1 The construction of KLF8 overexpression stable cell lines and the influence of KLF8 on EMT in CRCTo establish stable cell lines, Lovo transfected with empty pcDNA 3.1 vector and pcDNA3.1-KLF8 were passaged 1:15 (vol/vol). After 48 hours, we cultured cells in RPMI 1640 medium supplemented with 600 μg/ml geneticin (G418) for another 2 weeks till selecting monoclonal cells. Cells were cultured in RPMI 1640 medium supplemented with 800 μg/ml geneticin for 4 weeks. The expression of KLF8 in stable cell lines was verified by western. And western blot was used to detect the expression of E-cadherin, Vimentin and N-cadherin in cells transfected with pcDNA-KLF8 or pcDNA3.1.1.2 The influence of KLF8 on invasion and metastasis of CRC.Cells transfected with empty pcDNA 3.1 vector or pcDNA3.1-KLF8 plated in 6-well plates with 100% confluence were wounded with a pipette tip at time 0. Media was changed to remove cell debris. Photographs were taken at time 12 and 24 to assess cell migration. Plate Cells transfected with empty pcDNA 3.1 vector or pcDNA3.1-KLF8 in transwell chamber coated with matrigel. There were media without FBS in chamber and media with 10%FBS out chamber. After culturing for 24 hours, we counted cells dyeing with crystal violet to assess the influence of KLF8 overexpression on invasion.1.3 The effect of rTGF-β1 on KLF8 expression and EMTFirstly, different concertrations of rTGF-β1 were added into cells after PBS washing, and then incubated cells for additional 48 hours for protein extraction, Western blot was performed to detect KLF8, E-cadherin and Vimentin expression. Secondly, mIgG、α-TGF-β1、mIgG+rTGF-β1 and rTGF-β1+α-TGF-β1were added into cells respectively. After 48 hours, protein was extracted for western blot to detect KLF8, E-cadherin and Vimentin expression. GAPDH was used as internal control. Thirdly Cells transfected with siRNA for 24hours were treated with rTGF-β1for additional 48 hours. Protein was extracted for westeren to detect KLF8 and Vimentin, a mesenchymal marker of EMT.2. The expression of KLF8 and FHL2 in primary colon cancer, metastatic cancer tissues and colorectal cancer cells2.1 The expression of KLF8 and FHL2 in primary colon cancer, metastatic cancer tissuesAt least 15 pairs Consecutive tissues of primary colon cancer and metastatic cancer tissues collected from same person were stained with KLF8 and FHL2 by IHC, respectively.2.2 The expression of KLF8 and FHL2 in colorectal cancer cellsProtein of colorectal cancer cells such as Lovo, SW1116, SW620, HT-29, DLD1, CaCo2, Sw480 was extracted for western blot to detect the expression of KLF8 and FHL2. The distribution of KLF8 and FHL2 in cells was detected by indirect immunofluorescent assay.3. Interaction between KLF8 and FHL23.1 The binding site of KLF8 in FHL2 promoter districtThe binding site of KLF8 in FHL2 promoter district was predicted by bioinformatics software. To obtain more evidence of KLF8 binding to FHL2 promoter, chip was used to detect the binding site of KLF8 in FHL2 promoter district.3.2 The activity of KLF8 binding site in FHL2 promoter was testified by Dual luciferase reporter gene detection assay.We inserted the promoter sequences of FHL25’upstream into pGL3-basic carrier to build plasmid pluc55, pluc201 and pluc498. Transfect plasmid to different KLF8 expressing state of colon cancer cells to program dual luciferase reporter gene detection assay.3.3 Site-Directed Mutagenesis AnalysisThe QuikChange Site-Directed Mutagenesis Kit was used to generate constructs with the mutation of the putative interferon regulatory element. PLuc-55 construct was PCR-amplified in the process using Pfu DNA polymerase with the primers. The product was treated with DpnI endonuclease. The vector DNAcarrying the desired mutations was proliferated in Epicurian coli XL 1-Blue supercompetent cells. Isolate and sequence plasmind DNA to verify the prospectedmutated sequence, and name it plucMT. The mutation plasmid was transfected transiently into cancer cells to Luciferase assay.4. The influence of down-regulated FHL2 on colorectal cancer EMT and ability of proliferation, invasion and metastasis induced by KLF84.1 The influence of down-regulated FHL2 on colorectal cancer EMT induced by KLF8Scr-siRNA and FHL2-siRNA were transfected to KLF8 overexpression stable cell lines. After 48 hours, protein was extracted for western blot to detect the expression of FHL2, E-cadherin, Vimentin and N-cadherin. The distribution of E-cadherin and Vimentin in Lovo-KLF8-ScrRNA and SW480-KLF8-FHL2siRNA was observed by indirect immunofluorescent assay.4.2 The influence of down-regulated FHL2 on ability of proliferation, invasion andmetastasis induced by KLF8(1) WST-1 experiment verify the influence of FHL2 on KLF8-mediated ability of proliferationScr-RNA and FHL2-siRNA were transfected to KLF8 overexpression stable cell lines. Cells plated in 96-well plate were incubated for another 24 hours after transfection Ohour,24hours,48hours,72hours. Measure the 450nm OD of cells after adding 10ul WST-1 for 4hours.(2) The influence of down-regulated FHL2 on ability of invasion and metastasis induced by KLF8KLF8 stable cell lines transfected with Scr-RNA or FHL2-siRNA plated in 6-well plates with 100% confluence were wounded with a pipette tip at time 0. Media was changed to remove cell debris. Photographs were taken at time 12 and 24 to assess cell migration. Plate KLF8 stable cell lines transfected with Scr-RNA or FHL2-siRNA in transwell chamber coated with matrigel. There were media without FBS in chamber and media with 10%FBS out chamber. After culturing for 24 hours, we counted cells dyeing with crystal violet to assess the influence of KLF8 overexpression on invasion.(3) The influence of FHL2 siRNA on the morphology of celles of KLF8 overexpression.The morphology of KLF8 stable cell lines transfected with Scr-RNA or FHL2-siRNA was observed in white light microscope. KLF8 stable cell lines transfected with Scr-RNA or FHL2-siRNA were incubated in cover glass.48 hours later, cells were dyed by Rhodamine and phalloidin and were observed in fluorescence microscope.5. The influence of FHL2siRNA on the ability of invasion and metastasis induced by KLF8 in vivo5.1 The establishment of the subcutaneous tumor modelPGCSIL-FHL2 small hairpin RNA, a FHL2-RNAi lentiviral vector was c onstructed, GFP-lentiviral vecto r(pGCSIL-GFP) was used as a negative contr ol. The lentiviral vectors and pHelper were co-transfected into KLF8 stable ce 11 lines. Then Lovo-pcDNA3.1、Lovo-pcDNA3.1-KLF8 and Lovo-pcDNA3.1-K LF8-FHL2siRNA cell were injected into nude mouse subcutaneous of 5 to 6 weeks.35days later, we compared the tumor size and the consecutive tissues of tumor were stained with Ki-67 and CD 105 by IHC.5.2 The construction of liver tumor metastasized by tumor under the spleen envelopeThe lentiviral vectors and pHelper were co-transfected into KLF8 stable cell lines.Then Lovo-pcDNA3.1, Lovo-pcDNA3.1-KLF8 and Lovo-pcDNA3.1-KLF8-FHL2 siRNA cell were injected under spleen envelope of nude mouse of 5 to 6 weeks.3 weeks later, nude mouse was put to death to observe the tumor size metastasized to liver, and RNA of tumor was extracted for qRT-PCR to verify the expression of E-cadherin.6. Statistical analysisResults obtained from IHC, Luciferase assay, PCR, adhesion, invasion, metastasis and gray value of western experiments were expressed as the means±D. If equal variances not assumed, Satterthwaite t test or Welch method was used to analyze the quantities data; if equal variances assumed, two-tailed Student’s test or one way ANOVA were performed with significant difference being considered if P values were less than 0.05. Results1. KL8 promoted EMT, invasion and metastasis.1.1 The effct of KLF8 overexpression on EMTNegative correlation between KLF8 and E-cadherin and positive correlation between KLF8 and Vimentin, N-cadherin were verified by western blot.1.2 The effct of KLF8 overexpression on invasion and metastasis.Wound-healing assay and invasion experiment indicated the positive correlation between the expression of KLF8 and the ability of CRC invasion and metastasis.1.3 The effect of rTGF-p 1 on KLF8 and EMTFirstly, we showed that rTGF-β 1 induced KLF8 in a dose-dependent manner, as determined by western blotting. rTGF-β1 markedly induced the expression of the EMT marker vimentin. Blocking TGF-β1 with its neutralizing antibody significantly suppressed endogenous KLF8 expression and TGF-β-induced KLF8 expression in Lovo cells. Pre-transfecting cells with KLF8 siRNA not only inhibited KLF8 expression but also suppressed endogenous and TGF-β1 induced vimentin expression.2. The relationship of KLF8 and FHL2 in colorectal cancer tissues and cells.2.1 The expression of KLF8 and FHL2 in primary colorectal cancer and metastatic tumorWe showed that both proteins were highly expressed by cancer cells, whereas their expressions in normal tissues were absent or extremely low by IHC. Observations using serial sections showed that KLF8 and FHL2 were distributed in both the nucleus and cytoplasm of the same cancer cells. We also showed that FHL2 and KLF8 were expressed at high and intermediate levels in serial sections of lymph node metastatic cancer tissues, respectively, in the cytoplasm and nucleus of cancer cells.2.2 The expression of KLF8 and FHL2 in CRCWe first checked the expression of KLF8 and FHL2 in CRC cell lines by western blot. KLF8 was expressed at a relatively high level in SW480, SW1116, Caco2 and SW620 cells and at a relatively low level in HT29, DLD1 and Lovo cells. The FHL2 expression pattern was very similar to that of KLF8 in the above 7 cell lines, except for SW480 cells. A two-color immunofluorescence assay showed that the endogenous KLF8 and FHL2 proteins localized to both the nucleus and the cytoplasm of Lovo cells.3. FHL2 was a direct target for transcriptional activation by KLF83.1 The binding site of KLF8 to FHL2 promoterWe scanned the promoter region (<500 bp) of human FHL2 for the GT-box consensus sequence and found three potential binding sites:-50 to-55 (GT-box 1),-196 to-201 (GT-box 2) and -493 to -498 (GT-box 3) from the transcription initiation site. We performed ChIP analysis using an anti-KLF8 antibody in Lovo cells. PCR amplification showed that a band of 195 bp, containing the first possible binding (-55 to -50) site was immunoprecipitated. No bands were evident in the immunoprecipitates for the other two possible binding sites (GT-box 2 and/or GT-box 3) or for the control IgG.3.2 Luciferase assay testify KLF8 binded to GT box1We then cloned the promoter regions GT-box 1, GT-box 2 and GT-box 3 of human FHL2 upstream of a luciferase gene in a reporter plasmid. Transient transfections were performed to investigate whether the FHL2 promoter containing only GT-box 1, the only GT-box 1 belonging to the FHL2 promoter, was activated by KLF8 overexpression. The luciferase activity increased compared with that of the vector. However, the reporters that contained GT-box 2 and/or GT-box 3 of FHL2 showed slightly higher or lower luciferase activity compared with that of the vectors.3.3 The effect of site-mutated plasmid on FHL2 promoterWe next mutated two nucleotides of the identified GT-box 1 binding region. These mutations greatly reduced the effect of KLF8 on FHL2 promoter activity. These results suggested that the proximal GT-box at-55 is the main KLF8-binding site in the FHL2 promoter.4. The effect of down-regulated FHL2 on KLF84.1 FHL2siRNA suppressed EMT induced by KLF8SiRNA-mediated repression of FHL2 was performed in Lovo-KLF8 cells and resulted in decreased vimentin expression as well as in the conversion from mesenchymal marker (vimentin and N-cadherin) to epithelial marker (E-cadherin) expression compared with the control (KLF8-src siRNA) cells. Immunofluorescence staining of E-cadherin and vimentin, visualized by microscopy, confirmed the EMT-associated shift in marker expression.4.2 FHL2siRNA suppressed the ability of invasion and metastasis induced by KLF8FHL2 downregulation decreased KLF8-mediated proliferation of Lovo cells, as shown using a WST-1 assay. We performed a wound-healing assay. Knockdown of FHL2 in KLF8-overexpressing cells led to a reversal of the decreased migration in vitro. Similarly, FHL2 downregulation in KLF8-overexpressing cells led to the reversal of the decreased invasion in vitro by 34.5%.4.3 Low expression of FHL2 changed morphologic features induced by KLF8The stable vector transfectants displayed a round or flat morphology with a short cytoplasmic process. However, the KLF8 transfectants exhibited a spindle-like, fibroblastic morphology, which is one of the main characteristics of EMT. Long or dendritic-like cytoplasmic processes were visible under a phase-contrast microscope. FHL2 knockdown in KLF8-overexpressing cells led to EMT reversion. Compared with the empty vector-expressing cells, overexpressing of KLF8 cell was present throughout the cytoplasm and at the rim zone of the protrusion. Moreover, filopodia and lamellipodia were identified as dynamic cellular features on the cell membrane surfaces that require actin polymerization by phalloidin staining.5. FHL2 siRNA blocked the ability of invasion and metastasis induced by KLF8 in vivo5.1 Low expression of FHL2 decreased the tumor size of nude mouse subcutaneousThe tumor volumes of the KLF8-overexpressing cells were markedly greater than those of the vector-expressing cells. KLF8 overexpression progressed from a pronounced increase in vector cells at day 21 to a 4-fold increase in the cancer area 35 days after injection. In contrast, tumors derived from FHL2 downregulation in KLF8-overexpressing cells were markedly smaller than those of the vector-treated mice at 3 to 5 weeks. Representative images of the tumors were stained by immunohistochemistry. The KLF8 stable-transfectant group showed significantly increased proliferation rates and tumor vessel density than those in the vector group, whereas knockdown of FHL2 inhibited the growth rate and tumor vessel density in the KLF8-overexpressing group.5.2 Low expression of FHL2 suppressed the metastasis ability induced by KLF8 in vivo.We injected metastatic cells into the livers of nude mice. The presence o f CRC metastasis in the liver was confirmed by histological analysis. The tu mor volumes of the KLF8-overexpressing group were significantly increased c ompared with those of the vector group (p<0.001 vs. vector), whereas the t umor volumes after FHL2 knockdown were greatly decreased compared with t hose in the KLF8-overexpressing group on day 23.Conclusion1. KLF8 promotes EMT and the activity of invision and metastasis in colorectal cancer.2. There was positive expression of KLF8 and FHL2 in colorectal cancer tissues and cells.3. Luciferase assay, CHIP assay and gene mutation verified that KLF8 binded to FHL2 promoter region.4. FHL2siRNA suppressed EMT and the ability of invasion and metastasis induced by KLF8, and change the cellular morphology.5. Experiments in vivo verify FHL2 regulated EMT and the invasion and metastasis induced by KLF8.Taken together, these results clearly indicated that the KLF8-FHL2 axis plays an important role in CRC development and metastasis.