Regulation Of MiR-new14 To Different Genotype Of IGF-1 Gene In Pigs And Its Effect On Proliferation And Apoptosis Of PK-15 Cells

Bama Minature pigs are characterized in short stature,slow growth,short spermization cycle,and large litter size.They are similar to human in terms of anatomy,physiology,It has become a non-rodent experimental model of large animals and human disease research model.Therefore,the Bama Minature pigs have a good prospect of biomedical application.IGF-1 is one of the important candidate genes affecting animal growth and body shape traits,and plays an important role in regulating the formation of porcine dwarf traits.Therefore,it is of great significance to study the molecular mechanism of the factors related to the regulation of IGF-1expression.In this experiment,we first screened one SNP locus of IGF-1 gene 3'UTR in Bama Minature pigs and Large White pigs as control,and analyzed its possible function.The results showed that there was one SNP site?rs34142920,c.674C>T?between the two pig breeds,of which T was the dominant allele of Large White pigs and C was the dominant allele of Bama Minature pigs.The mRNA secondary structure of different genotypes of IGF-1 gene 3'UTR was predicted by online software.The results showed that there were differences in the secondary structure and minimum free energy of mRNA between them.To observe whether there are differences in gene expression of different genotype 3'UTR,the dual luciferase reporter gene recombinant plasmid carrying two genotypes of IGF-1 gene 3'UTR?C/T?were constructed,named psiCHECK-3'UTR-B and psiCHECK-3'UTR-L.The recombinant plasmid was transfected into PK-15 cells for 48 hours,luciferase activity was detected.The results showed that the luciferase activity in the psi CHECK-3'UTR-B groups was reduced by 27.86%compared to the psi CHECK-3'UTR-L groups?p<0.05?.In order to further explore the molecular mechanism of the effect of SNP on the expression of IGF-1,it was found in the literature that the seed sequence of miR-new14 existed in pigs was complementary to the 3UTR of IGF-1 gene,and the SNP was located in the binding region between miR-new14 and of IGF-1 gene 3'UTR.Therefore,in order to verify whether the gene expression was affected by the difference of miR-new14 binding to different genotypes of IGF-1 gene 3'UTR,the mi R-new14/NC and psiCHECK-3'UTR-B/psi CHECK-3'UTR-L were co-transfected into PK-15 cells.The results showed that,compared with the miR-new14 NC co-transfected groups,the luciferase activity of the miR-new14groupswasinhibited,andtheluciferaseactivityinthe psi CHECK-3'UTR-B groups was significantly inhibited compared to the psi CHECK-3'UTR-L groups?p<0.05?.These suggested that miR-new14 caused differential gene expression by binding to different genotype of IGF-1 gene 3'UTR.To further investigate whether miR-new14 can regulate the expression of endogenous IGF-1 gene,miR-new14 was transfected into PK-15 cells alone and the expression of IGF-1mRNA and protein were detected by RT-qPCR and Western-Blot,respectively.It was found that the expression amounts of IGF-1 m RNA and protein of miR-new14groups were significantly lower than these in miR-new14 NC groups,which indicated that miR-new14 inhibited IGF-1 transcription and translation levels.In order to further clarify the effects and mechanisms of IGF-1 expression regulated by miR-new14 on cells proliferation and apoptosis.The proliferation and apoptosis of PK-15 cells transfected with miR-new14 were detected by CCK-8 and flow cytometry,and the expression of AKT and ERK in IGF-1/PI3K/Akt and IGF-1/MAPK/ERK signaling pathways associated with IGF-1 were detected.The results showed that,after 48-72h transfection,the OD₄₅₀50 value of miR-new14 groups was significantly lower than that of miR-new14 NC groups?p<0.05?,and the apoptotic result showed apoptotic rate of miR-new14 groups?18.62±2.0%?was significantly higher than that of miR-new14 NC groups?13.3±1.61%??p<0.05?.These indicated that overexpressed mi R-new14 inhibited the proliferation of PK-15 cells and promoted its apoptosis.The expression amounts of AKT and ERK mRNA in miR-new14 group were significantly lower than that in miR-new14 NC groups?p<0.05?.There was no significant difference in the total protein expression amounts of AKT and ERK between miR-new14 groups and miR-new14 NC groups,but the phosphorylation level of AKT and ERK protein of miR-new14 groups was significantly lower than that in miR-new14 NC group?p<0.05?.These indicated overexpression of miR-new14 down-regulated the transcription level of AKT and ERK and inhibited the protein phosphorylation level of AKT and ERK.In conclusion,one SNP in IGF-1 gene 3'UTR of Bama Minature pigs and Large White pigs could induce the difference of IGF-1 gene expression.The miR-new14regulated IGF-1 mRNA and protein levels,and caused different silencing efficiency of IGF-1 with 3'UTR of different genotypes.The miR-new14 inhibited the proliferation of PK-15 cells and promote its apoptosis.The mechanism may be that miR-new14 down-regulated the levels of AKT and ERK mRNA and inhibited the phosphorylation of AKT and ERK proteins.