5-hydroxy-l-tryptophan Regulates Intracellular Calcium Level Via MiR-99a-3p/ATP2B1 In Goat Mammary Epithelial Cells

Calcium is one of the most abundant mineral elements in animals.It has the functions of forming bones and teeth,maintaining excitability of muscles and nerves.5-Hydroxy-L-tryptophan（5-HTP）is the primary product that converts L-tryptophan into serotonin（5-HT）.5-HT was considered as a regulator of bone turnover in lactation,playing important roles in inducing bone regulating factor,maintaining bone density and inducing bone remodeling.Micro RNAs（miRNAs）are a class of natural noncoding small molecule RNAs,influencing a serious of physiological processes like calcium metabolism by regulating target gene.Plasma membrane calcium ATPase1（ATP2B1）is a high affinity calcium pumps which contributes to the maintenance of intracellular Ca²⁺homeostasis.In our early research,six healthy goats（1.5-years old）of similar body weight were divided into two groups equally:5-HTP-injected group and saline-injected group（solvent of 5-HTP,as the control group）.The goats were injected with 5-HTP or saline daily for 7days before delivery,and the blood samples on the day of delivery were collected to perform the whole transcriptome sequencing assay.Based on the previous findings,the miRNAs which are regulated by 5-HTP as well as related with calcium metabolism were screened.Then,the molecular mechanism about how 5-HTP regulates intracellular calcium level of goat mammary epithelial cells（GMECs）by miRNA and its target genes were verified via bioinformatics assay and molecular cell biology methods.This study mainly obtains the following results:1. miR-99a-3p was significantly up-regulated in goats after 5-HTP injection by analyzing the whole transcriptome sequencing data（p（27）0.05）.In addition,the experiment in vitro also found that 5-HTP treatment resulted in a significant increase of miR-99a-3p level in GMECs（p（27）0.05）.2. ATP2B1 is one of the target genes of miR-99a-3p by bioinformatics analysis.Inhibiting or over-expressing miR-99a-3p in GMECs could significantly increase/decrease ATP2B1 m RNA and protein expression（p（27）0.05）,proving that miR-99a-3p targeted to regulate ATP2B1 expression in GMECs.3. The level of intracellular calcium ions was significantly increased after decreasing ATP2B1 expression using the RNA interfering technology（p（27）0.05）.4. miR-99a-3p increased intracellular calcium ions level by reducing ATP2B1expression in GMECs（p（27）0.05）.In summary,we drew the conclusion that 5-HTP increased intracellular calcium ions level by regulating miR-99a-3p/ATP2B1 in GMECs.This may be one of the potential reasons why 5-HTP injection could increase the calcium concentration in goat milk,which will help to accumulate data for calcium metabolic balance of mammals.