Effects Of Foxc2Gene On Proliferation And Apoptosis Of3T3-L1Adipocyte And Research On Its Promoter Activity

Foxc2is a new found transcription factor that involving in a variety of activities inadipocyte. It plays a main role in regulating adipocyte differentiation and lipid metabolism.And it can also resist series of diseases including obesity, hyperlipidemia, diet-induced insulinresistance et al. Considering that Foxc2has proved to promote cell proliferation and inhibitapoptosis in neural crest cells and C2C12myoblasts, we speculate that it may have the samefunctions in adipocyte. What’s more, in recent years, studies have been focused on Foxc2gene’s functions on adipogenic differentiation and regulatory mechanism of metabolism,however, rare studies had focused on the transcriptional regulation of its expression. In thisstudy, in order to clarify the effects and mechanism of Foxc2on adipocyte proliferation andapoptosis, and further explore the mechanism of transcriptional regulation of Foxc2expression, we over-expressed and knock-down Foxc2gene in3T3-L1adipocytes andfurther validated the role of Foxc2in apoptosis by constructing serum-starved and palmiticacid-induced apoptosis models, We also cloned the1100bp promoter region from5’flankingof mouse Foxc2gene to preliminary explore its promoter activty. To conclude, we obtainedthe following results:1. Transfect3T3-L1adipocyte with Foxc2overexpression vector and Foxc2shRNA-interference vector, take empty vector transfection as control. Cell counting showedthat overexpression of Foxc2increased cell number while shRNA-interference of Foxc2decreased cell number（P<0.05）. Cell cycle analysis revealed that over-expressed Foxc2promoted the proportion of cells in S-phase and reduced that of G0/G1-phase. Overexpressionof Foxc2facilitated expression of Cyclin E and reduced expression of p27and p53; however,shRNA-interference of Foxc2had the opposite effect. As shows above, Foxc2can promote3T3-L1adipocyte proliferation. And Foxc2had no significant influence on phosphorylationof ERK1/2and Akt as putatived implies that more meticulous research should be done todiscover the pathways that involved.2. Transfect3T3-L1adipocyte with Foxc2overexpression vector and Foxc2shRNA-interference vector take empty vector transfection as control. JC-1staining shows that the membrane potential in over-expression group was increased and that in the interferencegroup was decreased (P <0.01); Tunel staining indicates that fragmentation of genomic DNAis serious in the interference group, but maintained a low level in over-expression group. WithRT-PCR and Western Blotting to detect cell apoptosis related genes,we found that theexpression of Bax was increased significantly,but the expression level of Bcl-2wassignificantly decreased, and the activity of Cleaved Caspase-9, Cleaved Caspase-3weresignificantly enhanced in the interference group(P<0.01); however the situation inover-expression group was contrary. The expression of Bcl-2was significantlyincreased,while the expression level of Bax and the activity of Cleaved Caspase-9、CleavedCaspase-3were significantly reduced (P <0.01). This study confirmed that Foxc2inhibitedapoptosis through mitochondrial pathway in adipocytes.24h serum-starve and0.2mMpalmitate-induce successfully caused apoptosis in3T3-L1adipocytes, and the reverse effectof Foxc2on adipocyte apoptosis of these two types of apoptosis further validate the role ofFoxc2in inhibition of adipocyte apoptosis.3. On the basis of data from NCBI, we found the promoter sequence (1100bp beforeTBS and9bp after TBS) of Foxc2gene. As predicted by Matlnspector we found358possibleFBS on it. According to distribution of the358possible FBS,4pairs of primes weresynthesised to clone4sequences of different length from Foxc2promoter, subsequently,4luciferase reporter vectors were constructed to detect the promoter activity. The results ofDual luciferase reporter showed that in3T3-L1adipocytes the activity of PGL3(-1100/+10)was the highest, that of PGL3(-264/+10) was the lowest, the activity of PGL3(-1100/+10)was significantly higher than PGL3(-712/+10)(P <0.01),that of PGL3(-264/+10) wassignificantly lower than PGL3(-483/+10)(P <0.01) and there was no significant differencebetween PGL3(-712/+10) and PGL3(-483/+10). These results indicated that there may besome potential positive-regulate transcription binding sites both between-264bp and-483bpand between-712bp and-1100bp, there may be some potential negative-regulate binding sitesbetween–483bp and-712bp. In HEK293T cells there was no significant difference among4promoter sequences of different length. That may be have some relation with the fact thatthere‘s no endogenous Foxc2expression in HEK293T cells.