| IntroductionGastrointestinal stromal tumors(GIST)are common mesenchymal tumors of the gastrointestinal tract with the incidence rate 2/100,000,which account gastrointestinal tract cancer of 1%-4%.There is no significant difference in race and gender.GIST appears in any part of the digestive tract,which commonly arise in the stomach and intestine.Colon/rectumi are the second predilection site,It also happens in the omentum,retroperitoneal mesangial and parenteral tract.On the past,the organization, biological behavior and the malignancy of GIST are not clear,which brings a lot of confusion to the clinical diagnosis and treatment.With the development of immunohistochemistry,molecular biology,gene engineering and the emergence of Imatinib mesylate(Glivec),the diagnosis and treatment of GIST have made great progress in recent years.Although,Compared with other tumor,GIST is still unpredictabily,the biological characteristics of GIST are difficult to assess.how to control the invasion and metastasis is the main problems to treat GIST.Carcinogenesis are controlled by many cell incidences and Physiological processes,cell's signal transduction pathway is one of important reasons.A lot of cell function such as Proliferation,differentiation,apoptosis and glucose transportation are regulated by Phosphatidylinositol 3-kinase(PI3K).during passed few years,study showed IA type PI3K and cellueoprotein kinase B(PKB or Akt)were very important in generation,development,treatment and recurrence of cancer,it had became a new target for the treatment of cancer.The further study of this pathway regulation mechanism and the relationship with other pathway is conduced to increase the recognition of the effect in human normal tumor.It regulates cell proliferation and survive,the unusual activiting of PI3K/Akt pathway not only can induce cell carcinogenesis but also have relation with tumor cell immigration adhesion tumor blood vessels generation and extracellular matrix degradation.PTEN is a tumor suppressing gene,the mutation of PTEN can cause the lose of cell generation negative control and spring PI3K/Akt pathway witch induce carcinogenesis process.PTEN is one of the easiest mutation target gene,the rate is common to P53.PI3K as a bridge molecular combined the signal in vitro with cell response,witch is not completely independent,but have important regulating role in cell apoptosis through the effect that a series of upstream or bypassing signal factors act on the downstream signals.Many studies had showed the anti-apoptosis function of PI3K and Akt.Akt mainly acts in the anti-apoptosis pathway,PI3K/Akt can inhibit cell apoptosis and introduce cell survive through many way.It controlled cell survive through direct or indirect involved in the transcription factors such as Forkhead,NF-KB,P53 and so on.The angiogenesis of tumor is a requirement of the transference of tumor.PI3K/Akt signal path takes part in the tumor angiogenesis mediated by several factors.HIF-1, being a crucial regulatory factor,plays an Important role in the angiogenesis and invasion of tumors.The function of HIF-â… is mainly due to the expression and activity of HIF-â… Î±subunit.The expression of HIF-â… Î±protein is regulated by cancer suppressor genes PTEN in PI3K/Akt signal path,promoting the transcription of hypoxia-reacted genes and arousing a series of adaptation for hypoxia.It is also concerned with the metabolism of tumors,dilation of vessels and angiogenesis et al.Invasion and transference of tumors is a complicated process containing the interaction between the tumor cells and host cells.The degree of invasion and transference of tumors is the major factor to the prognosis.But the molecular mechanism of GIST invasion and transference is still unknown.In recent studies, PI3K/Akt signal path was found activated consistantly.TGF-βcan up regulate the expression of uPAmRNA and its protein depending on the dosage and time,mainly mediated by the PI3K/Akt/NF-KB signal path.It has been identified that,Upa is the target gene of NF-KB.So it was confered that the activation of PI3K/Akt signal path leads to the up regulation of NF-KB,which induced the transference of tumor by targeting uPA.That is to say,uPA is mainly regulated by NF-KB,coming to take part in the invasion and transference of tumors. Recently,reports on PI3K/Akt signal path increased more,but few was found on their changes in GIST.We detected the mRNA changes of PI3K,Akt and PTEN,further discussing the changes of factors at expression level after PI3K/Akt signal path was activated.We discussed the relationship between theis expressions and the clinical pathologic characteristics and prognosis of GIST,in order to explore the functions in the occurrence and development of GIST,providing a basis for tumor targeting therapy in further.Materials and Methods1,SubjectsThe 124 fresh samples of GIST patients(64 males and 60 females)were acquired from the Shengjing Hospital affiliated to China Medical University from 1990 to 2007. All the patients were diagnosed by the examination of pathology after surgery removal. The clinical pathological materials were intact and the follow-up results were definite in all the 124 patients.The range of age of these patients was 9 to 79 years old and the mean age was 54.6 years old.The tumors of 62 patients(50.0%)were located in stomach,28(22.6%)in small intestine,14(11.3%)in colorectus,9(7.3%)in esophagus,and 11(8.9%)outside the gastrointestinal tract(4 in omentum,4 in mensentery,3 in retroperitoneum).The paraffin samples of 57 GIST patients(29 males and 28 females)were acquired.The range of age of these patients was 16 to 62 years old and the mean age was 55.8 years old.The tumors of 29 patients(50.9%)were located in stomach,13(22.8%)in small intestine,7(12.3%)in colorectus,3(5.3%)in esophagus,and 5(8.8%)outside the gastrointestinal tract(2 in omentum,2 in mensentery,1 in retroperitoneum).2,Reagents of molecular biologyPTEN(10P01):sc-73420;p-Akt1/2/3(Thr 308)-R:sc-16646-R;p-PI3-kinase p85α(Tyr 508):sc-12929;HIF-1α(3C144):sc-71247;NOS2(C-11):sc-7271; VEGF(C-1):sc-7269;uPA(H-140):sc-14019;MMP-2(8B4):sc-13595;MMP-9 (M-17):sc-6841;TGF-β1(500-M66):sc-65378(Santa Cruz);Second antibody marked by HRP(Beijing Zhongshan Company);Cellulose Nitrate Membrane(Takara Company);RT-PCR kit(Takara Company);DAB Substrate(Takara Company);DNA molecular weight marker(Takara Company);PCR primer(Takara Company);other were analytical reagents of China.3,mRNA levels in GIST group and control group detected by RT-PCRThe extraction of mRNA was followed the instruction of QuickPro micro mRNA Purification kit.The procedures included the preparation of samples,isolation of mRNA,washing of oligo(dT)fibrous particles binding mRNA with high salt buffer and low salt buffer respectively and the elution of mRNA.TaKaRa RNA PCR(AMV) V3.0 kit was used in reverse transcription and PCR reaction.The imaging of the reactive products was implemented after agarose gel electrophoresis.4,Expression of related factors in GIST group and control group detected by Western blottingThe homogenate was prepared after 1 ml cell lysis solution(Cell signaling Technology,Beverly,MA)and 1 mmol PMSF added to 0.5g GIST.The protein was extracted followed the regular steps and the concentration was detected.Before electrophoresis,the SDS buffer was added into the protein extraction,heated to boiling 5 min at 100℃,loaded after centrifugation,separated with 12%SDS-PAGE, transferred the protein to the Cellulose Nitrate Membrane and then incubated with the primary antibody.IgG coupled alkaline phosphatase used as the second antibody and O-dianidine andβ-naphthyl acid phosphate were used as the substrate for alkaline phosphatase for coloration.5,Expression ofHIF-1α,NOS2 and VEGF in GIST group and control group detected by immunohistochemistry.The GIST tissues were fixed by formalin and then 4μm sections were prepared after embedded by paraffin.After dewaxed,one drop or 50μl blocking serum was added on the slide.Wiping off the serum,one drop(or 50μl)primary antibody was added on each slide,and then incubated.Then one drop(or 50μl)EnvisionTMsecond antibody was added on each slide,and then incubated.Finally DAB coloration was implemented for 1 to 2 minutes. Results1,Expression of PI3K,Akt and PTEN on the levels of mRNA and protein in the GIST group and control group,and the correlation analysis among the three indexesThe up-regulated expression of PI3K mRNA presented with the increasing of NIH grades(p＜0.05).The expression rate of PI3K was not associated with gender,age and histological type,while was significantly associated with the invasion of mucosa and lymph node metastasis(p＜0.05).These indicate that the expression of PI3K is associated with the transformation and invasive metastasis of GIST cell and the product of PI3K may regulate cell growth and apoptosis.We also found that the up-regulated expression of Akt mRNA presented with the increasing of NIH grades except that between high risk group and moderate risk group.The expression rate of Akt was not associated with sex,age and histological classification,while was significantly associated with mucosa invasion and lymph metastasis.These indicate that the over-expression of Akt contribute the lymph metastasis of gastric cancer.In our study, the down-regulated expression of PTEN mRNA presented with the increasing of NIH grades(p＜0.05),and the deficient rate of PTEN was higher with the severer malignancy of the tumor.The expression rate of PTEN was not associated with sex, age and histological classification,while was closely associated with mucosa invasion and lymph metastasis.These indicate that the deficiency of PTEN protein is associated with the development of GIST.The results of Western blotting showed that the changes of all the three indexes on the level of protein coincided with the changes on the level of mRNA.However,the expression of Akt protein increased significantly in the tissue of GIST caused by the changes of translation and(or)post-translation.In the 124 GIST samples,the expression of PI3K protein was positively correlated with the expression of Akt protein(r=0.292,P=0.031).The expression of PI3K protein was negatively correlated with the expression of PTEN protein(r=-0.412,P=0.036).The expression of PTEN protein was negatively correlated with the expression of Akt protein(r=-0.386, P=0.024).These suggested that the main action substrate in the downstream of PI3K was Akt.The positive regulation of PI3K can activate Akt and activated Akt can activate or inhibit the expression of target protein by phosphorylation.Furthermore the proliferation,differentiation,apoptosis and migration were regulated.The expression of PTEN negatively correlated with PI3K and Akt suggest that the antagonist action may present between PI3K/Akt and PTEN in the development of GIST.2,Expression of NF-κB,XIAP,bcl-2 and survivin on the levels of mRNA and protein in the GIST group and control group,and the correlation analysis among the four indexesThe changes of the expression of NF-κB mRNA were closely associated with NIH grades,lymph node metastasis of tumors and the invasion of mucosa,but not associated with age,gender and histological type.The up-regulated expression of XIAP mRNA presented with the increasing of NIH grades and the expression of XIAP was negatively correlated with differentiation of GIST which showed that the lower the differentiation and the higher the expression rate of XIAP.The expression of XIAP was higher in the GIST patients with lymph node metastasis,and it was closely associated with the invasion of mucosa,but not with age,gender,histological type,metastasis, recurrence and invasion of peripheral tissues.The expression of Survivin mRNA were closely associated with NIH grades,lymph node metastasis of tumors and the invasion ofmucosa,but not associated with age,gender and histological type in the GIST group. Due to the regulation of NF-κB,the positive correlation occurred in the mRNA expression of the four indexes.3,The expression and correlation analysis of HIF-1α,NOS2 and VEGF between GIST and control groupThe positive expression of HIF-1αin nucleus and cytoplasm is yellow or brown particles.The expression of HIF-1αis weaker in normal GIST tissue.In our 124 cases, (-)46 cases,(+)7 cases,(++)31 cases,(+++)28 cases,(++++)13 cases.The total positve expression rate is 63.7%(79/124).The expression of HIF-1αis closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of HIF-1αinâ… andâ…¡is 46.2%and it is 68.4%inâ…¢andâ…£.The positive rate of HIF-1αin mucosal invasion and non-mucosal invasion are separately 88.2%and 54.4%. The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 90.0%and 51.2%.The expression of HIF-1αhas nothing to do with other clinical pathological features. NOS2 is mainly expressed in cytoplasm and distributed like particles.In our 124 cases of GIST,NOS2 is high expression(64.5%),the positive expression rate is much higher than normal GIST tissue(17.7%)and the difference is significant.The expression of NOS2 is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of NOS2 inâ… andâ…¡is 42.3%and 70.4%inâ…¢andâ…£.The positive rate of NOS2 in mucosal invasion and non-mucosal invasion are separately 88.2%and 55.7%.The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 95.0%and 50.0%.The expression of NOS2 has nothing to do with other clinical pathological features.So we concluded that NOS2 maybe involved in the whole process of GIST mucosal cancer.NOS2 is one of the dangerous factors and at the same time NOS2 is a reliable indicator of prognosis.VEGF is mainly expressed in cytoplasm of GIST and it is diffuse distribution.The positive expression rate of VEGF is 58.1%(72/124).The posive signal is located in cytoplasm and membrane of GIST cell.The expression of VEGF is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.The positive rate of VEGF inâ… andâ…¡is 46.2%and 61.2%inâ…¢andâ…£.The positive rate of VEGF in mucosal invasion and non-mucosal invasion are separately 79.4%and 50.0%.The positive rate in metastasis cases is much higher than non-metastaisis cases.They are 80.0%and 47.6%.The expression of VEGF has nothing to do with other clinical pathological features.The cytoplasm of vascular endothelial cells is stained into brown particles in GIST and malignant tissue.In our 124 cases of gastric cancer,the value of MVD(23.15±8.92) is higher than adjacent tissue(8.3±2.76).The difference is significant.So we can concluded that the angiogenensis of GIST tissue is more active than adjacent tissue. Positive CD117 is located in cell membrane,CD34 is located in cytoplasm,they are all brown paricles.The MVD value of positive expression is much higher than negative expression in HIF-1α,NOS2 and VEGF.And MVD is closely related to NH classification,mucosal invasion and lymphatic metastasis,MVD has nothing to do with other clinical pathological features.The expression of HIF-1αand NOS2 are of significant positive correlation (r=0.392,P=0.026).The expression of HIF-1αand VEGF are of significant positive correlation(r=0.368,P=0.034).The expression of NOS2 and VEGF are of significant positive correlation(r=0.452,P=0.021).4,The changing in mRNA and protein level and correlation analysis ofuPA,MMP-2,MMP-9 and TGF-β1 in GIST and control group.In the 124 cases of GIST,the expression of uPA,MMP-2,MMP-9 and TGF-β1 have no relationship with age,sex and histological type of patients.The expression of uPA,MMP-2,MMP-9 and TGF-β1 is significantly related to lymphatic metastasis,NH classification and mucosal invasion.The protein level is consistent to mRNA.TGF-β1 can mediate PI3K/Akt signal transduction pathway and increase the expression of uPA, they are of positive correlation(r=0.356,P=0.028).The expression of uPA and MMP-2 are of significant positive correlation(r=0.323,P=0.035).The expression of MMP-2 and MMP-9 are of significant positive correlation(r=0.495,P=0.042).So we can conclude that the role of them are of significant correlation in GIST occurrence and development.DiscussionDisorder of PI3K/AKt signal transduction pathway expressed in a wide range of human tumors.the mutation of this pathway will cause the disfunction or acquire some functions witch can cause cell transformation.PI3K/AKt signal transduction pathways regulate tumor cells' proliferation and survive.AKt which is the role of PI3K downstream target protein,and is the core of PI3K/AKT signal transduction pathway it is an important protein kinas of signal transduction pathway.PTEN is the first ever found a phosphates activity of tumor suppressor genes can be activated by controlling activity phosphatidyl 3,4,5-instill trisphosphate(PIP3)to control the activity of AKt. Thus,PTEN mutation can cause AKt loss the ability to control cell proliferation,then leads carcinogenesis.PTEN can be phosphorylated by PIP3,antagonistic activity of PI3K and reduce the concentration of intracellular PIP3,thereby inhibiting the activation AKt.In GIST,the NIH classification with its PI3K,AKt expression,and expression of tumor suppressor gene PTEN decrease.The positive expression rate of PI3K,AKt and PTEN have no relation with the sex,age,histology type,but have closely relation with tumor invasion,metastasis and mucosal involvement.PI3K/AKt and PTEN may have mutual antagonism effect during the development of GIST.We study the PI3K-AKt signal transduction pathway downstream apoptosis related factors expression in GIST and the relationship between the expression with GIST clinical pathological characteristics and the prognosis through the observation of NF-κB and apoptosis related factors such as XIAP,blc-2 and surviving mRNA changes in GIST paraffin block.In GIST the malignant level increase accompanied by the increase of PI3K and AKt activity,activated AKt can phosphorylated IKKa directly then activating IKK,through witch regulating NF-κB activity.AKt is the important regulation factor of NF-κB dependent transcription gene,it can stimulate tumor cell generation.PI3K-AKt signal transduction pathway activation can stimulate NF-κB mRNA,witch can go through into cell nucleus to regulate XIAP,bcl-2 and surviving mRNA expression in GIST.The expression of NF-κB mRNA and NH classification have closely relation with tumor invasion,metastasis and mucosal involvement,but have no relation with age,sex and histology type.XIAP mRNA expression up-regulate according to NH classification;XIAP expression has negative relation with GIST histological differentiation,that is XIAP expression level increase accompanied by the decrease of differentiation.The metastasis GIST patient express high level XIAP protein,and XIAP expression has relation with mucosal involvement,but has no relation with age,sex and histology type.Bcl-2 mRNA expression up-regulation has relation with NH classification and mucosal involvement,but has no relation with age, sex,histology type and invasion of around tissue.Survivin mRNA expression has relation with NH classification,tumor invasion and metastasis,mucosal involvement. But its expression has no relationship with age,sex and histological typeBecause of the role of regulation and control of NF-κB,the mRNA level between the target was a positive correlation Change.Tumor angiogenesis is a necessary condition for the transfer happen,PI3K/Ak signal transductional pathway is involved in the process of tumor angiogenesis that mediated by a variety of factors.As a tumor micro-environment of a critical regulatory factor,HIF-1 play a central role in promoting tumor angiogenesis and invasiveness of the tumor.PI3K/Akt signaling pathway can regulate HIF-1αprotein expression by regulation of PTEN tumor suppressor genet,and activation of AKt can enhance the stability of HIF-1α,thereby promote the transcription of hypoxia response genes,causing cells to hypoxia a series of adaptive responses.VEGF probably through the regulation of tumor angiogenesis,development and transfer plays an important role. In many solid tumors,the expression of VEGF and tumor angiogenesis was highly related to each other.VEGF are closely related with growth,metastasis and prognosis of tumor,therefore,take VEGF as target,inhibiting its expression,thereby inhibiting angiogenesis of tumor,blocking the transfer of nutrient sources and channels of tumor, to achieve the purpose of the treatment of tumor,has become the new strategy of treatment of cancer in recent years.VEGF may activate PI3K/AKt channels and induce NO synthesis,HIF-1α,NOS2,and VEGF can participate in the occurrence and development of GIST by the signal transduction pathway of PI3K/Akt.Therefore,we believe that expression of HIF-1α,NOS2,and VEGF may be the important target of malignant tumor's growth,invasion and metastasis.They can be used as targets,inhibit their expression,thereby inhibit angiogenesis of tumor,blocki the transfer of nutrient sources and channels of tumor,to achieve the purpose of the treatment of tumor.The activation of PI3K/AKt signal transduction pathway can activate the expression of HIF-1α,HIF-1α,NOS2,VEGF,MVD and GIST of the NIH classification,submucosal invasion of tumor has closely relationship with invasion and metastasis of tumor,HIF-1α,NOS2 and VEGF expression may be the important target malignant tumor growth,invasion and metastasis.Tumor's invasion and metastasis is a complex multi-step tumor cells and host stromal cells and the process of interaction,the degree of invasion and metastasis is a major factor affecting the prognosis,TGF-β1 exists in the tumor and some normal cells,can regulation of normal cells and transformed cell growth and differentiation.TGF-β1 mRNA expression in lesions with high levels of infiltration depth has a strong and extensive orientation and ability to transfer,TGF-β1 itself as a sport can also be factors,tumor cells to promote exercise and transfer. TGF-β1 can mediate PI3K/Akt signal transduction pathway to increases uPA,uPA is regulated by the PI3K/Akt signal transduction pathway-mediated of TGF-β1,further activation of MMP-2,uPA and MMP-2 expression was positively between related.At the same time,MMP-2 also has the activitical function of MMP-9.Prompting that both of them are closely related at the role of the development process of GIST,possible through the synergies promoting tumor incidence,tumor extracellular matrix degradation and tumor cell invasion and metastasis.This study shows that,the expression ofTGF-β1,uPA,MMP-2 and MMP-9 have no relationship with sex,age, and with the submucosal invasion of tumor,NIH grade,tumor invasion and metastasis-related,suggesting that TGF-β1,uPA,MMP-2 and MMP-9 involved in the tumor's invasion and metastasis.Conclusion1,In gastrointestinal stromal tumors,the tumor suppressor gene-PTEN is reduced by PI3K and Akt increasing.The positive expression rate of PTEN,PI3K and Akt have no relationship with sex,age and histological type.On the other hand it has relationship with lymphatic metastasis and mucosal invasion.PI3K/Akt and PTEN maybe have the role of mutual antagonism in GIST occurrence and development.The expression of PI3K and Akt are of significantly positive correlation(r=0.292,P=0.031).The expression of PI3K and PTEN are of significantly negetive correlation(r=-0.412, P=0.036).The expression of PTEN and Akt are of significantly negetive correlation (r=-0.386,P=0.024).2,In gastrointestinal stromal tumors,the activation of PI3K/Akt signal transduction pathway can further active NF-κB mRNA,and then it can enter nucleus and control the expression of apoptosis-related factor-XIAP,bcl-2 and survivin mRNA.The expression ofNF-κB,XIAP,bcl-2 and survivin mRNA is closely related to NH classification,mucosal invasion and lymphatic metastasis.They have no relationship with age,sex and histological type.Because of NF-κB regulation,the expression of them are of positive correlation.3,In rich blood supplying GIST,the activation of PI3K/Akt signal transduction pathway can further active HIF-1α,the expression of HIF-1α,NOS2,VEGF and MVD is closely related to NH classification,mucosal invasion and lymphatic metastasis of GIST.They have no relationship with age,sex and histological type.Our study shows that the expression of HIF-1α,NOS2 and VEGF are of significantly positive correlation (r1=0.368,r2=0.452,r3=0.392,P1,P2,P3＜0.05).So we can conclude that they have interdependent positve regulation action in GIST invasion,metastasis and angiogenesis. They are all involved in GIST occurrence and development.So HIF-1α,NOS2 and VEGF are important targets in malignant tumor growth,invasion and metastasis.4,We found that TGF-β1 can mediate PI3K/Akt signal transduction pathway and increase the expression of uPA,then it can active MMP-2 and MMP-9,and effectively degrade extracellular matrix.The expression of TGF-β1 is increasing with deepening the depth of invasion,enhancing the extent of lymphatic metastasis and progressing NH classification.Our study shows that the expression of TGF-β1,uPA,MMP-2 and MMP-9 have no relationship with sex,age and histological type,they are closely related to NH classification,mucosal invasion and lymphatic metastasis.So TGF-β1, uPA,MMP-2 and MMP-9 are involved in tumor invasion and metastasis.
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