MYCT1 Regulates Vascular Smooth Muscle Cells Ribosomal Protein Gene Expression And Cell Function

BackgroundSmooth muscle is widely distributed in the human body,such as the respiratory tract,digestive tract,blood vessels,urinary and reproductive systems.Vascular smooth muscle cells(vascular smooth muscle cells,VSMC)are mainly present in the vascular media,and their function is to maintain the normal physiological function of blood vessels,and also plays an important role in the body's metabolism and endocrine.The pathogenesis of many vascular diseases involves dysfunction of vascular smooth muscle cells.In the pathological process,vascular smooth muscle cells play an important role in the repair and repair of vascular diseases through their own proliferation,migration and synthesis of extracellular matrix,such as hypertension,vascular disease,restenosis after angioplasty,cerebral aneurysm,atherosclerosis,and repair after vascular wall damage,etc.As a very important organelle,ribosome is composed of ribosomal protein(RP)and ribosomal RNA(rRNA).Ribosomal proteins have two important functions:one is to participate in intracellular protein synthesis,and the other is to regulate cell growth,proliferation,differentiation,apoptosis and other functions.The latter is called extraribosomal function.Therefore,the change of ribosomal protein in VSMC may involve the occurrence and development of different vascular diseasesMYCT1 is the first c-Myc target gene found in laryngeal cancer cells and was previously named MTLC.The MYCT1 gene contains two exons and produces a 1006 bp transcript encoding a protein with 235 amino acid residues.Studies have confirmed that MYCT1 is widely expressed in normal tissues,and is lowly expressed in various malignant tumor tissues such as laryngeal cancer,liver cancer and gastric cancer.After overexpression of MYCT1,the proliferation ability,apoptosis,and invasion ability of cancer cells decreased.The expression of MYCT1 in non-metastatic cancer tissue is greater than that of metastatic cancer.Most of the previous studies on MYCT1 have explored the regulation of this gene on various cell functions in various tumor cells.However,at present,MYCT1 has not studied the relevant functions of VSMC.In view of the importance of VSMC and RP,this topic aims to explore whether MYCT1 affects the function of VSMC by affecting the expression of RP.The research contents of the subject include:(1)whether NOC18(simulation of nitration stress caused by nitric oxide)can affect the expression of RP by regulating the expression of MYCT1;(2)take pro-Collagenlal as an example to study the inhibition of MYCT1 expression(In turn,it affects the ribosome biosynthesis)whether it has an effect on the translation ability of VSMC protein;(3)To investigate whether MYCT1 has an effect on the proliferation,apoptosis,and migration function of VSMC.Contents and methods1.Whether stress stimulation(NOC18 mimics nitrosation stress)can affect RP expression by regulating MYCT1:in vitro culture of rat VSMC,give cells NOC18 stimulation,real-time quantitative PCR method to detect the expression of MYCT1 at the mRNA level;by lentivirus After transfection overexpression of MYCT1 or NOC18 stimulation,the expression change of RPmRNA was detected;after shRNA lentivirus transfection shRNA knocked down the expression of MYCT1,real-time quantitative PCR and Western blotting were used to detect the change of RP mRNA and protein expression levels.;2.Take collagen pro-Collagenlal as an example to investigate whether the inhibition of MYCT1 expression has an effect on the translation ability of VSMC extracellular matrix protein:Lentiviral transfection knocks down MYCT1,and compares pro-Collagen1?1 at the mRNA level and at the basic level after TGF-p1 stimulation.Whether there is a differential change in the expression level of the protein level(ELISA method)to indirectly clarify whether the decrease in MYCT1 expression reduces the ability of VSMC protein translation3.The effect of MYCT1 expression changes on the proliferation,apoptosis and migration of VSMC:after lentivirus transfection knocked down MYCT1,Cell Counting Kit-8 kit was used to detect the proliferation ability of VSMC;Caspase-Glo(?)3/7 test kit was used to detect Apoptosis ability of VSMC;cell migration test(Wound Healing)was used to detect the migration ability of VSMC.Results1.In rat VSMC,NOC18 can down-regulate MYCT1 and RP mRNA levels;after knocking down MYCT1,RP mRNA and protein levels were significantly reduced.After overexpressing MYCT1,the role of NOC18 in inhibiting RP expression was partially reversed.2.In rat VSMC,TGF-?1 up-regulates the mRNA level of pro-Collagenlal,and increases the protein level of pro-Collagenlal accordingly.In cells knocked down by MYCT1,TGF-?1 also up-regulated the mRNA and protein levels of pro-Collagen1?1.Compared with the empty vector control,the pro-Collagenlal mRNA level in MYCT1 shRNA-420 was up-regulated,but its protein level was down-regulated.3.In rat VSMC,after knocking down MYCT1,compared with the control group,the cell proliferation and migration capacity were weakened and apoptosis increased;whereas overexpression of MYCT1 in normal VSMC had no significant effect on cell proliferation and apoptosis.Conclusions1.Stress stimulation in VSMC can affect the expression of RP by regulating the expression of MYCT1.2.MYCT1 in VSMC may affect the ability of cells to synthesize(protein translate)matrix proteins by regulating RP expression.3.Changes in the expression of MYCT1 have an effect on the proliferation,migration,and apoptosis of VSMC;whether these effects of MYCT1 depend on its regulation of RP expression has further follow-up studies.4.Our results suggest that MYCT1 regulates the expression of RP in vascular smooth muscle cells,which in turn affects the ability of cells to synthesize matrix proteins.MYCT1 may become a new target for regulating the damage repair function of vascular smooth muscle cells.