The Identification And Verification Of The Target Genes Of MiRNA-141-3P And MiRNA-346 In Rat Anterior Pituirtary

Micro RNAs(mi RNAs) are a family of non-coding RNA with the length of 19-25 nucleotides. Mi RNAs are known to play important roles in the regulation of a varity of celluar processes by downregulating the expression of specific target m RNAs after binding to complementary sequences present in the m RNA 3′ untranslated region(3′ UTR). Mi RNAs also take part in many biological processes that occur during development and diseases,such as tumors, diseases of the cardiovascular system and nervous system disfunction. It’s reported, mi R-181 a contribute to gastric cancer and mi RNA-7 are associated with the development of the pituitary. Above all, the study mi RNA has become more and more important.Anterior pituitary is one of the most important endocrine organs,which secrets lots of hormones involved in growth, metabolic and reproduction. Anterior pituitary secrets kinds of gonadotropins including follicle-stimulating hormone(FSH) and luteinizing hormone(LH). They regulate the development of a variety of cells in the testis and the synthesis and secretiongonadal of steroid hormone, that play an important role to reproduction. However, the regulation of expression of follicle-stimulating hormone receptor(FSHR) is still unknown. Therefore, the identification of mi RNAs that targeting FSHR in the anterior pituitary will help us understand more about the regulation of anterior pituitary by FSH.Our study focus on exploring the interaction between mi RNA and FSHR. Male Wistar rat pituitary(clean grade) tissues were harvested in 21 days and 6 months. Total RNA were extracted. After using micro RNA array detection, we analysed the difference between mi RNAs in these two periods, and found that the expressions of mi RNA-141-3p and mi RNA-346 showed difference in different periods, the RT-PCR results confirmed our conclusion. Remarkably, we noticed that FSHR is the potential target of mi RNA-141-3p and mi RNA-346 identified using Target Scan software. We designed the primers to introduce mutation according to FSHR 3’UTR wildtype sequence, we changed CAGTGTT(78-85) into GTCACAA and changed GGCAGAC(127-133) into CCGTCTG. We acquired FSHR mutant 3’UTR from wildtype template by PCR and inserted into pmi R-RB-REPORT? dual luciferase report carrier. It’s known that 3’UTR contains mi RNA’s binding site.We co-tranfered mi RNA with luciferase reporter to identify the interaction between mi RNAs and their target.Our results showed that rno-mi R-141-3p mimic did not down-regulate FSHR reporter, while rno-mi R-346 mimic down-regulate FSHR reporter significantly. The down-regulation could be rescued by introducing mutant binding sites. We concluded that rno-mi R-346 could regulate the expression of FSHR by binding 3’UTR in vivo, while rno-mi R-141-3p was unable to regulate by the same way.In summary, we discovered that both mi RNA-141-3P and mi RNA-346 involved in the post-transcription regulation of FSHR. Meanwhile, our study provided a theoretical basis for further study the effect of mi R-141-3P and mi R-346 on pituitary development, hormone secretion and reproductive function.