| Tuberous Sclerosis(Tuberous Sclerosis Complex,TSC)is a multi-system involved autosomal dominant syndrome due to the TSC-1 or TSC-2 gene mutation,leading to abnormal expression of tumor suppressor factor hamartin and tuberin respectively,and then activating downstream m TORC1(Mechanistic target of rapamycin complex 1)pathway,with uncontrolled pathways of phosphatidylinositol 3-kinase(PI3K),extracellular signal-regulated kinase and phospho-S6 ribosomal protein,finally affecting cell growth,proliferation,differentiation,autophagy and apoptosis.Because of its extensive involvement of organizations and organs,the symptoms are complex,clinical performance can be "benign" tumor of the lung,heart,brain,kidney,skin,face and retina,eventually leading to multiple organ failure and death,being seriously harmful to human health.Lymphangioleiomyomatosis(LAM)is the main complication in the lung of TSC patients,frequently-occurring disease in childbearing or postmenopausal women.Its main characteristics are progressive dyspnea,hemoptysis,pneumothorax,chylothorax therefore lack of specificity.Clinical diagnosis depends on lung biopsy.The typical pathological feature is abnormal proliferation of smooth muscle cells invading the lung,involving the lymph-vessel,vessels and trachea,and then blocking the airway and lymphatic vessels and causing abnormally dilated lymphatic and pulmonary cystic,then leading to form nodules,widely cystic dilatation of alveoli and respiratory failure in its late stages.Given that LAM is the female-predominant disease,we suggest that estrogen may promote the progression of LAM,clinical treatment including ovariectomy,estrogen receptor antagonist,has achieved better clinical efficacy,but individual difference is large.Rapamycin,as the inhibitor targeting m TORC1,had been approved for treatment of the LAM by the United States Food and Drug Administration(USFDA or FDA)in May 2015,significantly improving lung function.However,subsequent clinical trials indicated that the lung function of LAM patients continued to deteriorate without rapamycin.Thus,we need to explore mechanism of LAM for more effective treatment,which has important clinical significance.At present,although the source of LAM cells is not known,the genetic analysis of patients with sporadic and relapse of LAM supported that LAM cells migrate to the target organ via the potential pathways.The LAM patients who received lung transplantation still relapsed.The more and more evidence-based medicine suggests that LAM may be the metastatic tumor disease.Insulin-like growth factor binding protein 2(Insulin-like growth factor binding protein 2,IGFBP2),belonged to IGFBP superfamily,with a high affinity for the insulin-like growth factor(IGF)-I and-Ⅱ,which is also involved in activating pathways including integrin,VEGF,NF-κB,ERK1/2,etc,being closely associated with the proliferation and metastasis of tumor,inducing tumor angiogenesis,tumor apoptosis and regulation of estrogen.It has been used as the tumor biomarker of therapeutic effect and prognosis in breast cancer,ovarian cancer,colorectal cancer,glioma,lung cancer,prostate cancer,gastric cancer,being expected to the candidate target for cancer treatment.Our study showed that the expression of IGFBP2 and ER-α in LAM tissue is high compared with normal lung tissue,suggesting that E2/ER-α mediating IGFBP2 may play an important role in the growth and metastasis of LAM,suggesting that E2 may promote the progression of LAM.This study aims to explore the mechanism of development and metastasis of LAM,and explore whether E2/ER-α mediating IGFBP2 is involved in the progression of LAM,then screen and identify the potentially therapeutic targets,improving the clinical efficacy of LAM.Additionally,analysis the clinical pathological significance and prognosis of IGFBP2 in LAM specimens and the correlation with the expression of ER-α and then explore the possible mechanism.The studies confirm that TSC2 gene deletion is key to the LAM pathogenesis.We have constructed successfully LAM primary cells(621-101 TSC2-and 621-103 TSC2+).In vitro,explore whether E2/ER-α modulates the expression of IGFBP2 in LAM patients cells by upregulating expression of ER-α,and explore possible mechanisms.Knockdown the expression of IGFBP2,evaluate its role of cell proliferation,apoptosis,the invasion and metastasis in LAM cell.Meanwhile,construction the CB17-SCID xenograft model of ELT3 cells(TSC2-),replacing 621-101 cell,that is difficult to form xenograft,further explore whether E2/ER-α mediating IGFBP2 promote the growth of LAM in vivo.Part 1 The expression of IGFBP2 and ER-α and correlation with clinical pathological significance in normal lung and LAM specimens Methods1.Western blot and Real time-PCR were used to detect the protein and m RNA expression of IGFBP2 and the protein of P-S6 in 21 specimens of normal lung and 21 specimens of LAM.2.Immunohistochemistry was used to detect the protein expression of IGFBP2,α-actin,ER-α and P-S6 in LAM tissues.3.Immunofluorescence was used to locate the expression of IGFBP2 and P-S6 in LAM tissues.4.Analyze the expression correlation between IGFBP2 and ER-α and the relationship with clinicopathological parameters.5.Statistical analysis: the data were evaluated using Graph Pad Prism 6.0 software.The Pearson’s χ~2 test was used to compare the categorical variables.The Spearman rank correlation coefficient analysis was used to assess the correlation between two ordinal variables.Student’s t-test was used to determine differences between the two groups.P value < 0.05 was considered as statistical significance.Results1.The results of Western blot and Real time PCR showed that the rate of relative expression increased by more than two times of IGFBP2 protein and m RNA in LAM tissue were higher compared with normal lung tissue,and the rate was 71.43%,14.29%,76.19% and 19.05%,respectively.The expression of P-S6 in LAM was significantly higher than normal lung(P<0.05).2.The result of immunofluorescence showed that expression of IGFBP2 was primarily located in the nucleus of LAM tissue,and P-S6 was primarily located in membrane and cytoplasm.3.According the results of IHC,the expression P-S6 and α-actin in the LAM tissue further indicated pathological diagnosis of LAM.The expression of IGFBP2 and ER-α was primarily located in the nucleus of LAM tissue,the expression of ER-α was higher compared with normal lung tissue.The positive expression rate of IGFBP2 in normal lung,LAM with negative and positive expression of ER-α was 4.76%,33.33% and 88.89%(P<0.05).4.IGFBP2 expression in the LAM tissue was showed no correlation with age,tumor size(P>0.05),but the correlation with lung function(FEV1),suggesting that IGFBP2 may be the potential biomarker of LAM.The positive correlation was found between the expression IGFBP2 and ER-α using Spearman rank correlation analysis(P<0.05).Conclusions1.The protein and m RNA expression of IGFBP2 was significantly higher in LAM tissues than in normal lung tissues,and the protein expression of IGFBP2 was primarily located in the nucleus.The high expression of IGFBP2 was close relations with pulmonary dysfunction,suggesting that IGFBP2 may be as a potential predictive biomarker for poor prognosis in LAM patients.2.The positive correlation was found between the expression IGFBP2 and ER-α in LAM tissues,suggesting that IGFBP2 may be involved in E2/ER-α promoting the development and metastasis of LAM.Part 2 E2/ER-α modulates the expression of IGFBP2 in LAM cell 621-101 Methods1.The IGFBP2 protein expression in Human LAM cell 621-101(TSC2-)and 621-103(TSC2+)were detected by Western blot.2.The 621-101 cell was transfected with p EGFP-C1-ER-α vector,and transfection efficiency was detected by Western blot.3.The IGFBP2 protein expression in control group,transfected ER-α group and transfected ER-α combined with E2 24 h group was detected by Western blot.4.Immunofluorescence was used to locate the IGFBP2 protein in control group,transfected ER-α group and transfected ER-α combined with E2 24 h group.5.Statistical analysis: The data were evaluated using Graph Pad Prism 6.0.The Pearson’s χ~2 test was used to compare the categorical variables.Student’s t-test was used to determine differences between the two groups.P value less than 0.05 was considered as statistical significance.Results1.The result of western blot showed that the expression of IGFBP2 protein was significantly increased in 621-101 cell compared with 621-103 cell.2.The result of western blot showed that the expression of ER-α protein was significantly increased in 621-101 transfected ER-α group compared with control group,and the difference was statistically significant(P<0.05).The 621-101-ER-α+ cell was successfully constructed for subsequent research.3.The result of western blot showed that the IGFBP2 protein expression of control group,transfected ER-α group and transfected ER-α combined with E2 24 h group was not statistically different(P > 0.05).4.The result of immunofluorescence showed that the expression of IGFBP2 protein was primarily distributed in the cell membrane and cytoplasm in transfected ER-α cells and control group while transfected ER-α combined with E2 24 h group was in the nucleus,suggesting that E2/ER-α can promote IGFBP2 to migrate into cell nucleus.5.The result of Western blot showed that the nuclear expression of IGFBP2 protein in transfected ER-α combined with E2 24 h group was higher(P<0.05),compared with control group and transfected ER-α group.Conclusions1.The expression of IGFBP2 protein was significantly increased in 621-101 cell compared with 621-103 cell,suggesting that TSC2 may modulates the expression of IGFBP2 in LAM cell 621-101.2.The 621-101 ER-α+ cell was successfully constructed.E2 may promote the nuclear expression of IGFBP2 in LAM cell 621-101 ER-α+,suggesting that E2/ER-α mediating IGFBP2 promoted the development and metastasis of LAM.Part 3 Knockdown expression of IGFBP2 by si RNA affects biological function of LAM 621-101 cell Methods1.621-101 cell was transfected with si RNA IGFBP2 and transfection efficiency was detected by western blot and real time PCR.2.Cell proliferation was detected using MTT method.3.Cell death was detected using PI exclusion assay.4.Transwell method was used to detect the migration and invasion of cells.5.The expression of p-ERK1/2 protein was detected by Western blot.6.The expression of p-ERK1/2,IGFBP2 and P-S6 was detected by Western blot in 621-101 cell after single or combined treatment with rapamycin and AZD6244.7.Statistical analysis: the data were evaluated using Graph Pad Prism 6.0.The Pearson’s χ~2 test was used to compare the categorical variables.Student’s t-test was used to determine differences between the two groups.P value less than 0.05 was considered as statistical significance.Results1.The expression of IGFBP2 protein and m RNA was decreased in si RNA transfection group compared with control group,and the difference was statistically significant(P< 0.05).2.Compared with the control group,E2 could increase the cell proliferation,and this effect was inhibited by knockdown IGFBP2(P< 0.05).3.Compared with the control group,E2 could decrease the cell death,and this effect was inhibited by knockdown IGFBP2(P< 0.05).4.Compared with the control group,the ability of cell migration and invasion was reduced in si RNA transfection group(P< 0.05).5.Compared with the control group,the expression of p-ERK1/2 was inhibited in si RNA transfection group.6.Rapamycin could reduce the expression of P-S6 protein,AZD6244 could reduce the expression p-ERK1/2 protein and increase the expression of p-S6.The single or combined treatment of Rapamycin and AZD6244 did not affect the expression of IGFBP2.Conclusions1.E2 could increase the cell proliferation and decrease the cell death,which was inhibited by knockdown expression of IGFBP2.Silencing IGFBP2 gene can also inhibit the invasion and metastasis of LAM cell.These results suggested that E2/ER-α mediating IGFBP2 promoted the development and metastasis of LAM.2.The IGFBP2 promoting the development of LAM may be related to activation of downstream p-ERK1/2 pathway.3.The single or combined treatment of rapamycin and AZD6244 did not affect the expression of IGFBP2,however,the expression of p-ERK1/2 was inhibited by knockdown the expression of IGFBP2,suggesting that p-ERK1/2 may be signaling pathways downstream of IGFBP2.Part 4 E2/ER-α mediating IGFBP2 promotes tumor growth of TSC2-ELT3 cell in nude mice Methods1.The nude mouse transplantation tumor model of TSC2-ELT3 cell was established,and the difference growth of nude was compared between E2 treatment group and the placebo group.2.Immunohistochemistry was used to detect the expression of IGFBP2 and α-actin in E2 treatment group and the placebo group.3.Immunofluorescence histochemical staining was used to detect the location of IGFBP2 and P-S6 protein expression in E2 treatment group and the placebo group.4.Western blot was used to detect the nucleus and cytoplasm expression of IGFBP2 in E2 treatment group and the placebo group.5.Statistical analysis: the data were evaluated using Graph Pad Prism 6.0.The Pearson’s χ~2 test was used to compare the categorical variables.Student’s t-test was used to determine differences between the two groups.P value less than 0.05 was considered as statistical significance.Results1.The growth of the transplanted nude was significantly increased in E2 treatment group compared with the placebo group,and the difference was statistically significant(P<0.05).2.The positive rate of IGFBP2 protein in E2 treatment group and the placebo group was 80% and 20%,according to the result of immunohistochemistry,and the differences was statistically significant(P<0.05).3.The protein expression of IGFBP2 was primarily located in nucleus and P-S6 was mainly in the cytoplasm and cell membrane according to the result of histochemical immunofluorescence,E2 can promote nucleus expression of IGFBP2.4.The result of western blot showed that the nucleus expression of IGFBP2 in E2 treatment group was higher compared to the placebo group(P<0.05).Conclusions1.E2 can increase the growth of the transplanted tumor and promote the nuclear expression of IGFBP2 protein in vivo.2.IGFBP2 may be the effector gene of E2/ER-α,suggesting that E2/ER-α mediating IGFBP2 may promote the development of LAM.Conclusions1.Our previous study showed E2 may promote the growth and progression of LAM.This study showed that the protein and m RNA expression of IGFBP2 was significantly higher in LAM tissues than in normal lung tissues,and the positive correlation was found between the nuclear expression IGFBP2 and ER-α in LAM tissues,suggesting that IGFBP2 may be involved in E2/ER-α promoting the development and metastasis of LAM.The high expression of IGFBP2 was close relations with pulmonary dysfunction,suggesting that IGFBP2 may be as a potential predictive biomarker for poor prognosis in LAM patients.2.621-101 ER-α+ cell cell was successfully constructed.E2/ER-α can regulate IGFBP2 nuclear expression,suggesting that E2/ER-α mediating IGFBP2 promoted the development and metastasis of LAM.3.E2 could increase the cell proliferation and decrease the cell death,which and was inhibited by knockdown expression of IGFBP2.Silencing IGFBP2 gene can also inhibit the invasion and metastasis of LAM cell.These results suggested that E2/ER-α mediating IGFBP2 promoted the development and metastasis of LAM.The IGFBP2 promoting the development of LAM may be related to activation of downstream p-ERK1/2 pathway.4.E2 can increase the growth of the transplanted tumor and promote the nuclear expression of IGFBP2 protein in vivo.IGFBP2 may be the effector gene of E2/ER-α,suggesting that E2/ER-α mediating IGFBP2 may promote the development of LAM. |
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