Gene Silence Of Mcpr1 By RNAi Influence The Biological Behavior Of Mouse Embryonic Mesenchymal Palatal Cells

Cleft palate (CP) is one of the most common congenital deformities and its incidence is especially high in China. The investigation of congenital deformity in China from 1996 to 2000 shows that congenital cleft palate is the No.1 congenital deformity .The treatment of cleft palate is operation so far. But the operation treatment can not completely improve the patients'phonetic function and maybe impede dental eruption; it can also harm the patients'psychological health. The etiology of cleft palate is complex. Many genes contribute to this disease. Teratogens affecting either of these processes would lead to the formation of smaller shelves that fail to make contact upon elevation, a prerequisite for their fusion, leading to CP formation. Gene expression regulation is involved in the whole process and develops a regulation net to play a role during embryonic development. Mcpr1 was a novel gene cloned by subtractive hybridization method in our laboratory, which was obtained by comparing palatal shelves of normal murine embryo with ones of cleft palate models induced by retinoic acid. It was registered in GenBank as AYO74887, and named mouse cleft palate related gene. cDNA of Mcpr1 is 711bp which covers the whole sequence of gene BC120606 cloned by Mammalian Gene Collection Program Team. mRNA length of Mcpr1 gene had been identified. The expression of Mcpr1 in both mRNA level and protein level is observed in some tissues, such as liver, heart, brain, spleen, kidney, lung and muscle. Both the secretory protein and recombinant protein of Mcpr1 could inhibit cell proliferation of murine embryonic palatal mesenchymal cells, and impede the progression of G1-S phase in cell cycle. These findings suggest that Mcpr1 might function as one of retinoic acid up-regulated genes to be involved in inhibiting cell proliferation during palatogenesis and retinoic acid-induced cleft palate by regulating proliferation and apoptosis of MEPMCs. This study includes two parts involved in revealing functions of Mcpr1 gene initially. We construct Mcpr1 shRNA eukaryotic expression vector, silence the expression of Mcpr1 and analyze influence the biological behavior of MEPMCs. So is the following.AIMS:1. To construct Mcpr1 shRNA eukaryotic expression vector;2. To Observe the effect of Mcpr1 gene silence on MEPMCs biological behavior;METHODS:1. According to the Mcpr1 cDNA on Gene bank, we design two different shRNA targeting the coding sequence of the Mcpr1, the pGensil1-Mcpr1 shRNA was constructed by inserting the designed shRNA to the eukaryotic expression vector pGenesil-1.The vector was identified by restriction endonuclease digestion and partial nucleotide sequencing;2. The MEPMCs was transfected by pGensil1-Mcpr1 shRNA,negative control vector and positive control vector. After 48hr,the Mcpr1 and GAPDH expression was detected by RT-PCR;3. MTT assay was carried out to observe the effect on proliferation ability;4. FCM assay was carried out to observe the cell cycle;5. Western Blotting analyzed the expressions of CDK2,Cyclin E and p21.RESULTS and CONCLUSIONS:1. It was verified by restriction endonuclease digestion and partial nucleotide sequencing that the constructed eukaryotic vector expressing shRNA of Mcpr1 was correct;2. The constructed eukaryotic vector pGenesil1-Mcpr1shRNA1,pGenesil1- Mcpr1shRNA2,pGenesil1-Mcpr1 negtive-shRNA and pGenesil1- GAPDHshRNA were successfully transfected into MEPMCs; vector pGenesil1-Mcpr1shRNA1,pGenesil1-Mcpr1shRNA2 could inhibit the mRNA expression of Mcpr1 but pGenesil1-Mcpr1 negtive-shRNA and pGenesil1-GAPDHshRNA could not. Vector pGenesil1-GAPDHshRNA could inhibit the mRNA expression of GAPDH.3. There was significant difference in MTT Cell Proliferation Assay among the MEPMCs / pGenesil1-Mcpr1shRNA1 groop,MEPMCs / pGenesil1- Mcpr1 negtive-shRNA groop. 4. FCM results indicated that the proliferation activity of MEPMCs transfected with pGenesil1-Mcpr1shRNA1 was obviously up-regulated than transfected with pGenesil1-Mcpr1 negtive-shRNA.Transfection with pGenesil1-Mcpr1shRNA1 promoted cell cycle transition from S phase to G1 phase.5. After transfection, Western Blotting results showed that the expression of CDK2 and Cyclin E were up-regulated, p21 was down-regulated.