Mir-148a-3p Regulates Starvation-induced Autophagy In Porcine Intestinal Epithelial Cells By Targeting MLST8

Autophagy is a highly conserved catabolic process in eukaryotic cells that degrades cytoplasmic constituents and sustain metabolic function and energy homeostasis.Autophagy is important for cell survival,homeostasis,and disease defense ect.During the early neonatal starvation period,autophagy can maintain cells homeostasis and protect cells from excessive apoptosis.The intestinal tract is a main organ system that digests and absorbs nutrients.Autophagy can maintain intestinal homeostasis and resist the invasion of harmful microorganisms.At birth,the environment changes drastically and the neonatal’s intestinal tract faces severe stress.miRNAs are a type of small,endogenous,non-coding RNAs that modulate the cleavage of mRNAs or inhibit their translation by pairing with target mRNAs and usually present in various body fluids and participate in many physiological processes.Studies have reported that breast milk is rich in miRNA,and these milk-derived miRNAs affect the growth and development of newborns.miR-148a-3p has a very high expression level in many mammalian milks.However,miR-148a-3p regulate intestinal development through autophagy in piglets has not yet been revealed.In this study,we used small RNA-seq to identify the difference of miRNAomes in Pre-group,Glu-group and Col-group,and combined the expression of various mammalian colostrum miRNAomes and pigeon milk miRNAome.We used miR-148a-3p as a research subject and performed functional validation experiments in IPEC-J2 cells.The main results of this study are as follows:(1)We collected small intestine tissues for HE staining and related measurements and found a significant increase in the villus height and crypt depth of the Col-group.At the same time,we detected the level of autophagy and the expression of apoptosis-related genes in intestinal tissues,and found Col-group had higher levels of autophagy and lower levels of apoptosis compared with the other two groups.(2)The small intestine tissues total RNA were extracted and sequenced.We identified a total of 655 mature miRNAs in the six libraries,which contained 385 known miRNA,170 conserved miRNA and 100 novel miRNA.The results of hierarchical clustering analysis are divided into two categories:prenatal(Pre-group)and postnatal(Glu-group and Col-group).The top 10 miRNA(Top 10)in each group were compared and analyzed.It was found that seven miRNA were shared by all three groups Top 10,including miR-148a-3p.And,the expression level of miR-148a-3p was significantly changed in the three groups,and the trend of change was also consistent with the level of intestinal autophagy.(3)We analyzed and compared the miRNAomes of several mammalian colostrums pigeon milk,and found the expression level of miR-148a-3p is higher.(4)The ex vivo everted gut sac method was used to detect that miRNA in milk can be absorbed by the intestine.(5)Starvation can induce autophagy in IPEC-J2 cells,and fter comprehensive consideration,3 hour was established as starvation treatment time.Western blot,GFP-LC3 and qRT-PCR methods were used to verify that miR-148a-3p can enhance starvation-induced autophagy in IPEC-J2 cells.(6)Luciferase reporter assay confirmed that mLST8 is target gene of miR-148a-3p,and miR-148a-3p regulates starvation-induced autophagy by affecting mTOR pathway.In conclusion,the results indicate that colostrum changes the intestinal miRNAome of piglets.miR-148a-3p,which is highly expressed in milk,can be absorbed by the small intestine of piglets,and enhanced intestinal autophagy by inhibiting the expression of the target gene mLST8 and regulating the mTOR signaling pathway,further relieving starvation stress on piglets’ intestines.