| Background & objective Gastric Cancer,(GC)is one of the most common malignant tumors in the digestive tract.The incidence and mortality rate of gastric cancer in China are the second place in the malignant tumors of China.According to our epidemiological statistics on gastric cancer,about 30-40% of patients have been diagnosed as advanced disease when they first go to hospital,and the 5-year survival rate is less than 10%.Despite the optimization of treatment and new targeting drugs,as well as the immunotherapy,the survival of patients with advanced gastric cancer have been improved.However,in general,the diagnosis and treatment of gastric cancer in my country is still less optimistic,the early diagnostic rate is low,and there is still a lack of effective prediction and treatment for advanced gastric cancer.Since treatment methods are limited for advanced gastric cancer patients,there is an urgent need to identify novel prognostic biomarkers that accurately predict cancer invasionand metastasis.WASF3 plays an important role in the invasion and metastation of tumors.However,the role of WASF3 in gastric cancer and its mechanism is not clear.This study aims to understand the significance of WASF3 expression in gastric cancer,and analyze the effect of WASF3 on the biological behavior of gastric cancer cells,as well as to explore the molecular mechanism of WASF3 in regulating biology behavior of gastric cancer cell.At last,we learned about the role of WASF3 in the sensitivity of gastric cancer to oxaliplatin,and the underlying mechanisms.This study is divided into four parts:Part one Expression of WASF3 in gastric cancer and its clinical significancePurposes To explore the prognostic value of WASF3 in gastric cancer,as well as to analyze the relationship between the expression of WASF3 and the pathological characteristics in gastric cancer.Methods 1.We obtained gastric cancer tissue samples with complete clinical data from 164 gastric cancer patients(46 patients for quantitative realtime PCR and 118 patients for immunohistochemistry)that underwent surgery.Gastric cancer was confirmed histologically in all patients.2.We investigated the co-expression of WASF3 and E-cadherin in 46 fresh tissues of gastric cancer by real-time PCR.3. Immunohistochemical methodology were used to analysis of the expression of WASF3 and E-cadherin in 118 gastric cancer tissues.4.Statistics software was used to analysis the prognostic value of WASF3 in gastric cancer,and we also analyze the relationship between the expression of WASF3 and the pathological characteristics in gastric cancer.The relationship between WASF3 and E-cadherin was also explored.Result 1.The q PCR analysis demonstrated high WASF3 and low E-cadherin m RNA expression in gastric cancer tissues than adjacent normal tissues(p<0.001).2.The immunohistochemical results showed that WASF3 is mainly expressed in cytoplasm,while E-Cadherin is mainly expressed in cell membrane.And the positive expression of WASF3 correlated with lymph nodeme tastasis,vessel involvement and TNM stages(p<0.001),while reduced E-cadherin expression was associated with lymph node metastasis(p<0.024),vessel involvement(p<0.001).3.WASF3 and E-cadherin were coexpressed in 23.7%(28/118)of the gastric cancer patients.Positive WASF3 expression was associated with reduced expression of E-cadherin(p=0.002).4.survival analysis found that patients with positive WASF3 expression showed poor prognosis than those with negative WASF3 expression(p<0.001).There was no correlation between E-cadherin expression and overall survival(p=0.062).5.in 56 patients with positive WASF3 expression,the prognosis of patients received oxaliplatin treatment was poor than those not with oxaliplatin treatment,there is a significant difference between the two groups(P = 0.032).6.Single-factor variance analysis showed that there are relationship between the expression of WASF3 and vascular involvement,lymph node metastasis,TNM stage(P <0.001),multi-factor analysis suggested that the expression of WASF3 is an independent prognostic factor in gastric cancer(P = 0.027).Conclusion The expression of WASF3 in gastric cancer tissue is significantly higher than adjacent norma ltissues.The high expression of WASF3 is related to lymph node metastasis and vascular involvement.High expression of WASF3 indicates a poor prognosis in patients with gastric cancer and also suggestes not sensitive to oxaliplatin treatment.Part two Effect of WASF3 on the biological behavior of gastric cancer cellsPurposes To investigate the effects of inhibiting or overexpressing the expression of WASF3 on the biological behavior in gastric cancer cell line.Cell growth,proliferation assay,cell invasion and migration were carried out.Methods 1.q PCR and Western Blot were used to detect the expression of WASF3 and E-cadherin in four gastric cancer cell lines.Then,we selected two gastric cancer cells MGC803 and MKN45 of which WASF3 was high expressed and low expressed,the two cells were subsequently used to cell experiments.2.sh WASF3 gastric cancer cells were conducted using short interference RNA technology,and WASF3-overexpressing gastric cancer cells were conducted with LV-WASF3.3.The relationship between WASF3 and the cell growth,proliferation was verified by CCK8 flat-cloning formation assay.4.Cell scratche test and Transwell assaywere used to investigate the relationshio of WASF3 and the ability of invasion and migration of gastric cancer cellst.5 Xenograft model of node mice was used to verify the relationship between WASF3 and the growth capacity of gastric cancer in vivo.Result 1.qPCR and Western Blot results showed that WASF3 is highly expressed in SGC7901 and MGC-803 cell lines and lowly expressed in the BGC823 and MKN-45 cell lines.Conversely,however,E-cadherin was lowly expressed in SGC7901 and MGC-803 cell lines and highly expressed in the BGC823 and MKN-45 cell lines(P <0.05),thus,MGC803 and MKN45 cells were choosed for the subsequent experiments.2.sh WASF3 gastric cancer cells(MGC803+sh WASF3)and WASF3-overexpression gastric cancer cells(MKN45+oe WASF3)were conducted successfully.3.CCK-8 and cloning formation results showed that the ability of growth and proliferation of gastric cancer cells with sh WASF3 is significantly inhibited compared with control group;however,the ability of growth and proliferation of oe WASF3 gastric cancer cells was faster than the control group(P <0.05).4.The cell scratch test and Transwell assay results suggested that the ability of migration and invasion of sh WASF3 gastric cancer cells is significantly inhibited compared with control group;however,the ability of growth and proliferation of oe WASF3 gastric cancer cells was enhanced than the control group(P <0.05).4,Xenograft model in nude mice showed that the tumor volume in sh WASF3 group was significantly smaller than the tumor volume in the control group,and the weight of the tumors was also less than that of the control group;However,the tumor volume in oe WASF3 group was significantly larger than the tumor volume in the control group,and the weight of the tumors was also heavier than that of the control group(P <0.05).Conculusion WASF3 can promote the growth and proliferation of gastric cancer cells in vitro and in vivo,it also can promote the invasion and migration of gastric cancer cellsPart three WASF3 regulates PI3K/AKT/NFκB signaling pathway by interacting with P85Purposes To investigate the molecular mechanism by which WASF3 promotes the growth,proliferation,invasive and migration of gastric cancer cells.Methods 1.Western blot was used to detect the expression of p85,an important molecule in the PI3 K signaling pathway,and the downstream kinases p-AKT,p-NFκB,effector EMT-related proteins E-cadherin,N-cadherin,and ICAM-1 in gastric cancer cells when the WASF3 is low expressed or overexpression.2.Using immunoprecipitation to verify whether WASF3 interacts with p85.3.WASF3 low expression and p85 overexpressing gastric cancer cells(MGC803+sh WASF3+oep85)were conducted by lentiviral packaging infection,while WASF3 overexpressing and p85 low expression gastric cancer cells(MKN45+oe WASF3+shp85)were also conducted.4.Using CCK8 and plate clone formation assay to detect whether WASF3 promotes gastric cancer cell growth and proliferation dependent on p85.5.Cell scratching assay and Transwell assay were used to detect whether WASF3 promotes gastric cancer cell migration and invasion also depends on p85.6.Using western blot to understand whether WASF3 regulation of PI3K/AKT/NFκB signaling pathway is dependent on p85.7.Immunoprecipitation was used to preliminarily explore the possible mechanism of WASF3-p85 interaction.Results 1.Western blot results showed that when compared with the control group,the expression levels of p85,p-AKT,and p-NFκB were decreased after inhibition of WASF3 expression,and the expression levels of the effector molecule EMT-related protein E-cadherin were increased,while the expression of N-cadherin and ICAM-1 were decreased,and AKT,NFκB total protein were not significantly changed.However,after making WASF3 overexpression,the expression levels of each of the above signaling pathway proteins were reversed.2.The immunoprecipitation results revealed that there was an interaction between WASF3 and p85.3.Stable transduced gastric cancer MGC803 cells with WASF3 low expression p85 overexpression(MGC803+sh WASF3+oep85)and gastric cancer MKN45 cells with WASF3 overexpression p85 low expression(MKN45+oe WASF3+shp85)were successfully constructed.4.CCK8 and plate clone formation assays showed that the growth and proliferation ability of gastric cancer cells didn’t change insignificantly after simultaneous regulation of WASF3 and p85 expression compared to the control group,and this result indicated that WASF3 promotes the growth and proliferation of gastric cancer cells dependent on p85.5.Cell scratching assay and Transwell invasion assay showed that the migration and invasion ability of gastric cancer cells did not change significantly compared to the control group after simultaneous regulation of WASF3 and p85 expression,which revealed that WASF3 promoted migration and invasion of gastric cancer cells also dependent on p85.6.Western blot results suggested that after simultaneous regulation of WASF3 and p85 expression,the related proteins of the PI3K/AKT/NFκB signaling pathway did not change significantly compared to the control group,indicating that WASF3 regulation of PI3K/AKT/NFκB signaling pathway was dependent on p85.7.The immunoprecipitation results reveal that the mechanism of the interaction between WASF3 and p85 might be that WASF3 could affect the binding of p110 to p85Conclusion WASF3 promotes the growth proliferation,invasive and migration of gastric cancer cells by affecting the binding of p110 to p85,which in turn activates the PI3K/AKT/NFκB signaling pathway to promote EMT.Part four Study on the role and mechanism of WASF3 reduces chemosensitivity of gastric cancer cells to oxaliplatin by reducing DNA damagePurposes To investigate the effect of WASF3 gene on the chemosensitivity of gastric cancer cells and to gain insight into its mechanism of action.Methods 1.To construct gastric cancer cells with stable low and overexpression of WASF3 by using lentivirus infection.2.Applying CCK-8 method and plate clone formation assay to investigate whether WASF3 gene affects the effect of oxaliplatin on the growth and proliferation of gastric cancer cells.3.Using cell scratching assay and Transwell invasion assay to verify whether WASF3 affects oxaliplatin platinum on the invasion and migration ability of gastric cancer cells.4.To investigated the mechanism of the effect of WASF3 on oxaliplatin on gastric cancer cells,Western blot was used to detect the expression of EMT-related proteins E-cadherin and N-cadherin.5.Using comet assay to further verify whether WASF3 affects DNA damage by oxaliplatin on gastric cancer cells.Result 1.The results of CCK8,plate clone formation assay,cell scratch assay and Transwell invasion experiments showed that the inhibitory effects of oxaliplatin on the cell growth,proliferation,invasion and migration of gastric cancer MGC803 cells were further increased after inhibition of WASF3 expression.However,after making WASF3 overexpression,the effects of cell growth,proliferation,invasion and migration of oxaliplatin on gastric cancer MKN45 cells were decreased.2.Western blot results showed that after inhibition of WASF3 expression,the expression levels of E-cadherin were significantly increased compared to the control group,while the expression levels of N-cadherin were significantly decreased compared to the control group;when WASF3 was made to be overexpressed,the expression levels of both Ecadherin expression levels were both significantly decreased compared with the control group,while N-cadherin expression levels were significantly increased compared with the control group.3.The results of the comet assay showed that the degree of DAN damage in gastric cancer cells induced by oxaliplatin increased when the expression of WASF3 was inhibited,and the degree of DNA damage in gastric cancer cells induced by oxaliplatin was attenuated when WAFSF3 was made to be overexpressed,Conclusion WASF3 may reduce DNA damage by increasing the occurrence of EMT,thereby inhibiting the effects of oxaliplatin on the cell growth,proliferation,invasive and migration of gastric cancer cells.Together with the above experimental results,this study confirmed that high expression of WASF3 was associated with poor prognosis of gastric cancer.WASF3 promoted the ability cell growth,proliferation,as well as invasion and migration of gastric cancer cells,and WASF3 can resisted the effect of oxaliplatin on gastric cancer cells.Mechanistic exploration suggested that WASF3 may affect the binding of p110 to p85,which in turn activates the PI3K/AKT/NFκB signaling pathway to promote EMT,thereby promoting the growth proliferation and invasion and migration of gastric cancer cells.WASF3 may also reduce DNA damage by increasing the occurrence of EMT,thus inhibiting the effects of oxaliplatin on gastric cancer cells. |
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