The Experimental Study On The Expression Of RKIP Protein In GIST And Its Mechanism Of Tumor Proliferation And Metastasis Through ERK/MAPK Pathway

Research Background GISTs(gastrointestinal stromal tumor)is the most common gastrointestinal mesenchymal tumor,had "leiomyoma,leiomyosarcoma and schwannoma" as a diagnostic classification.In 1983,Mazur et al.Used the results of immunohistochemistry and electron microscopy to first present the concept of gastrointestinal stromal tumors.Further study found that: about more than half of GISTs occurred with the C-kit gene and PDGFRA gene mutation,which provides an important direction for the gene-targeted treatment of GISTs.Small molecule tyrosine kinase inhibitors,represented by imatinib,can achieve quite a definite therapeutic effect,but some patients still show drug resistance.GISTs such gastrointestinal tumors often exhibit biological behavior that is inconsistent with their apparent morphology,which tends to be more characteristic of malignant tumors in the trend of invasion and proliferation: such as easy invasion and destruction of the gastrointestinal tract,blood vessels and other tissues,prone to occur Distant metastasis,rapid growth rate,resistance to Anti-tumor drugs and so on.This also makes us must pay attention to the mechanisms of proliferation,invasion and apoptosis in order to obtain more accurate diagnosis of GISTs,to make more timely and effective treatment.MAPK pathway(Mitogen-activaed protein kinase)is one of the most profound studied signal transduction pathways.It widely exists in various human cells and is associated with many tumorigenesis and tumor-related development.The pathway consists of multiple parallel triplex signals,and now six MAPK family subsets have been identified in mammals: extracellular signal-regulated kinase(ERK1 / 2),C-Jun N-terminal kinase 1/2/3(JNK1 / 2/3),p38 MAPK,ERK7 / 8,ERK3 / 4 and ERK5.The firstly discovered and most deeply studied MAPK pathway is the Raf-MEK-ERK(ERK MAPK)pathway.Among them,ERK1 / 2(extracellular signal-regulated protein kinase 1/2)is MAPK,which is the core molecule in the signaling pathway,which forms an efficient and accurate signal to the upstream and downstream effector molecules;and MEK1 / 2 is MAPKK(MAPK kinase),which activates two sites of Thr and Tyr on ERK1 / 2 protein to phosphorylate ERK1 / 2 in activated state;Raf-1 protein is upstream Of MAPKKK(MAPKK Kinase),can be obtained by phosphorylation of MEK1 / 2,and then make the entire pathway activated.The pathway is regulated by the cell cycle and extracellular stimuli,which regulate the downstream of many protein kinases,phospholipases and transcription factors,play an important biological function.RKIP protein is a natural inhibitor of Raf-1 protein,which exists widely in vivo.RKIP protein can regulate many cell-mediated pathways including Raf-MEK-ERK pathway(ERK MAPK),beta adrenergic pathway and NF?B pathways.RKIP protein is both a kinase inhibitor and a phosphorylation target.It has important and complex functions of regulating tumor growth,metastasis,affecting cell cycle and apoptosis.Recent articles on RKIP proteins have been reported in large amount of articles,such as gastric cancer,bowel cancer,esophageal cancer,gynecologic oncology,and the like.However,there are only a few references on the expression of RKIP protein in gastrointestinal stromal tumors.What kind of function does the RKIP play in the GISTs neoplastic generation and metastasis? What is the relationship between the RKIP expression and GIST clinical data? How is the prognosis of GIST affected? How Does RKIP Affect GIST Tumor Cell Biology? The above problems are all problems that we urgently need to solve.In this experiment: 1 The expression of RKIP in gastrointestinal stromal tumors was studied by immunohistochemistry to explore the clinical significance of GIST expression and its effect on prognosis;2 By in vitro experiments,The biological characteristics of GIST-T1 cells in each group after the stable transfection with CRISPR plasmid were observed and the biological characteristics of the cells were analyzed.The biological effects of RKIP protein on tumor cells were analyzed.3 Immunohistochemistry and Western blotting was used to detect the expression of a key protein in the ERK / MAPK pathway and important functional protein molecules in the downstream of the pathway.The mechanism of RKIP on the proliferation and metastasis of tumor cells was analyzed.Through the above studies,provide the basis for the diagnosis of GIST,prognosis assessment and new targets for cancer treatment.Objective:1.In this experiment,we will study the expression of RKIP in gastrointestinal stromal tumors by immunohistochemistry to explore the clinical significance of GIST expression and its prognosis.The research will provide evidence and support for the diagnosis of GIST,Prognostic evaluation and new tumor treatment targets 2.To observe the biological characteristics of proliferation,metastasis,apoptosis and cell cycle of GIST-T1 cells in each group after stable transfection with CRISPR plasmid.And observe the biological effects of RKIP protein on GIST-T1 tumor cells in vitro.3.RKIP is an inhibitor of Raf-1 protein,which itself is directly involved in the regulation of MAPK pathway downstream sites(such as ERK \ MEK)function.This part of the experiment will focus on the RKIP protein in the ERK \ MAPK pathway on GISTs proliferation and metastasis and its mechanism of action,and analysis of its downstream target protein function.Methods: 1.The study included 63 cases of paraffin-embedded specimens of surgically resected and pathologically confirmed clinical specimens from Shengjing Hospital Affiliated to China Medical University.All the cases are clinically and pathologically complete dated from January 2011 to January 2015.The expression of RKIP protein was analyzed by immunohistochemistry.The Kaplan-Meiier method was used to calculate the overall survival of 60 patients followed up for survival analysis.The prognostic significance of each index was analyzed by COX multiple regression to further clarify the RKIP The value of protein in the diagnosis and prognosis of GISTs.2.The GIST-T1 cell line was transfected with CRISPR plasmid and screened by puromycin.The cells were divided into RKIP OE group(RKIP(+)group),Control / Vector group and control group(Mock group).The expression of RKIP m RNA was detected by real-time PCR.The expression of RKIP protein in each group was detected by WB method.The transfection efficiency was further verified and confirmed.To further observe the biological characteristics of GIST-T1 cells,such as proliferation,metastasis,cell cycle and other biological characteristics after stable transfection in each group,the following jobs were done: 1.The proliferation of GIST-T1 cells was detected by CCK-8 assay;2.platelet colony formation assay to verify cell proliferation and malignancy;3 Transwell chamber experiment to detect the invasion and migration of GIST-T1 cells in each group; 4 flow cytometry cell cycle assay to analysis cell proliferation in GIST-T1 cells;Flow cytometry was used to detect the apoptosis of GIST-T1 cells in each group.3.1.Immunohistochemistry was used to analyze the expression of P-ERK and P-MEK,an important protein downstream of the MAPK pathway,in 63 cases of the first part of the cases,and the correlation with the expression of Cyclin D,And cell migration related protein MMP-2 expression and analysis of its relationship with clinical data,RKIP coexpression and prognosis;4.Western Blotting analysis of the protein samples.The expression of RKIP,ERK \ P-ERK,MEK \ P-MEK,Cyclin-D,MMP-2 and MMP-9 in GIST-T1 cells of each group(RKIP OE group,CONTROL \ Vector group and MOCK group),Try to discuss its regulatory mechanism.RESULTS: 1.In GIST tissues,RKIP protein was positively expressed in the cytoplasm and cell membrane with brownish-yellow or brown granules.RKIP protein positive expression was found in 34(54%)of the 63 specimens in this experiment,and the total negative expression was 29 Example(46%).The RKIP positive expression is related with GIST tumor size,NIH grade and mucosal invasion.The RKIP and age,gender,tumor location,how many mitotic figures were not related.Kaplan-Meier method was used to draw the survival curves related to RKIP differential expression.The results showed that the 1,3,5-year survival rates of RKIP positive group were 94.4%,89.2% and 80.5% respectively.The survival rates of RKIP negative group at 1,3 and 5 years were only 88.6%,68.2% and 48.2%,respectively.Comparing with the RKIP negative Group,RKIP high expression group is correlated with a better survival rate,the difference was significant.(Log-Rank analysis,P = 0.0015).The results of COX multivariate analysis showed that the prognosis of patients with GISTs was related to NIH grade(P = 0.037)and Exp(B)was 3.664,indicating that NIH risk grade was an important factor to evaluate the prognosis of patients.However,the expression of RKIP correlated with the prognosis of patients(P = 0.122).The Exp(B)value was 2.855,suggesting that RKIP expression may have some reference value for the survival of GIST.2.Compared RKIP m RNA expression levels in three groups of cells: Compared with m RNA level of MOCK group(GIST-T1 in negative control group),m RNA expression in Control / Vector group(empty plasmid transfected GIST-T1)1.712 ± 0.261 fold,but the expression of RKIP(+)was 3.344 ± 0.545 times(P <0.05),and the expression of RKIP(+)/ CONTROL was significantly higher than that of the first two groups / MOCK group(1.651 ± 0.063)compared with CONTROL / Vector group(1.261 ± 0.095,P <0.05)and MOCK group(normalized to 1.000,P < 0.05),which was consistent with the result of qRT-PCR,which indicated that the transfection efficiency of RKIP plasmid was higher and a stable transfected GIST-T1 cell line was established.3.The growth curve of CCK-8 assay showed that the experiment The growth of GIST-T1 cells in the control group(MOCK group)and control group(CONTROL \ Vector)was significantly slower than that in the control group(RKIP up-transfection group),and the growth rate was significantly different from the third day(P <0.05)4.Colony formation assay was performed to detect the formation of single cells in different groups of GIST-T1 cells Further assessment of tumor cell proliferation and malignancy.The results showed that the number of colonies in RKIP transfection group(46.67 ± 8.02)was significantly lower than that in MOCK blank control group(79.33 ± 3.51)(P = 0.031),but no significant difference compared with Vector / Control group(47.67 ± 4.04)P = 0.802).The above results showed that the upregulation of RKIP protein could inhibit the colony-forming ability of GIST-T1 cells to a certain extent;5.Transwell chamber results showed that the number of overlying cells in RKIP(+)group was(170.00 ± 24.98)(P = 0.047),but significantly decreased compared with the MOCK group(204.00 ± 26.00)(P = 0.014),indicating that RKIP protein expression affects cell invasion and migration ability,but the transfection process May play a role in cell viability,the difference tends to decrease;6.flow cytometry can be used to analyze cell cycle and apoptosis,the cycle distribution of each group of cells transfected and the proportion of apoptosis statistics are as follows.The results showed that the ratio of G0 / G1 phase(70.60%)in GIST-T1 cells(RKIP + group)transfected with RKIP protein was significantly higher than that in the other two groups(MOCK: 65.77% and CONTROL / Vector: 61.97%(P = 0.017 and 0.001).However,there was no significant difference between G2 / M phase and S phase(P> 0.05).This shows that RKIP protein can inhibit tumor cells in the G0 / G1 phase and thus inhibit tumor cell growth and proliferation,but not directly affect cell division and DNA replication.In terms of apoptotic ratio,the proportion of D4 quadrant in RKIP up-regulated group(6.40% ± 0.23%)was significantly higher than that in control group(MOCK group: 2.33% ± 0.37%,CONTROL / Vector group: 1.03% ± 0.15% The high expression of RKIP protein can promote the apoptosis of GIST-T1 tumor cells.7.The positive expression of P-ERK was related to the number of nuclear fission,tumor size and NIH grade in GISTs.The 1-year,3-year and 5-year survival rates of P-ERK positive cases were 94.12%,73.53%,63.13% The 1-year,3-year and 5-year survival rates of ERK negative patients were 96.15%,88.46% and 83.44% respectively.The 1-year,3-year and 5-year survival rates of P-ERK positive patients were significantly lower than those of negative patients.The positive expression of P-MEK was related to the tumor size of GISTs(P = 0.048,<2.0cm,> 10.0cm between groups),but had no correlation with prognosis(P = 0.32)(P = 0.001).The positive expression of Cyclin-D1 was related to mitotic numbers,tumor size and NIH grade in GISTs.The 1-year,3-year and 5-year survival rates of Cyclin-D1 positive group were significantly lower than those of negative group;8.Protein co-expression analysis: RKIP protein was positively correlated with P-MEK(r =-0.359,p = 0.004),P-ERK)Expression was negatively correlated.RKIP protein and MMP-2 protein expression,there is a negative trend,but not statistically significant(r =-0.14,p = 0.275).There was a positive correlation between the expression of P-ERK protein and the downstream target protein Cyclin-D1(r = 0.360,p = 0.004),but not with MMP-2 expression(r = 0.218,p = 0.086)9.COX multi-factor prognosis analysis of RKIP,P-ERK,P-MEK,Cyclin-D1,MMP-2 and OS: By age,gender,NIH stage,the expression of each protein,mitosis and tumor size,Multiple factor analysis COX analysis showed that NIH could be an independent factor to predict the prognosis of GISTs(P = 0.027,Exp(B)= 4.182).Although RKIP,P-MEK,PERK and Cycin-D1 have a higher influence weight on OS(Exp(B)= 1.370,2.112,1.450,2.018),they can not be used as independent factors that influence prognosis.Statistical significance(P = 0.751 \ 0.257 \ 0.671 \ 0.470);10.Western Blotting analysis: After RKIP protein was up-regulated,Raf-1 protein expression was inhibited,p-MEK1 / 2 protein expression was decreased,p-ERK1 / 2 was decreased,while downstream target proteins such as C-Myc and Cyclin-D1 expression Inhibition,MMP-2 protein expression decreased.Conclusion: 1.The expression of RKIP protein in GISTs correlated with tumor size,NIH stage and invasiveness of the mucosa(invasion degree),and each index suggested that the higher the degree of malignancy was,the lower or more loss of RKIP expression was.Compared with other factors(age,sex,Tumor location,etc.)are not relevant.2.RKIP overexpression is positively correlated with the survival of patients with GISTs,which has some implications for the prognosis of patients.3.RKIP expression has certain reference value for the survival of GIST,but it can not be used as an independent factor to evaluate the prognosis of GISTs.4.RKIP protein has an inhibitory effect on the proliferation of GIST-T1 cell line 5.RKIP overexpression can inhibit the migration and metastasis of GIST-T1 cells 6.RKIP protein inhibits(high expression of RKIP protein)cell proliferation and promotes apoptosis.7.In GIST tumor cells: P-ERK protein may be related to tumor growth and proliferation(high expression promote tumor growth),and affect the degree of malignancy and prognosis;P-MEK1 / 2 protein affects gastrointestinal stromal tumor In the GISTs,MMP-2 protein affected the metastasis and invasion of GISTs,and the expression of Cyclin-D1 protein affected tumor growth,differentiation and prognosis.8.In GISTs,RKIP protein expression was negatively correlated with the expression of p-ERK1 / 2,p-MEK1 / 2 and Cyclin-D1(P <0.05).To a certain extent,it is understood that as the RKIP protein is overexpressed,the downregulation of p-ERK1 / 2,p-MEK1 / 2 and Cyclin-D1 protein will be reduced,thus affecting the relevant biological activity of tumor cells.9.Upregulation of RKIP protein in the ERK / MAPK pathway inhibited the expression of Raf-1 protein and down-regulated the expression of p-MEK1 / 2 protein,which in turn decreased the activation of ERK protein(p-ERK1 / 2 decreased)Such as C-Myc and Cyclin-D1 expression is inhibited,so that tumor cells appear inhibition of G1 phase,slow down the proliferation,apoptosis can also increase.The MMP-2 protein also has a decreased expression under the regulation of P-MEK-AP1(c-fos / c-Jun)-MMPs pathway,and then negatively controls the ability of tumor metastasis and invasion.However,in GISTs,the low expression or complete inactivation of RKIP protein appears,which is the possible mechanism of the proliferation,migration and progression of GISTs.