Studies On Screening Of Differentially Expressed Proteins In Gastric Carcinoma With Various Degrees Of Differentiation And On Expression And Mechanism Of Related Protein Serpin B1

Gastric carcinoma is the most common malignant tumor, and it is thesecond-leading cancer killer in the world, and gastric carcinoma is the leadingcause of death in malignant tumors in China. Even if patients receive standardradical resection and standardized postoperative treatment,5-year survival rateof advanced gastric carcinoma is still at around30%to40%. There are manyfactors affecting the prognosis of gastric carcinoma, in which the degrees ofdifferentiation of tumor cells play an important role. Different degrees ofdifferentiation of gastric carcinoma cells have different abilities ofproliferation and differentiation as well as invasion and metastasis. Thedegrees of differentiation of the tumor cells can also be changed with theprogress of the tumor; in the process if certain genes or signaling pathwayscan be intervened to achieve purposes of inhibition or even reversal, it willplay an active role in the prognosis of patients, which is the original intentionof this experimental design.Proteomics along with its related technologies, an emerging branch ofmolecular biology in recent years, focuses on studies of the proteome encodedby genes, and makes complete analysis of the composition, expression levelsand modification state of the intracellular proteins with dynamic changes, andunderstands the interactions and associations among proteins, thus revealingprotein function and its relationship with laws of the cellular activities as wellas regulation mechanisms of gene expression as a new research field. Its coretechnology is protein separation techniques based on two-dimensionalpolyacrylamide gel electrophoresis (2-DE) and protein identificationtechnology based on mass spectrometry (MS) and bioinformatics, which haveadvantages of high throughput and sensitivity, high resolution, repeatability and high automation, and are ideal for large-scale parallel separation andanalysis of protein; with the corresponding analysis software and database,large amounts of data can be processed in a short period of time.Precisely because of this, the application of proteomics in cancer researchhas become a research focus, and proteomics researches on cells, tissue, serumand gastric juice of patients with gastric carcinoma have been reported in largenumbers, covering many aspects like the diagnosis, treatment and functionalmechanism. The study found that certain genes of gastric carcinoma and theirexpression products changed with the development of the disease andinfluenced a series of processes such as tumor invasion and metastasis throughspecific channels. On the study of tissues, mostly gastric carcinoma tissueswere compared with normal tissues. For example, Jing and others discoverednuclear matrix proteins hnRNP-A2/B1were highly expressed in gastriccarcinoma tissues, which might influence the interactions between certaincancer-promoting genes and tumor suppressor genes, so as to affect thedevelopment of gastric carcinoma, and it is one of the potential diagnosticmarkers for gastric carcinoma; Cai and others found that the gastric cardiacancer was prone to metastasis, which might be associated with abnormalitiesof certain enzymes in the glucose metabolism, leading to significantlyincreased amount of pyruvic acid into carboxylic acid cycle and promoting thegrowth and metastasis of tumor cells; inhibition of this pathway might providepotential targets for the treatment of gastric carcinoma. In addition, there havebeen few reports of differentially expressed proteins in different differentiatedtissues. So on the basis of the preliminary work of this experiment, furtherisolation and identification of differentially expressed proteins were made invarious degrees of differentiation of gastric carcinoma, which had advancescharacter and clinical value.In the first and the second part of the experiment, the advancedtwo-dimensional fluorescence difference gel electrophoresis (2D-DIGE) wasemployed together with LTQ ion trap mass spectrometer to screen and identifydifferentially expressed proteins in gastric adenocarcinoma tissue with various degrees of differentiation; six kinds of proteins that might be associated withtumor differentiation were identified after removal of structural proteins andnon-specific functional proteins, namely, serine protease inhibitor, clade B,member1(Serpin B1), calphobindin annexin A3, transcription factorNm23-H1, Adenine Phosphoribosyltransferase (APRT), glutathioneS-transferase enzymeπ-1(GST-π-1), antimicrobial peptides Dermcidin-lL,which laid the foundation for future research. Expressions of Serpin B1,annexin A3, Nm23-H1and APRT in different differentiated gastric carcinomatissues were validated in the third part. Considering that there were fewresearches on Serpin B1in tumor study and that it might be related with tumorinvasion and metastasis ability, so in the fourth to the fifth part in-depth studywas carried out on Serpin B1, which further validated the Serpin B1expression in gastric carcinoma cells and tissues; the relationship between thebiological behavior of gastric carcinoma and Serpin B1was explored and apreliminary study was conducted on the influence mechanism of Serpin B1oninvasion ability of human gastric carcinoma cell. The results showed that thepositive expression rate of Serpin B1in gastric adenocarcinoma wassignificantly higher than that in normal gastric tissue and was associated withsome clinical and biological behavior, indicating that the Serpin B1might beinvolved in tumor progression through some of the pathways and had potentiallinks with the degree of malignancy; serpin B1-siRNA transfected into gastriccarcinoma cells BGC823significantly inhibited Serpin B1protein expressionand might reduce gastric carcinoma cell invasion and migration; this processmight be achieved by regulating the expression of certain factors.Although the present study demonstrated that Serpin B1expressionimpacted invasion and metastasis ability of the gastric carcinoma cells, but thedevelopment and progression of malignant tumors were the results ofinteractions between a variety of genes and proteins and it was difficult tofully explain the complex tumor biological behavior with a single gene. In thefuture research, we will keep on testing the overall protein expression ofdifferent samples with proteomics technology, and the study will be not only on tissues and cells but also on blood and body fluids of patients with differentdifferentiated gastric carcinoma and metastasis potential; the proteinexpression differences and their relationship will be compared to clarifypossible mechanisms of tumor occurrence and development, and its functionswill undergo in-depth study; this comprehensive, dynamic and quantitativeanalysis will have a potentially significant value in elucidation of the tumorpathogenesis, determination of the treatment effect and assessment ofprognosis.The specific contents of the five parts in this subject are as follows:Part Ⅰ: Differentially expressed proteins in different differentiated gastriccarcinoma separated by2D-DIGEObjective: To separate proteins in different differentiated gastriccarcinoma, find the differences, and analyze the function of these proteins andtheir relationship, and to attempt to achieve the purpose of:(1) searching forpotential diagnostic indicators;(2) providing a theoretical basis for tumormechanism study;(3) possibly reversing tumor malignant behavior byinterfering with the expression of these proteins;(4) taking it as the moleculartarget of the drug, blocking the relevant signaling pathways, controlling genemutation, preventing tumor angiogenesis and inhibiting tumor cell growth andproliferation so as to treatment tumors. In addition, through this experimentthe practical value of2D-DIGE technology in the gastric carcinoma study willbe verified.Methods: Different differentiated gastric carcinoma tissues served as thesubjects;2D-DIGE method was applied to the purified protein for proteinseparation; after electrophoresis and then fluorescence scanning, Deep Purplewas employed for staining; Tphoon TRIO was used to scan, and DeCyderDifferential Analysis Software was used to analyze the scanned digital imagesto find significant differences; the pick list was generated and channeled intothe Ettan spot picker to pick spots.Results: According to different staining, there were some signiciantdifferences in the different differentiated gastric carcinoma tissues; some of the specific proteins had significantly higher expression in poorlydifferentiated adenocarcinoma, while others in well-differentiatedadenocarcinoma had significantly higher expression. There were some spotspresented with the coincidence of red and green fluorescence, which wereregarded to be expressed in different differentiated gastric carcinoma; theseproteins did not belong to the differentially expressed proteins in differentdifferentiated gastric carcinoma tissues, but there might be specific spotspresent in gastric carcinoma,which would be still of potential value for futureexperiments. Thirty-nine protein spots with the significant differences werecut and obtained with2D-DIGE.Conclusions:(1) As a separation technology,2D-DIGE is applied todifferent differentiated gastric carcinoma tissues for differential proteinseparation and the experiment has the advantages of high sensitivity, accuracy,good repeatability and clinical practicability, but experimental process iscomplex and highly specialized and with high cost, so more time is stillneeded to get it widely used in the clinical studies.(2) In the results of thisexperiment a number of spots with significant differences are separated, whichshows there are differences in protein expression in different differentiatedgastric carcinoma tissues, which is worthy of further study.Part Ⅱ: The application of liquid chromatography-mass spectrometry totechnical appraisal of differentially expressed proteins isolated with2D-DIGEObjective: To identify differentially expressed proteins isolated with2D-DIGE by employing LTQ ion trap mass spectrometer with highthroughput and high speed together with the corresponding analysis softwareand database; to screen preliminarily differentially expressed protein identified;to remove structural proteins, non-functional protein, proteins with extremelylarge or extremely small molecular weight as well as very acid or very alkalineproteins, to have preliminary knowledge of functions of proteins that meet therequirements and to select a meaningful spots of difference as the objects forfurther research. Methods: The in-gel enzymolysis method was applied to protein spotsisolated in the first part of the experiment to get the samples to be tested.Top-down technology and LTQ XL ion trap mass spectrometer were appliedto differentially expressed proteins and nanoESI-LC-MS/MS analysis methodwas used to carry on proteomics research. LC-MS/MS data were in Datadependent mode and the operating software was The Xcalibur2.0Software.The SEQUEST computing method in BioWorks3.3.1software was appliedfor database retrieval and the human protein sequence database was used inthe protein database with FASTA form; all NCBI protein data were used togenerate the library so as to determine the false positive rate.Results:(1) Thirty-nine protein spots with the significant differenceswere cut and obtained with2D-DIGE and a total of48kinds of proteins wereidentified; by functional analysis six kinds that might be related to tumordifferentiation were identified after screening, namely, Serpin B1,calphobindin III annexin A3, transcription factor Nm23-H1, GST-π-1, APRTand antimicrobial peptides Dermcidin-lL.(2) Gel figures were compared, onlyto find Serpin B1and annexin A3had high expression in poorly differentiatedgastric adenocarcinoma, while expressions of the APRT, Nm23-H1, GST-π-1and Dermcidin-lL were higher in differentiated gastric adenocarcinoma.Conclusions:(1)2D-DIGE, LTQ XL ion trap mass spectrometer andnanoESI-LC-MS/MS analysis methods are used to carry on proteomicsresearch; the analysis method is ideal for large-scale parallel separation andanalysis of clinical protein samples; the mass spectrometer has the advantageof high automation, high throughput, high-speed scanning and large-scale datacan be processed in a short time.(2) Expressions of Serpin B1and annexin A3are enhanced in poorly differentiated gastric adenocarcinoma while APRT,Nm23-H1, GST-π-1and Dermcidin-lL have enhanced expression indifferentiated gastric adenocarcinoma, suggesting that the expression of theseproteins are closely related with the degree of differentiation, and may directlyor indirectly affect gastric carcinoma invasion and metastasis and othercharacteristics, which is worthy of further study.(3) Preliminary analysis shows that these proteins are of important value in signal transduction ofgastric carcinoma cell, maintenance of cell morphology and normal defensefunction, promotion of cell division and proliferation and apoptosis as well asinvasion and metastasis.(4) In48kinds of proteins identified, some arestructural proteins or functionally non-specific proteins while thecorresponding proteins in some different spots are not identified; six differentpoints are left after functional analysis, which may be related withmodification of protein phosphorylation and methylation in the experiment; orafter enzymolysis these proteins do not form the ideal fragment suitable formass spectrometry identification, which might also be related with theSEQUEST computing method.Part Ⅲ: Selected differentially expressed protein in differentdifferentiated gastric carcinoma tissues by Western blot and QPCRvalidationObjective: To validate the expressions of Serpin B1, calphobindin IIIannexin A3, the transcription factor Nm23-H1, APRT identified in gastriccarcinoma and to observe whether expressions of these factors in the tissuesare consistent with the results of proteomics identification.Methods:(1) QPCR method was applied to verify factors detected on themRNA levels; GAPDH served as an internal reference gene to calculaterelative quantitative value(RQ value) of the target gene expression; aftercomparison, there was statistically significant difference (P<0.01or0.05).(2)Total protein was extracted and Western blot method was used to verifydetected differentially expressed proteins; GAPDH served as an internalreference and the integrated optical density values (IOD values) of proteinbands was measured with the BioRad image analysis system; the ratio of thetarget protein to reference protein in IOD values indicated expression levels ofthe target protein.Results:(1) Verification results of QPCR: verification results of mRNAexpression level of four differentially expressed proteins in differentdifferentiated gastric carcinoma tissues showed that Serpin B1and calphobindin III annexin A3had enhanced expression in poorly differentiatedgastric adenocarcinoma tissue while the transcription factor Nm23-H1andAPRT had enhanced expression in differentiated gastric adenocarcinomaincreased (P<0.01), which were consistent with results in proteomics.(2)Verification results from Western blot analysis: Expression levels of fourdifferentially different proteins were different in different differentiated gastriccarcinoma tissues. Serpin B1and calphobindin III annexin A3had enhancedexpression in poorly differentiated gastric adenocarcinoma tissues while thetranscription factor Nm23-H1and APRT had enhanced expression indifferentiated gastric adenocarcinoma, which were consistent with the resultsin proteomics.Conclusions:(1) QPCR and Western blot analysis are used to verifymultiple differentiation-related proteins isolated in preliminary experimentswith proteomic technologies, and it is found that on both mRNA and proteinlevel the results are consistent with those in proteomics, indicating the highercredibility of this study and that the protein spots are directly related todifferentiation of gastric carcinoma.(2) Preliminary analysis of differentiallydifferent proteins shows that these proteins are of great value in signaltransduction of human gastric carcinoma cell, maintenance of cell morphologyand normal defense function, promotion of cell division and proliferation andapoptosis as well as invasion and metastasis, and it involves other biologicalproperties of gastric carcinoma cells. Different expressions of these proteinsare closely related with differentiation phenotype of gastric carcinoma cells. Infurther studies it may be possible to find the key differentiation factors; theintervention of the expression of these factors may help inhibit or even reversethe differentiation of gastric carcinoma cells and with it as the target todevelop therapeutic drugs, patient outcomes are expected to get improved.Part Ⅳ: Serpin B1expression in gastric carcinoma tissues and itsrelationship with biological behavior of gastric carcinomaObjective: To detect whether there are differences between the SerpinB1expression in gastric adenocarcinoma and that in normal gastric tissue; to analyze Serpin B1expression in gastric adenocarcinoma and its relationshipwith clinical biological behavior; to discuss the practical value of Serpin B1ingastric carcinoma diagnosis, efficacy monitoring and prognostic evaluationand intrinsic link between Serpin B1and tumor progression, laying afoundation for further research, and to finish preliminary analysis of thepossibility of Serpin B1as therapeutic target in gastric carcinoma.Methods: Eighty cases of gastric adenocarcinoma with complete clinicaland pathological data served as the experimental group while40cases ofnormal gastric tissue served as a control group; with reference to the2010version of WHO clinical and pathological classification and staging ofstomach tumor to categorize the clinical and pathological data;immunohistochemistry method was used for detection Serpin B1expression inboth gastric adenocarcinoma and gastric normal tissue; statistical softwareSPSS13.0was used to analyze the relationship between Serpin B1expressionand clinicopathological parameters of gastric carcinoma.Results:(1) The positive expression rate of Serpin B1in gastricadenocarcinoma was71.25%, while expression rate of the normal tissue was15%, which showed statistically significant difference (P<0.01).(2) Serpin B1expression had no significant correlation with patient's age, gender, tumor size,location, distant metastases (P>0.05), but was closely related with the depth oftumor invasion, degree of differentiation, lymph node metastasis and clinicalstage (P<0.01or0.05).Conclusions:(1) The positive expression rate of the protein Serpin B1ingastric adenocarcinoma is significantly higher than that in normal gastrictissue, indicating a certain degree of specificity of Serpin B1in gastriccarcinoma, which can be served as gastric carcinoma tumor markers forfurther clinical studies.(2) Serpin B1expression is closely related with depthof invasion, degree of differentiation, lymph node metastasis, clinical stage oftumor and so on, suggesting that the positive Serpin B1expression may beassociated with tumor characteristics such as invasion and metastasis. Somefunctions with Serpin B1involved may accelerate the progress of the disease, so interference with Serpin B1expression is expected to serve as a means oftargeted therapy of gastric carcinoma.Part Ⅴ: The influence mechanism of Serpin B1in cell invasion ability inhuman gastric carcinomaObjective: To investigate Serpin B1expression in different differentiatedgastric carcinoma tissues and cells and its influence on gastric carcinoma cellinvasion and to analyze its mechanisms.Methods: Western blot method was employed for detection of Serpin B1expression in gastric carcinoma tissue, adjacent tissues and gastric carcinomacell lines; siRNA was synthesized specific to Serpin B1and then transfectedinto gastric carcinoma cell lines BGC823; cell wound healing assay andTranswell chamber invasion assay were used to observe the influence ofSerpin B1-siRNA after being transfected into gastric carcinoma cells BGC823on invasion and migration activity; expression changes in the invasion andmigration-related protein expression of MMP-2, MMP-9, ICAM-1andTIMP-1were detected before and after transfection.Results:(1) Serpin B1expression in gastric adenocarcinoma tissues andcells was significantly higher than the expression in adjacent tissues, and itwas significantly increased (P<0.01) with decreasing degree of differentiation.(2) Serpin B1-siRNA after transfection into BGC823cells significantlyinhibited Serpin B1expression, and the inhibitory effect was the most obviousin the dose concentration of80nmol/l and the action time of48hours.(3)Serpin B1-siRNA obviously inhibited the invasion and migration activity ofgastric carcinoma cell line BGC823.(4) In48h after Serpin B1-siRNA wastransfected into the gastric carcinoma cell line BGC823, theinvasion-associated protein expression of MMP-2, of MMP-9, ICAM-1andthe mRNA as well as protein expression and activity were significantlylowered, while TIMP-1mRNA and protein expression were significantlyenhanced.Conclusions:(1) There are different Serpin B1expressions in differentdifferentiated gastric carcinoma tissues and cells.(2) After transfection into BGC823cells Serpin B1-siRNA significantly inhibits Serpin B1expression,and greatly reduces invasion and migration ability of gastric carcinoma cells.(3) Reduced invasion and migration activity of Serpin B1-siRNA aftertransfection into gastric carcinoma cells may be caused by regulation ofbalance among the ICAM-1, MMP-2, MMP-9and TIMP-1.Based on the five parts of this research, we could draw conclusions asfollows:1.2D-DIGE, the LTQ ion trap mass spectrometer and nanoESI-LC-MS/MSanalysis method are ideal for large-scale parallel separation and analysis ofthe clinical protein samples in proteomics research, and the experiment hasadvantages of highly automation, high throughput, high sensitivity,accuracy, good repeatability and practicability. But the wholeexperimental process is more complex, highly specialized and with highcost, so more time is still needed to make it widely used in the clinicalresearch process.2. There are significant differences in protein expressions in differentdifferentiated gastric carcinoma tissues. In this study48proteins areidentified from39differentially expressed protein spots; no data areobtained from some protein spots with the mass spectrometer method,which may be influenced by phosphorylation and methylation of proteinsduring the experimental process; after removal of structural proteins andfunctionally non-specific proteins, six kinds of differentiation-relatedproteins are identified and their expressions were different in differentdifferentiated gastric carcinoma tissues. QPCR and Western blot analysisare applied to verify four kinds, and the result is consistent with that inproteomics, indicating the higher credibility of this study and the identifiedproteins are indeed closely related with tumor differentiation.3. Serpin B1and annexin A3have enhanced expression in poorlydifferentiated gastric adenocarcinoma, while APRT, Nm23-H1, GST-π-1and Dermcidin-lL have enhanced expression in well-differentiated gastricadenocarcinoma. Preliminary analysis of differentially different proteins shows that these proteins are of great value in signal transduction ofhuman gastric carcinoma cell, maintenance of cell morphology and normaldefense function, promotion of cell division and proliferation andapoptosis as well as invasion and metastasis, and it involves otherbiological properties of gastric carcinoma cells. The intervention of theexpression of these factors may help inhibit or even reverse thedifferentiation of gastric carcinoma cells and with it as the target todevelop therapeutic drugs patient outcomes were expected to getimproved.4. The positive expression rate of Serpin B1in gastric adenocarcinoma issignificantly higher than that in normal gastric tissue, indicating a certaindegree of specificity of Serpin B1expression in gastric carcinoma, whichcan be served as a gastric carcinoma tumor markers for further clinicalstudies. Serpin B1expression is closely related with tumor depth ofinvasion, degree of differentiation, lymph node metastasis, clinical stageand so on, suggesting that the positive Serpin B1expression may beassociated with tumor characteristics such as invasion and metastasis.Some functions with Serpin B1involved may accelerate the progress ofthe disease, so interference with the Serpin B1expression is expected toserve as a means of targeted therapy of gastric carcinoma.5. QPCR and Western blot analysis are applied to verify the Serpin B1expression in different differentiated gastric carcinoma cell lines such asBGC823, SGC7901and MKN28, and the results further illustrate theconsistency of Serpin B1expression in both gastric carcinoma tissues andcells; its expression is increased significantly in poorly differentiatedtissues and cells; it can be used as the specific protein in gastric carcinomadiagnosis and differentiation judgment.6. After transfection into gastric carcinoma cells BGC823Serpin B1, SerpinB1-siRNA significantly inhibits protein expression, and can effectivelyreduce gastric carcinoma cell invasion and migration. Although theresearch is still at a preliminary stage, to some extent it indicates its clinical value and this treatment may help inhibit the malignant behaviorof gastric carcinoma and improve survival in patients.7. Serpin B1-siRNA transfected into gastric carcinoma cells causes thereduced activity of migration and invasion, which may function byregulating the balance between matrix metalloproteinase and theirinhibitors and by interfering with cell adhesion molecules. Yet the exactmechanisms still needs further study.