Mechanism Of Sp1-miR-3178-TRIOBP Axis In Regulation Of Metastasis

Cancer has always been one of the main threats to public health.The latest statistics show that until 2014,the incidence of cancer in China has reached nearly 0.2%,that is,200 new cancer cases per one hundred thousand people every year.Meanwhile,cancer related mortality rate is up to 0.1%,that is,100 people die of cancer every year per hundred thousand people.Metastasis is attributable to about 90% of cancer related casualties,causing more threats to patients’ health and life than primary tumors.Metastasis is a complex multistep cascade,beginning with invasion of tumor cells to surrounding tissues and organs.Then cancer cells enter the blood vessel or lymph tube,and reach remote organs with the aid of circulation system.Under suitable conditions,circulating tumor cells extravasate vessels and proliferate to form new colonies.People have been studying metastasis for decades,but still many metastasis-related mechanisms are unclear.Sp1,one of the first discovered transcription factors,is widely involved in various physiological processes including proliferation,differentiation,metabolism,apoptosis and so on,and its dysregulation is associated with cancer progression.Sp1 is also involved in tumorigenesis and metastasis by targeting oncogenes and tumor suppressors such as TP53 and p21.There are more than 12 thousand predicted Sp1 binding sites,most ongoing researches on Sp1 target genes are focused on protein coding genes,while more than 97% of the human genome are non-coding genes.So,it is of no doubt that Sp1 regulate various physiological and pathological processes by targeting a large number of non-coding genes.Micro RNA is conserved small non-coding RNA with length of only 19~22 nt.Complementary binding between micro RNA seed sequence and 3’ UTR of target m RNA causes translation suppression or degradation of m RNA.Micro RNA has been found to be involved in the regulation of various physiological processes and is closely related to tumorigenesis and metastasis.In this study,we first discovered that proteasome inhibitor treatment of LNCa P cells lead to decrease of SP1 expression and up-regulation of series of mi RNAs,among which mi R-3178 was the most up-regulated.In cancer cell lines,expressions of SP1 and mi R-3178 were negatively correlated,and overexpression of SP1 suppressed mi R-3178 expression.Using bioinformatic prediction methods,we found that mi R-3178 promoter region contains three potential Sp1 binding sites,which are located at-799 ~-788 bp,-97 ~-88 bp,and-82 ~-73 bp upstream of transcription starting site,respectively.Using luciferase reporter assay system and Ch IP experiment,we found that Sp1 inhibited mi R-3178 transcriptional activity by binding to the-82 ~-73 bp site.We then compared SP1 expression between primary tumor and metastasis tumor tissues using data downloaded from Oncomine and GEO,and found that SP1 is up regulated in metastatic tissues.Expression of mi R-3178 negatively correlated with metastatic potential of cancer cell lines.Besides,scratch assay and Transwell experiment demonstrated that endogenous mi R-3178 suppresses migration and invasion of cancer cells.In vivo study using nude mice metastasis model and in vivo imaging system showed systematic lung,liver and lymph node metastasis of prostate cancer cells injected through tail vein,and mi R-3178 over expression dramatically suppressed metastasis,as indicated by fluorescence signals,as well as H&E and DAPI staining,demonstrating that mi R-3178 inhibit in vivo metastasis of cancer cells.TRIOB and F-actin binding protein(TRIOBP)regulates migration and invasion by promoting pseudopodia formation.There are mainly three isoforms of TRIOBP,namely TRIOBP-5/4/1.Among all isoforms,TRIOBP-1 is most highly expressed in cell lines in this study,whereas TRIOBP-5 has much lower expression and no TRIOBP-4 was detected.Using online mi RNA target prediction data bases,we found that predicted binding site of mi R-3178 located on 3’ UTR of TRIOBP m RNA.Luciferase reporter assay also demonstrated that mi R-3178 suppressed luciferase activity by binding to TRIOBP 3’ UTR region.Besides,TRIOBP is overexpressed in highly metastatic cancer cell lines compared with lowly metastatic cancer cell lines,and alteration of mi R-3178 expression using specific mimics or inhibitors lead to according down-/up-regulation of TRIOBP m RNA and protein level.Besides,TRIOBP-1 recovered migration and invasion ability which was suppressed by mi R-3178 overexpression,indicating that mi R-3178 suppresses metastasis via targeting TRIOBP.At last,we further confirmed through Ch IP experiment that Sp1 could not regulate TRIOBP transcription by binding to its promoter region.In sum,this study revealed role of Sp1-mi R-3178-TRIOBP axis in regulation of metastasis.Through series of experiments,we proved that mi R-3178 suppressed cancer cell migration and invasion by targeting TRIOBP.Sp1 suppresses mi R-3178 transcription by binding to its promoter region.Taken that Sp1 is up-regulated in many kinds of cancers,this our results indicated potential use of mi R-3178 to suppress metastasis in Sp1 overexpressed cancers.