Nuclei Transcription Factor SRF From In Vivo Screening Expedites Tumor Metastasis By Modulating Expression Of MiR-199a In Human Gastric Cancer

Metastasis is a complicated process including lots of change in genes and proteins, such as transcription factors (TFs). After tumors exist in a dormant state without invasion and metastasis activity for months to years, a shift of the balance to the tumor metastatic and invasive state appears. The switch to the metastasis phenotype also permits pre-symptomatic dormant cancers to become metastasis and invasion. This perturbation in tumor is achieved by some TFs targeting cancer cells as the effectors cells. So far, although some TFs, such as AP-1, NF-kB, Sp1, EGR, CREB, ATFs, were thought to be associated with tumor invasion and metastasis, the regulatory mechanisms of these TFs were still poorly understood, especially in gastric cancer (GC), one of the most common cancers and lethal malignancies in the world. We sought to investigate the molecular mechanisms mediating the switch by TFs related to the metastatic phenotype in GC.【Objectives】1. To screen the TFs involved in metastasis of GC; 2. To study the relationship between SRF and invasion and metastasis of GC by expressional and functional experiment;3. To find downstream effectors,including miRNAs and to examine the possible pathway and mechanisms of metastasis in GC.【Methods】1. We performed a high-throughput TF microarray twice to profile the signatures of active TFs in human GC highly metastatic cell line GC9811-P, which was screened from parent lower potential metastasis GC9811 in vivo in our lab, and then verified the selected TFs by ELISA;2. We tested the subcellular location and expression of actived SRF protein in GC metastatic tissues and GC cell lines with different potential metastasis by Western blot and Immunohistochemistry.3. We found or constructed the full-length vector, deletion mutants and siRNA vector of SRF;4. We tested the effects of SRF on the adhesive, invasive and in vivo metastatic abilities in GC cell lines, including GC9811, GC9811-P and SGC7901, by migration assay, invasion assay, adhesion assay and tail vein metastatic assay in vitro and in vivo;5. We studied the expression levels of SRF, miR-199a-5p and miR-199a-3p in different transfectants GC cell lines and the fresh GC metastatic tissues by real time PCR;6. We tested the direct bind of SRF with promoter of miR-199a gene by CHIP and evaluated the activity by dual luciferase reporter assay. Then we tested the effects of miR-199a-5p and miR-199a-3p on the adhesive, invasive and in vivo metastatic ablilities in GC cell lines, including GC9811, GC9811-P and SGC7901,by migration assay, invasion assay and adhesion assay respectively. 【Results】1. Elucidation of metastasis-related TFs by high-throughput profiling of the activities transcription factors assay.To analyze differentially active TFs profile changed in the switch of gastric cancer, we compared GC9811 and GC9811-P cells by using high-throughput profiling of the activities TFs assay twice. We found 11 TFs were significantly up-regulated and 9 down-regulated. Interestingly, among these candidates of TFs involved in metastasis, AP1, NF-KB, ATFs, Sp1, CREB and EGR, which were expressed at apparent differential activity in metastastic cells, were classic molecules in tumor metastasis. We chose SRF, one of these, for the further work.2. The active SRF expression in human GC metastatic tissues and cells lines.To confirm the signature data on the phosphorylated protein level, we tested the expression of p-SRF in human gastric tissue in vivo by high density human GC metastasis microarray. We analyzed 25 normal gastric tissues (NG), 25 gastritis (without intestinalisation), 21 chronic atrophic gastritis with intestinalisation (CG), 28 GC, 26 metastatic GC (MC) in lymph node, and 22 MC in distant metastasis. Intriguingly, the p-SRF, activation of SRF, in human samples paralleled the phenotypic observation of enhanced metastasis in tumors from NG, to GC, to MC tissue samples. In detail, p-SRF was largely staining expressed in the nuclei in the metastatic tissues. To our surprised, p-SRF was also expressed in cytoplasm in same cases of the GC and MC tissues, indicating that SRF was activated in GC tissues, and then transposited into nuclei in MC tissues. To further validate the role and location of SRF protein in GC cells metastasis, we tested the expression of SRF in the nuclear and cytoplasm protein respectively in fresh human metastasis GC specimens from 6 individuals. Intriguingly, the comparison between NG, GC and MC samples showed obvious up-regulation of SRF expression in the nuclear of cancer cells in MC tissues, but SRF was only found in cytoplasm in NC and part of GC tissues. It observed cellular translocation of SRF in tumor and their metastasis tissues demonstrated the enhanced phenotypic of metastasis in GC tissues. We could assume that the active SRF in the pro-metastasis tissues prepareed a translocation in the cytoplasm for the future development of cancer metastasis. And when the active SRF was trans-posited into the nuclei, the metastasis happened. Taken together, our findings thus provided the clinical evidence for the concept of the metastasis switch of cancer development and suggested a key role for the translocation of active SRF in cancer development process.We then assessed the role of SRF in the two pair of GC cell lines lower metastasis GC9811 and the high positive GC9811-P by Western blot, the gastric cancer cells. SRF and p-SRF were over-expressed in high potential metastatic cells than lower metastatic cells. This phenomenon could also be found in colon cancer cells. The translocation of SRF in the two cell lines was also tested by Western blot and Immunofluorescence. All these revealed that SRF protein was predominantly located in cytoplasm in GC9811 cells but was activated and made the transposition into the nuclear in GC9811-P cells when endogenous SRF was examined with SRF antibody.3. SRF promoted adhesive, invasive and metastatic abilities of GC cells in vitro and in vivo.Short interfering RNA (siRNA) oligonucleotides were introduced into GC and depleted the expression of SRF in the highly metastatic GC9811-P cells and SGC7901 cells for metastasis assay in vitro and in vivo. Depletion of SRF significantly impaired the ability of GC9811-P cells to migrate and invade through collagen-coated membranes or membrances without collagen towards serum-containing medium in a modified Boyden chamber assay, compared with the control GC9811-P-vector. The scratch-wound assay showed that depletion of SRF from GC9811-P cells indicated substantially reducation of migration. The strikingly similar results were obtained in SGC7901 cell lines. Moreover, GC9811-P cells and the control cells showed a mild impairment of adhesion 30 min after plating, but the phenotypes were transient and the cells assumed a fully spread morphology on either matrix by 4 hours after plating. In contrast, the GC9811-P-SRF-siRNA cells showed decreased adhesion after 4 hours. The effects of SRF depletion on SGC7901 were strikingly similar in GC9811-P. Taken together, these results implicated that SRF was in control of the adhesion of gastric cells. Oppositely, overexpression of SRF protein in GC9811cells markedly improved the metastases in the assays of the adhesion, invasion and metastasis in vitro. Conclusively, at least in certain contexts, SRF were required for the invasion, metastasis and adhesion in these GC cell lines, and attenuation of SRF led to suppression of the metastasis. Tail vein metastatic assay in nude mice was further adopted to examine the in vivo metastatic abilities of GC9811-P SRF-siRNA . Compared with control cells transfected with empty vector, GC9811-P SRF-siRNA cells led to significantly less visible tumors in liver and lung surface. Both in vitro invasion assay and in vivo nude mice assay suggested that SRF had a potential to promote metastasis of GC. However, SRF could not affect proliferation and apoptosis of GC cells.4. SRF enhanced metastasis of GC cells by modulating miR-199aResults by bioinformatics analysis and real time PCR showed that some CArG element-contained microRNAs (miRNAs), such as miR-199a-3p and miR-199a-5p, highly expressed in GC metastasis tissues and cell lines, were considered as direct target of SRF. The directly regulation between SRF and miR-199a-1 and miR-199a-2 cluster, which encoded miR-199a-3p and miR-199a-5p, are further verified by CHIP and Luciferase reporter assays. To the intriguing, the translocation in nuclei of SRF could increase the expression of miR-199a-5p and miR-199a-3p. However, restoration of miR-199a-5p, but not miR-199a-3p, could significantly promote metastasis of GC cells, whereas silencing it could remarkably suppress GC cells metastasis.【Conclusion】Overall, by systemically identifing series of TFs participated in the metastasis of human GC, and uncovering that the translocation of SRF are strong positive correlation with miR-199a in this process, we for the first time indicate that nuclei SRF has the ability to expedite GC metastasis by modulating of miR-199a, which may represent as a new potential therapeutic target for GC treatment.