Mechanism Of Rapamycin Regulating IGFBP3 Inhibiting The Growth And Metastasis Of Lymphangioleiomyomatosis

Background and ObjectiveTuberous Sclerosis Complex(TSC)is a rare,multisystemic autosomal dominant genetic disease characterized by benign congenital tumors in multiple organs.Tumors differentiated by epithelioid cells around blood vessels,including vascular smooth muscle lipoma(AML)and lymphangioleiomyomatosis(LAM).TSC is caused by the mutation of TSC-1(coding for the protein of the unconstructed protein)or TSC-2(coding for the potato protein),resulting in the abnormal expression of the encoded protein,which Activated the downstream rapamycin target protein complex 1(mechanistic target of rapamycin complex 1,mTOR1)pathway,resulting in the abnormality of multiple signaling pathways,resulting in changes in biological functions such as cell proliferation,differentiation,and apoptosis.TSC is a multi-systemic systemic disease,and eventually lead to multiple organ failure and death.Pulmonary lymphangioleiomyomatosis(LAM)is the main complication of TSC in the lungs.The incidence of pulmonary cystic lesions in female TSC patients is about 26%-38%.LAM occurs in women of childbearing age or premenopausal women and is characterized by proliferation of atypical muscle cells(LAM cells)around the airways,blood vessels,and lymphatics.LAM may be sporadic(S-LAM)or associated with tuberous sclerosis syndrome(TSC-LAM).Disease progression can lead to blockage of blood vessels and airways,cyst formation,and lung destruction.The main clinical features of LAM include progressive dyspnea,pneumothorax,chylothorax,and abdominal tumors,such as angiomyolipoma and lymphangioleiomyoma,and occasionally hemoptysis.LAM cells have histologically smooth muscle characteristics and can exhibit TSC1 or TSC2 gene mutations.The study found that the key to the pathogenesis of LAM is that mutations in the TSC2 gene cause abnormal activation of the mTORl pathway.Rpamycin has been approved by the US Food and Drug Administration(FDA)for the treatment of LAM as a targeted inhibitor of mTOR1.It can significantly improve the lung function of LAM patients.Therefore,it is of great clinical significance to continue to explore the mechanism of the occurrence and development of LAM diseases and explore more effective treatments.At present,the detailed pathogenesis of LAM is not fully understood.Insulin-like growth factor binding protein 3 is an important member of the IGFBP protein family and is the highest content of IGF binding protein in serum.It binds insulin,IGF-? and ? in the circulation,and participates in Activation of VEGF,NF-?B and other pathways,thereby regulating the survival,differentiation,migration and invasion of tumor cells.Recent studies have confirmed the role of IGFBPs in the diagnosis and prognosis prediction of solid tumors including rectal cancer,ovarian cancer,and pancreatic cancer.Although IGFBP3 plays an important role in tumorigenesis,the mechanism of IGFBP3 in LAM and other hormone-dependent tumors is still unknown.Therefore,IGFBP3 is expected to become one of the most promising candidate targets for cancer treatment.Through previous research,we found that compared with normal lung tissue,IGFBP3 and P-S6 are highly expressed in LAM tissue.After treatment with sirolimus(rapamycin analog),IGFBP3 and P-S6 are expressed.reduce.It suggests that rapamycin can control the mechanism of LAM by targeting the mTORl pathway to regulate IGFBP3 and P-S6.Screen and identify potential effective therapeutic targets.Studies have confirmed that the key to the pathogenesis of LAM lies in the deletion of the TSC gene.In the early stage,human LAM primary cells(621-101 TSC2-and 621-103 TSC2+cells)were successfully constructed.Through in vitro experimental studies,in complete medium containing 10%FBS Culture the cells separately under the condition of the medium without serum,observe the growth and appreciation of the cells,after intervening with rapamycin,look at the expression of IGFBP3 and P-S6 proteins in the cells,and explore their possible mechanisms After silencing IGFBP3 gene expression,the effects of IGFBP3 on the biological functions of human LAM cells such as proliferation,invasion and metastasis were evaluated.Part1 Expression of IGFBP3 and P-S6 in normal lung tissue and LAM tissueMethodsThe pathological changes of LAM tissues and normal lung tissues were observed under HE staining microscope.The expression of IGFBP3 and p-s6 in LAM tissues was detected by immunohistochemical method.The protein expression in LAM tissues was changed after treatment with sirolimus(rapamycin analogue).Results1.The results of immunohistochemical:According to LAM specific marker proteins smooth muscle actin and P-S6 positive expression in LAM tissues to further evaluate the pathological diagnosis of LAM.Phospho-S6 positive cell clusters can be seen due to activation of the mTOR pathway.After sirolimus treatment,the mTOR pathway is inhibited and Phospho-S6 is negative.2.Previous studies have found that IGFBP2 is highly expressed in LAM patients.In the patients in this study,we found that the previous IGFBP2 was positively expressed in LAM tissues?The study found that IGFBP3 was positively expressed in these two patients.SummaryThe expression of IGFBP3 and P-S6 is positive in LAM tissues,and P-S6 is negative after sirolimus treatment.Because the high expression of IGFBP3 is related to the decrease of lung function in LAM patients,it may be an important predictor of clinical progress and prognosis in LAM patients Potential biological indicators.Part 2 Rapamycin regulates the expression of IGFBP3 and P-S6 in human LAM cells 621-101 and 621-103Methods1.The CCK8 method was used to detect the growth curves of 621-101 and 621-103 cells in two cases containing 10%FBS medium and serum-free medium respectively.2.Detect the cell cycle of 621-101 and 621-103 cells in two cases containing 10%FBS medium and serum-free medium respectively.3.Western blot method was used to detect the expression of secreted proteins in the supernatants of 621-101 and 621-103 cells.4.Western blot detected changes in the expression of 621-101 and 621-103 cells under the influence of rapamycin,and compared with serum-free medium culture.5.PT-PCR was used to detect the effect of rapamycin on IGFBP3 mRNA expression in 621-101 and 621-103 cells,and compared with serum-free medium culture.6.Statistical analysis:Statistical analysis:Graph Pad Prism 8.0 software is used for all data analysis and statistics.The measurement data is represented by x±s,and independent sample t test statistics are used to determine the difference between the two groups of averages;?=0.05 is the significance test level.Results1.The results of Western blot showed that 621-101 cells secreted more protein IGFBP3 than 621-103 cells.2.The results of Western blot showed that rapamycin significantly inhibited the expression of P-S6 protein in 621-101 and 621-103 cells,and the same conclusion was reached when compared with cells cultured in serum-free medium.However,rapamycin inhibited IGFBP3 in 621-101 cells more strongly than 621-103 cells.3.The results of RT-PCR showed that in 621-101 cells,cells were cultured in complete medium containing 10%FBS,and rapamycin in 621-101 cells inhibited the expression of IGFBP3 mRNA stronger than cells cultured without serum(P<0.05).There was no significant difference in the effect of rapamycin on the expression of IGFBP3 gene between the 621-103 cells and serum culture cells.Summary1.High expression of IGFBP3 in human LAM cells,rapamycin can inhibit the expression of IGFBP3 in 621-101,and the inhibition effect is more significant under sufficient nutrition,as an inhibitor of mTOR pathway,it may participate in the regulation of IGFBP3,Inhibited the expression of IGFBP3.2.P-S6 is highly expressed in human LAM cells.Rapamycin can inhibit P-S6 in 621-101 and 621-103.As an inhibitor of the mTOR pathway,it also participates in the regulation of P-S6.Part III The effect of silenced IGFBP3 gene expression on the biological function of TSC2-cellsMethods1.Transfect 621-101 cells to obtain cells that silence the expression of IGFBP3 gene,and use WB to detect the transfection efficiency.2.After successful transfection,use cck-8(draw growth curve)to detect the growth of cells.3.After successful transfection,Transwell method was used to detect changes in cell migration and invasion ability.4.Statistical analysis:Graph Pad Prism 8.0 software was used for all data correlation analysis and statistics.Independent sample t test statistics were used to determine the difference between the two groups of means;with ?=0.05 as the significance test level.Results1.Compared with the control group,the expression of IGFBP3 protein in the siRNA transfection group decreased.2.Compared with the control group,knockdown IGFBP3 expression can inhibit cell proliferation(P<0.05).3.Compared with the control group,the Transwell method detects the reduction of cell migration and invasion ability after knockdown IGFBP3 expression(P<0.05).SummaryAfter knockdown IGFBP3,the cell's ability to increase value,migration and invasion is weakened,and the ability to express IGFBP3 is weakened,indicating that IGFBP3 is involved in the occurrence and development of LAM disease.The mTOR pathway inhibitor rapamycin can inhibit its value-added and protein expression capabilities,indicating that rapam Amycin inhibits the expression of IGFBP3 by targeting the mTOR pathway.Conclusions1.This study found that compared with normal lung tissue,IGFBP3 and P-S6 are highly expressed in LAM tissue,and the high expression of IGFBP3 is closely related to the decrease of lung function.Rapamycin can inhibit the expression of IGFBP3 and P-S6.2.The mTOR1 pathway inhibitor rapamycin can inhibit the expression of IGFBP3 and P-S6 in 621-101 cells,and silencing the expression of IGFBP3 can inhibit the proliferation,invasion and migration of cells,suggesting that IGFBP3 is the cause of LAM disease in the mTOR pathway The important protein,rapamycin,inhibits the progression of LAM disease by inhibiting the expression of IGFBP3.