| IntrodutionGastric cancer(GC)is a disease with high morbidity and mortality worldwide,among which nearly half of Gastric cancer occurs in China.Therefore,the effective treatment of gastric cancer is a problem that cannot be ignored in China.The pathogenesis of gastric cancer is still not clear,and the treatment of gastric cancer mainly includes endoscopic treatment,surgical treatment,chemoradiotherapy and so on.However,the overall situation is still not optimistic,only 40%of gastric cancer patients can reach the 5-year overall survival.Therefore,it is urgent for us to further clarify the pathogenesis and molecular mechanism of gastric cancer,so as to provide more effective therapeutic targets for the gastric cancer treatment.The protein 4.1 subfamily consists of four members:4.1B,4.1R,4.1G and 4.1N,which are encoded by four homologous gene sequences.All the protein 4.1 subfamily members have three highly conserved domains:FERM domain(four-1 protein,Ezrin,Radixin,Moesin),spectrin-actin-binding domain(SABD)at the amino terminus,and carboxy-terminal domain(CTD)at the carboxy-terminal terminus.The FERM domain is the largest conservative domain.The Erythrocyte membrane Protein Band 4.1 Like 3(EPB41L3)is a member of protein 4.1 subfamily.4.1B gene is located on chromosome 17 in mice,but it is located on chromosome 18p11.3 in human.At present,many studies had found protein 4.1B expression were decreased or deficiency in various tumors.There were also a few studies about protein 4.1B and gastric cancer.However,these studies did not elaborate on the specific role of 4.1B protein in the process of gastric cancer and the internal mechanism.Tumor cells are characterized by abnormal proliferation,migration and enhanced invasion ability.The purpose of this study was to investigate the role of protein 4.1B in proliferation,migration and invasion of gastric cancer.Then,the mechanism about protein 4.1B inhibits the proliferation ability of gastric cancer cells was further investigated by detecting EGFR/MAPK and PI3K/AKT pathways.Epithelial-to-mesenchymal transition(EMT)means that the polarized epithelial cells transform into mesenchymal cells.After transformation,the adhesion ability between cells are decreased,making the cells easier to migrate.Recently,many studies had shown that there was a close relationship between EMT and tumor cells invasion and metastasis.We first investigated the effect of protein 4.1B on gastric cancer cells adhesion,migration and invasion,and then observed the expression of EMT-related proteins E-cadherin,MMP-2 and MMP-9 and so on.Part I Protein 4.1B expression level in gastric cancer mucosa and its effect to the prognosisAims:Gastric cancer is a disease with high morbidity and mortality worldwide,making it to be the second leading cause of cancer-related deaths and the fourth cancer incidence in the world.At present,many studies have shown that the expression of protein 4.1B is decreased or absent in various tumors.There are also a few studies about protein 4.1 B and gastric cancer,but these studies are not enough on internal mechanism.In this part,we first observed the expression of protein 4.1B of gastric mucosal tissues in gastric cancer patients and the normal control.Then we further investigated whether the protein 4.1B expression level could affect the prognosis of gastric cancer patients as an independent factor(DFS and OS).Materials and methods:1.We chose 102 paraffin-embedded specimens of gastric resection of gastric cancer from January 2011 to January 2013 at the department of pathology of the Fifth Affiliated Hospital of Zhengzhou University.Among them,49 cases were under 65 years old and 53 cases were over 65 years old.There were 59 males and 43 females.None of the patients received preoperative chemo-radiotherapy.The corresponding para-cancer control samples were at least 5cm away from the cancers,and all cancers and para-cancer normal gastric mucosa were diagnosed by HE staining and histopathology.Of the 102 samples,only 10 were highly differentiated,34 were moderately differentiated,and 58 were poorly differentiated or undifferentiated.This study was approved by the Fifth Affiliated Hospital of Zhengzhou University medical ethics committee,and the patients or their families signed the informed consent.2.Hematoxylin-eosin staining(HE)staining was performed on all gastric cancer tissue specimens and normal adjacent control group specimens to observe the structure of tissue cells.3.Immunohistochemistry(IHC)staining was performed on all gastric cancer tissues and para-cancer control samples,so as to observe whether there was any difference in protein 4.1 B levels between gastric cancer tissue and normal para-cancer control group;And whether this difference has a certain correlation with the severity of gastric cancer.4.According to the IHC results,we used the following criteria:using 10 x 40 optical microscope,randomly selected at least 10 view and 100 cells each view,and counting cell cytoplasm membrane staining positive judgment,0 point:positive cells 5%or less;1 point:positive cells 5%to 25%;2 points:positive cells 25%?50%;3 points:positive cells 50%?75%;4 points:positive cells more than 75%.According to the cell membrane and cytoplasm staining degree background color is 0 point,1 point for mild staining,2 points for moderate staining and 3 points for severe staining.4-7 points mean positive and 0-3 points mean negative.The gastric cancer group and the normal control group were divided into negative and positive groups according to the degree of protein 4.1B expression.Then,we analyzed whether there was statistically significant between gastric cancer group and the normal control group by?2 test.5.If there was significant difference of protein 4.1B expression level between the gastric cancer group and control group,we would further use ?2 test to observe whether there was a statistically significant difference in the clinical indicators between the 4.1B protein positive group and the negative group.6.We followed up the gastric cancer patient survival(DFS and OS),and evaluated the average survival between protein 4.1B positive and negative groups by Kaplan-Meier survival analysis.7.If there is a difference in survival(DFS and OS)between the positive group and the negative group of gastric cancer patients with protein 4.1B expression,we intend to further observe whether the expression level of protein 4.1B can affect survival(DFS and OS)of gastric cancer patients as an independent factor through univariate and multivariate COX regression analysis.Results:1.According to the IHC and table 1,we found that the expression level of protein 4.1 B in gastric mucosa of normal control group was significantly higher than that of gastric cancer patients(?2=32.23;P<0.001),and the expression level of protein 4.1B was negatively correlated with the severity of gastric cancer patients.2.Table 2 results showed that the expression level of 4.1B was negatively correlated with tumor volume,tumor differentiation degree,number of lymph node metastasis and the whole TNM grading(P<0.05).There was no significant correlation with age,sex,helicobacter pylori infection,tumor location,WHO classification,vascular invasion,tumor invasion depth and nerve metastasis.3.We analyzed the DFS and OS of the patients by Kaplan-Meier analysis and log-rank test,and found that the 5-year DFS of the patients of 4.1B positive group was 22 months,while the 4.1B negative group was only 15 months.The 5-year OS of 4.1B positive group was 51 months,but negative group was only 33 months.Therefore,we can get a conclusion that the expression level of 4.1B affects the gastric cancer patients DFS and OS.(P<0.001).4.Univariate and multivariate COX regression analysis was used to analyze the DFS and OS of the remaining gastric cancer 95 patients.We found univariate COX regression analysis showed that the expression level of 4.1B protein,tumor size,lymph node metastasis(N0 vs.N1+N2+N3)and TNM grade(?+? vs.?)were the factors influencing the prognosis of gastric cancer patients.Then,we further found that expression level of 4.1B,tumor volume and total TNM grade(?+? vs.?)were independent factors influencing DFS and OS in gastric cancer patients through multivariate COX regression analysis.Conclusions:The expression level of protein 4.1 B in gastric cancer mucosa was decreased or absent compared with the normal control group,and the 4.1B expression level was negatively correlated with the disease severity.Protein 4.1B expression level can affect the DFS and OS of gastric cancer patients as an independent factor.Part ? Protein 4.1 expression in GC cell lines and its relationship with cells proliferationAims:In this part,we intend to observe the expression level of protein 4.1B in MGC-803 and MKN-45 cell lines,and the effect of protein 4.1B on proliferation capacity of this two GC cell lines.We investigated the effect of 4.1B on GC cells proliferation by overexpressing the 4.1 B in MGC-803 cells and knocking out it in MKN-45 cells.Finally,subcutaneous tumor-bearing experiments in nude mice were conducted to observe the effect of 4.1B in vivo.Further,the changes of EGFR/MAPK pathway and PI3K/AKT pathway were detected to understand the internal mechanism of 4.1B on proliferation of GC cells.Materials and methods:1.GC cell lines MGC-803 and MKN-45 were cultured in vitro to extract the total proteins of them,and then the expression of protein 4.1B of the two GC cell lines was detected by western blotting.2.GC cell lines MGC-803 and MKN-45 were planted in a 10cm diameter petri dish according to 0.5 ×106/dish,and triplicate was set in each group.Then,cell counts were performed on day 3,day 5,and day 7,respectively.Each group was counted at least three times,and the mean value was taken.Finally,GraphPad Prism 7 and SPSS20.0 were used to analyze the data.3.After overexpression protein 4.1B in MGC-803 cells,we sorted 4.1B-GFP cells by flow cytometry.Then G418 was added to continue the screening.Western blotting and confocal microscopy were used to determine the proportion of target cells above 90%.Then we investigated the proliferation of 4.1B overexpression group(4.1B-GFP)and the control group(GFP-CON(4.1B-/-)(same as step 2).4.4.1B was knocked out in MKN-45 cells by Crisper cas9.Puromycin was added to screen the mono-clones.We checked whether the knock out was successful by Western blotting.Then we investigated the proliferation of 4.1B knockout group(4.1B KO)and the control group(MKN-45(CON(4.1B+/+))(same as step 2).5.Twenty 5-week-old Balb/c mice were selected,and 4.1B overexpression group(4.1B-GFP)and the control group(GFP-CON(4.1B-/-),4.1B knockout group(4.1B KO)and the control group(MKN-45(CON(4.1B+/+))were subcutaneously injected into 5 mice each group,respectively.After 1 week,we can observe subcutaneous tumor formation by eyes.The tumors size was recorded with a vernier caliper every other day.The tumor volume was calculated according to the formula:volume=short diameter of tumor body2 × long diameter of tumor ×0.5.When the maximum diameter approached 20mm,the nude mice were sacrificed.The tumors were weighed as soon as possible,and data were accurately recorded.Finally,GraphPad Prism 7 and SPSS20.0 were used to analyze the data.6.4.1B overexpression group(4.1B-GFP)and the control group(GFP-CON(4.1B-/-)MGC-803 cells protein were extracted,and the influence of 4.1B on EGFR/MAPK and PI3K/AKT signaling pathways was detected by western blotting.7.4.1B knockout group(4.1B KO)and the control group(MKN-45(CON(4.1B+/+))MKN-45 cells total protein were extracted,and the influence of 4.1B on EGFR/MAPK and PI3K/AKT signaling pathways was detected by western blotting.Results:1.Western blotting showed that protein 4.1B was absent in MGC-803 cells,while it was expressed strongly in MKN-45 cells.2.Cell proliferation curve showed that MKN-45 proliferated slower than MGC-803 cells,and the difference was statistically significant(P<0.05).3.The proliferation of MGC-803 was inhibited after 4.1B overexpression(P<0.001).However,the proliferation capacity of MKN-45 increased significantly after 4.1B was knocked out(P<0.001).4.Tumor-bearing experiments in nude mice showed that the volume(P<0.05)and weight(P<0.001)of tumor-forming subcutaneous tumors in nude mice after overexpression of 4.1B protein were significantly lower than those in the negative control group(4.1B-/-).When the 4.1B gene of MKN-45 was knocked out(4.1B KO),the subcutaneous tumor volume(P<0.001)and weight(P<0.05)of nude mice were significantly increased compared with the control group MKN-45(CON(4.1B+/+)).5.Western blotting results showed that the protein expression levels of p-ERK(P<0.01)and p-AKT(P<0.05)in the 4.1B overexpressed group(4.1B-GFP)were decreased compared with those in the control group(GFP-CON(4.1B-/-)cells,while the total expression levels of ERK and AKT were not significantly different.Both total EGFR(P<0.01)and phosphorylated EGFR(P<0.01)were reduced.There was no difference in protein levels between MAPK/JNK and MAPK/p38.6.Western blotting results showed that the protein expression levels of p-ERK(P<0.01)and p-AKT(P<0.01)in MKN-45 cells of the 4.1B knockout group(4.1B KO)and the control group(CON(4.1B+/+)were increased,while the total expression levels of ERK and AKT were not significantly different.Both total EGFR(P<0.01)and phosphorylated EGFR(P<0.001)were significantly increased.The protein levels of MAPK/JNK and MAPK/p38 pathways also showed no difference.Conclusions:Protein 4.1B could inhibit the proliferation of gastric cancer cells in vivo and vitro through EGFR/MAPK/ERK1/2 and EGFR/PI3K/AKT pathways.Part ? The mechanism of 4.1B to inhibit the proliferation of GC cellsAims:The results of part ? and part ? showed that the expression of 4.1B protein in GC patients and normal control group was different.The expression level of 4.1B was much different in different proliferation capacity MGC-803 and MKN-45 cells.In addition,we further verified that 4.1B had a certain inhibitory effect on the proliferation ability of GC cells through the tumor-bearing experiments in nude mice.Then,we verified 4.1B inhibited GC cells proliferation through EGFR/MAPK/ERK1/2 and EGFR/PI3K/AKT pathways by Western blotting.However,we found that 4.1B can affect total protein EGFR level,so our purpose in this section is to further explore how 4.1B protein affects total protein EGFR level.Materials and methods:1.Using wild-type and 4.1B gene knockout C57BL/6 mouse models,we isolated the embryos at day 13.5 of gestation.Mouse embryonic fibroblasts(MEFs)were isolated and immobilized in vitro.We checked the proliferation of wild-type MEF cells(4.1B+/+)and 4.1B KO MEF cells(4.1B-/-)using the method same as part ?.2.Extracted the total proteins of wild-type MEF cells(4.1B+/+)and 4.1B KO MEF cells(4.1B-/-),and detect the changes of EGFR/MAPK and EGFR/PI3K/AKT signaling proteins by western blotting.3.EGFR inhibitor erlotinib was added to observe the proliferation of wild-type MEF cells(4.1B+/+)and 4.1B KO MEF cells(4.1B-/-)by cell count We further clarify whether protein 4.1B affects cell proliferation through the EGFR pathway.4.Real-time PCR was used to observe whether EGFR changed at mRNA level with the change of 4.1B in MGC-803,MKN-45 and MEF cell lines.To determine whether EGFR protein changes are related to its synthesis or degradation.5.Observe the changes of mRNA level and protein level of EGFR transcription factor Spl in MGC-803,MKN-45 and MEF cell lines with the changes of 4.1B protein by real-time PCR and western blotting.6.By interfering with the synthesis of Spl in MKN-45(4.1B KO)and MEF(4.1B KO)cells with siRNA,we observed that the synthesis of EGFR was down-regulated.To prove that Spl is indeed a key transcription factor for EGFR synthesis.7.Cell immunofluorescence and protein co-precipitation were used to observe the co-localization and interaction between protein 4.1 B and protein EGFR.8.Designed polypeptide fragments of EGFR intracellular segment and protein 4.1B domains,and to determine which domain of 4.1B bond to which peptide fragment of EGFR protein to inhibit cell proliferation by pull-down technology.Results:1.The proliferation capacity of 4.1B KO MEF cells(4.1B-/-)was significantly increased compared with that of wild-type MEF cells(4.1B+/+),and the difference was statistically significant(P<0.01).2.Western blotting results of wild-type MEF cells(4.1B +/+)and 4.1B KO MEF cells(4.1B-/-)were the same as those of GC cell lines MGC-803 and MKN-45 through EGFR/MAPK/ERK1/2 and EGFR/PI3K/AKT pathways.3.The proliferation capacity of 4.1 B KO MEF cells decreased significantly after adding EGFR inhibitor erlotinib,which was close to that of wild-type MEF cells,and this suggested that the function of 4.1B worked through EGFR protein.4.Real-time PCR results showed that the mRNA level of EGFR also changed with the change of protein 4.1B:the EGFR mRNA level of 4.1B expression group MGC-803(4.1B-GFP)(P<0.01),MKN-45(4.1B+/+)(P<0.001)and wild-type MEF(P<0.05)was significantly decreased.5.Real-time PCR and western blotting results showed that EGFR transcription factor Sp1 changed with the change of 4.1B.EGFR transcription factor Sp1 decreased in 4.1B expression group MGC-803(4.1B-GFP),MKN-45(4.1B+/+)and wild-type MEF both in mRNA level and protein level compared with that of the 4.1 B deficient group.6.Si RNA interference showed that expression level of EGFR decreased after Sp1 was knocked down in MKN-45(4.1B KO)and MEF(4.1B KO)cells.This result proved that transcription factor Sp1 is key for EGFR protein synthesis,and that 4.1 B must act through this pathway to affect cell proliferation.7.Cellular immunofluorescence revealed protein 4.1B and protein EGFR were co-localized,and immunoprecipitation revealed the interaction between protein4A1B and protein EGFR.8.The pull-down results clearly showed that the FERM domain of protein 4.1B could combine with the starting position 13 amino acid(P13)at the intracellular membrane of EGFR protein to play a role in regulating cell proliferation.Conclusions:The FERM domain of protein 4.1B inhibited the activation of EGFR by combining with the P13(R644-R656)of protein EGFR,thereby inhibiting the dimerization of the intracellular membrane region(JM)of protein EGFR.Reduced activation of EGFR down-regulated the activation of EGFR transcription factor Spl by down-regulating the MAPK/-ERK1/2 and PI3K/AKT pathways,resulting in a decrease of total protein EGFR.This inevitably leaded to a decrease of total protein EGFR and its phosphorylation level,thus forming a negative feedback to affect the cell proliferation.Part ? The effect and mechanism of protein 4.1B on GC cells migration and invasionAims:We discussed the inhibitory effect of 4.1B on the proliferation of GC cells and its internal mechanism in the first three parts.In this part,we intended to observe the effect of 4.1B on the migration and invasion of GC cells and its internal mechanism.Materials and methods:1.Investigated the adhesion ability changes of 4.1B overexpressed group(4.1B-GFP)and the control group(GFP-CON(4.1B-/-))MGC-803 cells and 4.1B knockout group(4.1B KO)and the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1B expression.The crystal violet absorbance was detected at 560nm by the full-automatic multifunctional microplate reader EnsightTM(Perkin Elmer).The data was analyzed by GraphPad Prism 7 and SPSS20.0.2.Investigated the adhesion ability changes of 4.1B overexpressed group(4.1B-GFP)and the control group(GFP-CON(4.1B-/-)MGC-803 cells and 4.1B knockout group(4.1B KO)and the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1B expression.Zeiss LSM880 laser confocal scanning microscope was used to observe the cells.The data was analyzed by Image J,GraphPad Prism 7 and SPSS20.0.3.Investigated the migration ability changes of 4.1B overexpressed group(4.1B-GFP),the control group(GFP-CON(4.1B-/-)MGC-803 cells and 4.1B knockout group(4.1B KO),the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1B expression by wound healing and transwell migration experiments.The data was analyzed by Image J,GraphPad Prism 7 and SPSS20.0.4.Investigated the invasion ability changes of 4.1B overexpressed group(4.1B-GFP),the control group(GFP-CON(4.1B-/-))MGC-803 cells and 4.1B knockout group(4.1B KO),the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1 B expression by transwell invasion experiments.The data was analyzed by Image J,GraphPad Prism 7 and SPSS20.0.5.Investigated the hematogenous metastasis ability changes of 4.1B overexpressed group(4.1B-GFP),the control group(GFP-CON(4.1B-/-))MGC-803 cells and 4.1B knockout group(4.1B KO),the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1B expression by mice tail vein injection experiment.transwell invasion experiments.The data was analyzed by GraphPad Prism 7 and SPSS20.0.6.Observed the EMT related proteins of 4.1B overexpressed group(4.1B-GFP),the control group(GFP-CON(4.1B-/-))MGC-803 cells and 4.1B knockout group(4.1B KO),the control group(CON(4.1B+/+))MKN-45 cells with variation of protein 4.1B expression by western blotting.Results:1.The results of cells adhesion experiments showed that 4.1B overexpression would increase the adhesion rate and spreading area of MGC-803 cells.After the knockout 4.1B of MKN-45 cells,the adhesion rate and spreading area decreased.2.The results of cells migration and invasion experiments showed that 4.1B overexpression could inhibit the migration and invasion ability of MGC-803 cells.However,after the knockout 4.1B of MKN-45 cells,the migration and invasion ability of MKN-45(4.1B KO)increased,3.The results of mice tail vein injection showed that 4.1B overexpression could inhibit the formation of metastatic tumor of MGC-803 cells.However,after the knockout 4.1B of MKN-45 cells,the transfer ability of MKN-45(4.1B KO)was increased.4.Western blotting results showed that 4.1B could up-regulate the expression levels of ?-catenin and E-cadherin;while it inhibited the expression levels of MMP-2 and MMP-9.Conclusions:4.1B inhibited EMT by down-regulating the activation of EGFR/MAPK/ERK1/2 andEGFR/PI3K/AKT signaling pathway to inhibit the migration and invasion of GC cells. |
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