Cloning And Preliminary Functional Analysis Of FST Gene In Apoptosis Induced By Cold In Min Pig

Follistatin(FST) is a single-chain glycoprotein, named because of a strong inhibitory effect on FSH,the original study on follistatin was mostly concentrated on its effect on the reproductive system. Studies have shown that, FST could interact with several TGF-β superfamily members, and played an important role in the regulation of cell proliferation, fat cells and neural cells differentiation, regulating energy metabolism and so on besides the reproductive system. In this experiment, we set FST as the research object which was selected from the Min pig cold chip. The basic sequence information of Min pig FST gene was got by Cloning and sequencing method, then we detected the core promoter of FST by luciferase reporter and its methylation status was also detected. Subsequently, FST stable expression and interference of porcine fibroblast cell line were constructed. We explored the function in cold induced apoptosis of the FST, then predicted the pathway by the digital gene expression profiling method. The results were as follows:(1) Successfully obtain the Min pig 5’ flanking sequences of 1800 bp by PCR amplification,similarity is 99% compared with NCBI sequence. We use liver tissue cDNA as template cloning the full-length FST CDS of Min pig, there are 5 base differences compared with NCBI sequence, and caused3 sense mutations. Nested PCR was used to get the part of FST 3’UTR sequence of Min pig, a has-miR-320 c binding site was predicted by PITA.(2) We constructed 10 deletion recombinants with luciferase reporeter system, the promoter acativity of FST 5’ flanking 1800 bp was analyzed by dual-luciferase reproter assay system in PK15 cells,the core promoter of porcine FST was located at the-298~-291 bp upstream of FST translation start site ATG(A is 0 site). Constructed the MZF1 transcription element binding region alone, the FST gene promoter activity was significantly decreased. Then, the MZF1-GFP fusion vector was constructed and transfected, we found that the over-expression of MZF1 transcription factor can significantly promote the expression of FST gene.(3) The ear tissues of four local pig living different latitude(temperature) were treated by bisulfite,and the methylation status of their FST core promoter region were detected. We found that, the CpG island methylation status was at extremely low levels. Subsequently, the methylation status of the fibroblast was detected during cold induced process, found a similar situation in methylation status of the region, at a extremely low level.(4) We detected the porcine fibroblast apoptosis at 4℃ condition for 2h, 5h, 16 h, 20 h, 24 h and 40 h and then we made statistical analysis. In the first 2h cells were not found obvious apoptosis, it was named induction period. Cells showed significant apoptosis at 2h~5h period. The degree of cold inducedapoptosis was proportional to time, almost all cells lysed at 40 h.(5) Real-time PCR and Western blot analysis to detect the expression of FST at the process of 4℃cold induced. We found that, the rising expression of FST was similar with the changes that glucose starvation induced apoptosis, suggesting FST gene played an important role in the process of resistance cell apoptosis in cold induced. The changes of Bcl-2, Bcl-2l1 and Bax which were closely related to apoptosis were detected by Real-time PCR, found all the gene expression downward trend, Bcl-2 gene significantly down regulated at 4℃ after 24 h treatment.(6) Primary cultured porcine fetal fibroblast cells, constructed pc DNA3.1(+)-FST eukaryotic expression vector and RNA interference vector pSilencer-FR1 vector, successfully established stable FST gene over-expression and interference porcine fetal fibroblast cell line by transfecting and G418 selecting.(7) Identified the FST effect on cell proliferation by MTT. We found that over-expression of FST gene can significantly promote the proliferation of fibroblast. After FST gene interfering, cells proliferated slowly compared with normal cells, but the difference was not significant compared with the negative control group.(8) There was low degree cell apoptosis in FST over-expression group compared with the blank group and interference group at 4℃ cold induced for 5h. Microscopic morphology observation indicated that FST gene interference group appeared lager, local cavitation and other apoptosis phenomenon.Real-time PCR detected the expression of Bcl-2, Bcl-2l1 and Bax of FST over-expression and interference group in the process of cold induced at 4℃, found that compared with the corresponding time point control group and negative group cells, over-expression of FST can promote Bcl-2 and Bcl-2l1 genes’ expression and down regulated expression of Bax. On further analysis,however, it was noticed that the percentage of Bcl-2/Bax were higher in FST over-expression than that in blank group and FST interference group.(9) Differentially expressed genes between FST gene stable over-expression and FST gene stable interference were detected by DGE. Then the GO analysis and pathway enrichment were carried on. It is found that the two pathways related to apoptosis were all point to the p53 signal pathway. We speculated that FST involved in the regulation of p53 signaling pathway, function in cells apoptosis in cold induced.