| Kirsten rat sarcoma viral oncogene homolog（Kras）is a proto-oncogene that encodes the small GTPase transductor protein KRAS,which has been found to promote cytokine secretion,cell survival,and chemotaxis.However,its effects on preadipocyte differentiation and lipid accumulation are unclear.In this study,the effects of KRAS on proliferation,autophagy,and adipogenic differentiation as well as its potential mechanisms were analyzed in the 3T3-L1 and C2C12 cell lines.First,in order to explore the effects of KRAS on the proliferation in 3T3-L1 and C2C12 cells,the proliferation ability changes were detected using Ed U and CCK-8methods.The m RNA expression levels of proliferation-related genes Mtor,Pcna,and Myc were detected by q RT-PCR after si-KRAS treatment.The results showed that in3T3-L1 and C2C12 cells,the proportion of Ed U-positive nuclei in the KRAS inhibition group was 0.89±0.07-fold and 0.90±0.05-fold of that in the NC group,respectively.The CCK-8 assay results also showed that the proliferation capacity in si-KRAS group was 0.82±0.02-fold and 0.80±0.02-fold of that in the NC group in3T3-L1 and C2C12 cells,respectively.The m RNA levels of Mtor,Pcna,and Myc were also decreased in 3T3-L1 and C2C12 cells after KRAS inhibition.Then,we investigated the effects of KRAS on autophagy in 3T3-L1 and C2C12cells.After inhibition of KRAS,the expression of LC3B in cytoplasm was detected by immunofluorescence;the levels of LC3B-II/LC3B-I was detected by Western blot;the m RNA levels of autophagy related genes Beclin 1 and Atg7 were detected by q RT-PCR.The results indicated that,in 3T3-L1 and C2C12 cells,the fluorescence intensity levels of LC3B in si-KRAS group were 1.78±0.11-fold and 1.38±0.04-fold of those in the NC group,respectively.The protein levels of LC3B-II/LC3B-I in the si-KRAS group were 2.25±0.06-fold and 1.84±0.14-fold of those in the NC group,respectively.Morever,the m RNA levels of Lc3b,Beclin1,and Atg7 were also increased in 3T3-L1 and C2C12 cells with KRAS inhibition.Finally,we investigated the effects and portential mechanisms of KRAS on adipogenic differentiation in 3T3-L1 and C2C12 cells.After KRAS inhibition,adipogenic differentiation,lipid accumulation and m RNA levels of adipogenesis-related genes Dgat1,Scd1,C/ebpβ,and Hmgr were determined by oil red O（ORO）staining,triglyceride level assay,and q RT-PCR,respectively.The phosphorylation levels of MAPKs（ERKs,JNKs,and p38）and PI3K were detected by Western blot.In addition,the potential mechanism of KRAS on lipid formation was investigated using MAPKs activator ANI and PI3K activator 740 Y-P,respectively.The results showed that compared to the NC group,the si-KRAS-treated group in3T3-L1 and C2C12 cells had significantly higher triglycerides levels（1.24±0.02times and 1.24±0.01 times,respectively）.The level of ORO OD520 value and Dgat1,Scd1,and C/ebpβm RNA expression were also increased after KRAS inhibition.And both in 3T3-L1 and C2C12 cells,the phosphorylation levels of ERK1/2,JNK1/2/3,p38,and PI3K were decreased with KRAS inhibition.Moreover,MAPK activator ANI can alleviate the increased lipid accumulation induced by KRAS inhibition,conversely,740 Y-P further improve lipid accumulation.In conclusion,this study indicated that KRAS could regulate cell proliferation,autophagy,and adipogenic differentiation in 3T3-L1 and C2C12 cells.These results will contribute to explore the process and mechanism of fat formation and accumulation at the molecular level as well as providing a theoretical basis for regulating the nutrition and composition of meat as well as improving meat quality. |
