Preliminary Functional Studies Of Scaffold Attachment Factor-B (SAFB) In Colorectal Cancer

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.Scaffold attachment factor-B1 (SAFB)was identified as a nuclear matrix-associated protein. SAFB is part of a protein family with at least two other family members, SAFB2 and the SAFB-like transcriptional modulator SLTM.It has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis.and has since been implicated in a number of fundamental cellular processes, including RNA splicing, the regulation of transcription and the cellular response to stress. Sequence analysis shows that SAFB and a more recently identified homologue SAFB2 contain several functional domains including an N-terminal DNA binding region (SAP/SAF-box), nuclear localization signal, a central RNA recognition motif (RRM) and Glu/Arg, Ser/Lys and Gly-rich protein interaction regions. The presence of these highly conserved domains may reflect that SAFB plays a role in transcription and/or that SAFB proteins will have multiple cellular functions, a conclusion partially borne out by the findings of three independent groups that identified SAFB according to different biological actions. The protein was first identified as SAF-B, a protein that binds scaffold or matrix attachment region (S/MAR) DNA, and then as HET, a protein that down regulates the transcription of the hsp27 gene by binding its promoter. Then, in a two-hybrid screen, a protein that associated with hnRNP A1 and was recruited to stress bodies was identified and shown to be identical to SAF-B and HET.SAFB has been shown to interact with a number of alternative RNA splicing regulators including hnRNP A1 and hnRNP D, Tra2, SF2/ASF,9G8, SRp30c, YT521B, SRp86, T-STAR (also known as SLM2) as well as protein kinases that phosphorylate splicing regulators (SRPK1 and CLK2).SAFB also interacts with several nuclear hormone receptors including the estrogen receptor ER (a transcription factor).Over the last few years, it has become clear that the function of SAFB during apoptosis and present evidence to show that it migrates to the nucleolus, is cleaved and forms a distinct peri-nucleolar body via its coiled-coiled domains during the cell death process. SAFB moved into the nucleolus 15 min after the induction of apoptosis and before the release of cytochrome c into the cytoplasm. Two hours later SAFB formed a peri-nucleolar ring-like structure and this occurred after cytochrome c release and before PARP cleavage. In addition, SAFB cleavage was shown to be mediated by caspase-3 and occurred after the formation of the peri-nucleolar ring and after cleavage of PARP (characteristic of proteins having a direct role in apoptosis).In this study, we aim to clarify the possible role of SAFB gene in the proliferation invasion of CRC. It will be helpful to understand the molecular basis of CRC, and establish SAFB as a new target for early diagnostic markers and novel therapeutic strategies.METHODS1.Expression of SAFB in colorectal carcinomas tissue and cells lines and its clinical significanceThe expressions of SAFB gene in eight colorectal carcinoma cell lines and 28 CRC and paired nomal colorectal mucosae were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot..The expression of SAFB was examined in specimens of 283 CRC,114 paired nomal colorectal mucosae,120 regional lymphatic metastases by immunohistochemical (S-P) method.2.Effect of SAFB overexpression on the biological behaviors of human CRCFull length cDNA of SAFB was amplified by RT-PCR with total RNA extracted from the human nomal colorectal mucosae as template, and cloned into eukaryotic expression vector pcDNA3.1(-).The recombinant plasmid of pcDNA3.1(-)/SAFB was identified by restriction endonuclease analysis and DNA sequencing. The pcDNA3.1(-)/SAFB plasmid was transfected into SW480 cells. SAFB expression in transfectant was determined by real-time reverse transcription polymerase chain reaction (PCR) analysis and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, in vitro invasion assay.3.Effect of SAFB knockdown on the biological behaviors of human CRCA double stranded DNA fragment for RNA interference on human SAFB gene was cloned into retrovirus vector pSUPER Retro, and the constructed recombinant retrovirus vector pSUPER/shRNA-SAFB was packaged in 293FT cells. The obtained recombinant retrovirus was infected to CRC cell lines, and the stable clones were screened with puromycin, in which the transcription level of SAFB mRNA and expression level of SAFB protein were determined by real-time fluorescent quantitative PCR and Western blot. MTT assay, plate colony formation assay were used to assess the functional effects of SAFB silencing on tumor cell proliferation.4. Expression of cadherin related signaling molecules in SW620 cells with stable knock-down of SAFB geneCadherin related signaling molecules including E-cadherin,α-cantenin,β-catenin,γ-catenin, GSK-3β, Phosphorylated GSK-3β, AKT, Phosphorylated AKT, P38 MAPK, Phosphorylated P38 MAPK were investigated by immunoblot and E-Cadherin,Slug,Snail,MTA by real-time reverse transcription polymerase chain reaction (PCR) analysis.RESULTSThe main results and findings are as follows:1.Expression of SAFB in CRC tissue and cells linesSAFB was expressed heterogeneously among the normal colorectal mucosae, carcinomas and lymphatic metastasis of colorectal carcinoma(χ2=271.214, P=0.000). SAFB was higher expressed in carcinomas compared with normal colorectal epithelium (Z=-4.608, P=0.000). Furthermore, the SAFB expression beteen in colorectal carcinoma with lymph node metastasis and in colorectal carcinoma without lymph node metastasis, there is no significant differnces. (Z=-0.466, P=0.642). As revealed by RT-PCR analysis, a higher level of SAFB expression was seen in the colorectal carcinoma but not in the normal colorectal mucosae. The western blotting analysis also showed that. Furthur more, the SAFB expression in SW620 cell lines was higher than in the DLD-1, SW480-M5, LS174T and HT29, the lowest in the SW480,HCT116,colo205.2.Construction and identification of human SAFB eukaryotic expression vectors and effect of SAFB overexpression on the biological behaviors of human CRC.Recombinant plasmid pcDNA3.1 (-)/SAFB was proved to be successfully constructed by restriction enzyme digestion analysis with ECoR I, BamH I and DNA sequencing, and the sequence of target gene was completely correct. Western blotting and real-time reverse transcription polymerase chain reaction (PCR) analysis showed that the protein level of SAFB in pcDNA3.1(-)/SAFB transfection group was higher than that in untransfected group and pcDNA3.1(-) transfection group, but there was no significant difference between the latter two groups. The data proved that SAFB was overexpressed in pcDNA3.1(-)/SAFB transfected SW480 cell line.pcDNA3.1(-)/SAFB cells showed a significantly enhanced proliferation compared with the pcDNA3.1(-)/vector cells as determined by in vitro MTT assay (F=71.944, P<0.01)3.Down-regulation of SAFB expression by RNAi and effect of SAFB silencing on the biological behaviors of human CRCSequencing result proved that recombinant retrovirus vector pSUPER/ shRNA-SAFB was constructed correctly. Stable clones were screened from HCT116,SW480 and SW620 cells infected with recombinant retrovirus. Both the transcription level of SAFB mRNA and expression level of SAFB protein in the three CRC cells infected with pSUPER/shRNA-SAFB were significantly lower than those in negative control and normal control cells. And we found clone 2 exhibited a dramatic knock down of SAFB protein expression (93%) (Designated SW620/ shRNASAFB).Two group of SW620/shRNASAFB cells showed a significantly reduced proliferation compared with SW620/pSUPER cells as determined by in vitro MTT assay. In addition, SW620/shRNASAFB cells had a significant reduction in their ability to form colonies in plate as compared with SW620/pSUPER cells.4. The role of SAFB in regulation of cadherin related signaling pathways of colorectal cancer Knockdown of SAFB induces epithelial mesenchymal transition by up-regulation of E-cadherin and down-relation of Slug, Snail, and MTA. Phosphorylated GSK-3βand phosphorylated P38 MAPK are involved in cadherin related signal pathway and regulation of SAFB.CONCLUSION1. SAFB gene plays an important role in proliferation. Clinically it may be a useful indicator of the tumor progression and metastasis in CRC.2. SAFB induces epithelial mesenchymal transition by down-regulation of E-cadherin.3. Phosphorylated GSK-3βand phosphorylated P38 MAPK are involved in cadherin related signaling pathway and regulation of SAFB. These data will be helpful to elucidate the molecular mechanism of SAFB and provide new clues.