Investigating Tumor Growth Of Gastric Cancer In Nude Mice Model By The Quantitative Parameters Of MRI And The Mechanism Of ARHGEF10L In Gastric Tumorigenesis

Part 1 Investigating tumor growth of gastric cancer in nude mice model by the quantitative parameters of magnetic resonance imagingBackgroundGastric cancer is malignant tumor which originated in the gastric mucosa,and is the first incidence of malignant tumor in our country.In our country,gastric cancer has obvious regional differences,and the incidence rate of gastric cancer in the northwest region is significantly higher than that in the eastern region.The age of gastric cancer is more than 50 years old,and the ratio of male to female is about 2:1.Because of the change of diet structure,the increase of work pressure and the infection of Helicobacter pylori,gastric cancer presents the younger tendency.At present,the early diagnosis rate of the gastric cancer in China is still low.There are many factors,steps and stages in the carcinogenesis process of gastric cancer.The changes of oncogenes,tumor suppressor genes,apoptosis related genes,metastasis related genes and the forms of gene changes are also various.With the development of MR imaging technology,more and more applications have been used in the diagnosis of tumor.Diffusion-weighted imaging(DWI)uses diffusion sensitive gradient field(b value)to observe the diffusion of water molecules and calculate the apparent diffusion coefficients(ADC).The larger ADC value indicated the stronger the diffusion ability of water molecules,on the contrary,the lower ADC value indicated that the diffusion is limited.Therefore,the ADC value can be used to quantitatively reflect the microstructure changes of tissues.Intravoxel incoherent motion(IVIM)is a multi-b-value DWI imaging,and it has been paid more and more attention and widely used in clinic diagnosis in recent years.In biological tissues,there in two factors including the water diffusion and the blood perfusion in IVIM.IVIM provided quantitative parameters for the diffusion of water molecules in tumor tissue(D),reflected the perfusion of tumor tissue(D*,f),predicted the growth activity of tumor tissue,and evaluated the therapeutic effect of tumor.In this study,ARHGEF10L(Rho guanine nucleoide exchange factor 10 like protein)belonged to the GEF family protein,which activated RhoA from inactive GDP RhoA state to active GTP RhoA state,thus playing a variety of biological functions.It had been reported that ARHGEF10L was located in chromosome 1p36 region and was closely related to ovarian cancer,liver cancer and other tumors.Due to the high cell composition and the low water content,the difusion of water molecules was limited in gastric cancer tissues.By reducing the expression of ARHGEF10L,the number,the density and the blood supply of tumor tissues would change.In the past,IVIM was mainly used in the study of human tumors,such as breast cancer and liver cancer.The purpose of this study was to observe the effect of ARHGEF10L on the growth activity of gastric cancer tissue in nude mice,and explore the application value of quantitative parameters(ADC,D,D*,f)of magnetic resonance molecular functional imaging to evaluate the growth of nude mice tumor,and provide a new evaluation method for the basic research of tumor.ObjectiveThe quantitative parameters of MR molecular functional was used to evaluate the growth activity of gastric cancer in nude mice,to investigate the effect of ARHGEF10L on the growth of gastric cancer cells.Methods1.BALB/c(Vital River,Beijing,China)nude mice with four to five weeks were chosen to generate the nude mice model.The nude mice were randomly divided into the experimental group(n=10)and the control group(n=10).SGC7901 cells transfected with the lentivirus packing shARHGEF10L were subcutaneously injected into the right flank of mice to generate the nude mice model in gastric cancer.2.Using GE discovery 750 3.0T MRI scanner and small animal coil,we performed multi-b-value DWI scanning on tumor bearing nude mice,and selected 10 b-values 10,20,30,40,50,80,100,150,200,400s/mm2,and used axial spin echo sequence for scanning.The multi-b-value DWI images were analyzed by GE post-processing workstation AW 4.6 software system,and the quantitative parameters ADC value,D value,D*value and F value were measured.SPSS 17.0 software was used for statistical analysis,and the normal distribution and homogeneity of variance were detected.Independent sample t test was used to compare the difference between the two groups.P<0.05 was considered to be statistically significant.ResultsThe ADC values of the control group and the shARHGEF10L group were 2.39±0.24×10-4 mm2/s and 4.13±0.21×10-4 mm2/s,P<0.001,and the difference between the two groups was statistically significant.The lower ADC value of the control group indicated that the water molecular diffusion was obviously limited,suggesting that ARHGEF10L may promote the growth of gastric cancer cells.The D value of the control group and the shARHGEF10L group were 6.81±0.21×10-3mm2/s and 8.03±0.13×10-3 mm2/s,P<0.001.The difference was statistically significant between the two groups.The lower D value in the control group indicated that the real water molecule diffusion was obviously limited,which also suggested that ARHGEF10L might promote the growth and growth of gastric cancer cells.The tumor D*values of the control group and the shARHGEF10L group were 4.37±0.15×10-2 mm2/s and 4.55±0.17×10-2 mm2/s,P>0.05;the f values of the control group and the shARHGEF10L group were 0.21±0.02 and 0.17±0.01,P>0.05.There was no significant difference in D*value and F value between two groups.Conclusions1.The ADC value and D value of IVIM quantitative parameters can effectively evaluate the tumor growth of gastric cancer in nude mice.2.The quantitative parameters of MR molecular functional imaging suggested that the ARHGEF10L could promote the growth of gastric cancer cells.Part 2 Investigating the Mechanism of ARHGEF10L in Gastric TumorigenesisBackgroundAs a member of the RhoGEF family,the gene ARHGEF10L was located on chromosome 1p36 and coded GrinchGEF protein which induced GDP/GTP exchange at RhoA.ARHGEF10L could further involve in a variety of biological functions such as cell proliferation,cell migration and cell adhesion.Two SNPs rs2256787 and rs10788679 in ARHGEF10L gene were associated with increased endometrioid epithelial ovarian cancer risk and invasive serous epithelial ovarian cancer risk.The recent literature demonstrated that two SNPs rs2244444 and rs 12732894 in the ARHGEF10L-encoding gene is genetically susceptible to liver tumor.The increased expression of ARHGEF10L protein contributes to increased cell proliferation and migration in liver tumorigenesis.ARHGEF10L overexpression stimulated the GTP-RhoA-ROCK1-phospho ERM signaling.However,the specific mechanism of ARHGEF 10L involving in gastric cancer pathogenesis process is still unknown.ObjectiveIn this study,we investigated ARHGEF 10L expression in gastric cancer tissues,observed the effects of ARHGEF 10L protein expression on cell proliferation,cell migration and epithelial-mesenchymal transition(EMT).Then we further determined the possible molecular signaling pathway of ARHGEF10L participating in gastric carcinogenesis process.Methods1.Solid gastric cancer tissue samples(n=6)and their peritumoral tissues(n=6)were collected at the Shandong Provincial Qianfoshan Hospital and proteins were extracted from the tumor tissues.Western blot was performed to detect the protein expression of ARHGEF10L in a panel of GC tissue and the statistical analysis was carried on.2.The GC cell line SGC7901 cells were the epithelioid adherent cells and cultured.These plasmids including pcDNA3.1-RFP and pcDNA3.1-ARHGEF10L-RFP were transfected into SGC7901 cells using PolyJetTM In Vitro DNA(SignaGen,USA)according to the manufacturer’s protocol.The pcDNA 3.1-RFP expression vector without the cDNA insert was used as control plasmids(mock).After 72h,proteins were extracted from the cultured cells and western blot analysis was used to detect the ARHGEF10Lprotein level.Cell Counting Kit-8(CCK-8)assays,migration assays and tumor cell tube-like structure formation assays were measured to detect the effects of ARHGEF10L overexpression on cell proliferation and cell migration.The statistical analysis was carried on.3.The GC cell line SGC7901 cells were cultured.The two siRNAs including Allstars and Anti-ARHGEF10L siRNA were transfected into SGC7901 cells using PepMuteTM siRNA Transfection Reagent according to the manufacturer’s protocol.Allstars siRNA,which has no target sequence in human genes,was used as a control.After 72h,proteins were extracted from the cultured cells and western blot analysis was used to detect the ARHGEF10Lprotein level.Cell Counting Kit-8(CCK-8)assays,migration assays and tumor cell tube-like structure formation assays were measured to detect the effects of ARHGEF10L on cell proliferation and cell migration.The statistical analysis was carried on.4.The ARHGEF10L protein was inhibited or overexpressed in GC cell line SGC7901 cells,respectively.Seventy-two hours after the transfection,western blot analysis was used to detect the protein level of epithelial cell marker protein(E-cadherin)and mesenchymal cell marker protein(N-cadherin、Slug).The statistical analysis was performed.5.GC cell line SGC7901 were transfected with pcDNA3.1-RFP and pcDNA3.1-ARHGEF10L-RFP using PolyJetTM In Vitro DNA.Using Rho Activation Assay Kit,we performed a Rho activation assay to determine the protein level of GTP-RhoA,ROCK1 and pERM.The Rho activation assay was determined the effect of ARHGEF10L expression on the Rho pathway and the statistical analysis was performed.6.GC cell line SGC7901 were transfected with pcDNA3.1-RFP and pcDNA3.1-ARHGEF10L-RFP.48 hours after the transfection,RNA-sequencing analysis(OE Biotech Company)was used to determining the possible downstream pathway of ARHGEF10L.Results1.Compared to the peritumoral tissue samples(n=6),the gastric tumors tissue samples(n=6)showed significantly increased ARHGEF10L protein expression.And the statistical analysis was significant.2.SGC7901 cells were transfected with ARHGEF10Lexpressing plasmids or the blank vectors(Mock).Western blotting detected higher protein level of ARHGEF10L in the transfected SGC7901 cells than in the cells transfected with the blank vectors.Compared to the control cells transfected with blank vectors,the cells transfected with the ARHGEF10L-expressing plasmids showed increased cell proliferation.The transwell assay showed enhanced SGC7901 cell migration in the cells transfected with ARHGEF10L-expressing plasmids compared with the control.The results of the tumor cell tube formation assay showed many more tube-like structures in the SGC7901 cells transfected with ARHGEF10L-expressing plasmids than that in the controls.The statistical analysis was significant.3.The SGC7901 cells were transfected with anti-ARHGEF10L siRNA or Allstars siRNA.Western blotting showed a decreased protein level of ARHGEF10L in the cells with transfection of anti-ARHGEF10L siRNA compared with that in the cells with transfection of Allstars siRNA.CCK-8 assay showed that the ability of cell proliferation was suppressed in SGC7901 cells with transfection of anti-ARHGEF10L siRNA compared with the controls.Transwell assay showed that the ability of cell migration was inhibited in the SGC7901 cells with transfection of anti-ARHGEF10L siRNA.Tumor cell tube formation assay showed less tube-like structures in the cells with transfection of anti-ARHGEF10L siRNA compared with the control.The statistical analysis was significant.4.Compared to the control cells,SGC7901 cells with ARHGEF10L-expressing plasmids showed significantly increased expression of N-cadherin and Slug,and the decreased expression of E-cadherin.On the other hand,SGC7901 cells with transfection of anti-ARHGEF10L siRNA suggested the downregulated expression of N-cadherin and Slug,and the upregulated expression of E-cadherin.The statistical analysis was significant.5.We found that the level of GTP-RhoA was significantly increased in SGC7901 cells overexpressing ARHGEF10L,indicating the activation of GTP-RhoA.ROCK1 was correspondingly increased in SGC7901 cells overexpressing ARHGEF10L.Western blotting also detected an increased expression of phospho-ERM protein in SGC7901 cells overexpressing ARHGEF10L.The statistical analysis was significant.6.Using RNA-sequencing analysis,SGC7901 cells with transfection with ARHGEF10Lexpressing plasmids detected significantly increased expression of HSPA6 compared with the control cells.Conclusions1.The gastric tumors tissue samples showed increased ARHGEF10L protein expression,suggesting that ARHGEF10L was associated with the pathogenic process of gastric tumors.2.Cell Counting Kit-8 assays,transwell assays and tumor cell tube formation assays indicating that overexpression of ARHGEF10L elevated cell proliferation,migration and tube-like structure formation ability in SGC7901 cells.Meanwhile,knocking-down ARHGEF10L expression suppressed cell proliferation,migration and tube-like structure formation ability in SGC7901 cells.3.Compared to the control cells,SGC7901 cells with ARHGEF10L-expressing plasmids showed significantly increased expression of N-cadherin and Slug,and the decreased expression of E-cadherin,inducing the occurrence of epithelial mesenchymal transformation.4.In gastric cancer,overexpression of ARHGEF10L protein could contribute to the tumorigenesis of gastric cancer through the GTP-RhoA-ROCK1-pERM signaling.And overexpression of ARHGEF10L protein in SGC7901 cells promoted significantly increased expression of HSPA6.