| Background:Gastric cancer(GC)is a malignant tumor with high morbidity and mortality worldwide,especially in China.The death rate of GC in China accounts for about 45% of all deaths worldwide.Although the morbidity and mortality of GC have decreased in the past decades,it is still a great threat to the health of people.Currently,an increasing number of studies have been focused on the occurrence and development of GC,and the diagnosis and treatment methods of GC have also been greatly improved.However,the mechanism of invasion,growth and metastasis of GC still remains unclear.In recent years,more and more studies have been reported on the involvement of adhesion molecules in malignant tumors,and the researches with respect to adhesion molecules involved in the progress of GC have become a hot spot.Vinculin is a cytoplasmic actin binding protein,which is enriched in cell-cell and cell-matrix adhesion.It participates in many physiological processes,such as cell adhesion,extension,migration,cell morphology maintenance,signal transduction and so on.Previous studies showed that Vinculin is abnormally expressed in many highly malignant and metastatic tumors,or is involved in other regulatory pathways that affect the occurrence and development of malignant tumors.However,the role of Vinculin in GC has not been fully clarified.It has been reported that celecoxib can inhibit the occurrence and development of GC by inhibiting the expression of Vinculin,which indirectly suggests that Vinculin can promote the occurrence and development of GC.Therefore,it is necessary to further explore the role of Vinculin in the occurrence and development of GC.Objectives:The expression of Vinculin in GC tissues and its relationship with clinicopathological parameters including prognosis,age,sex,tumor differentiation,TNM stage and tumor size were studied in this paper,as well as the impact o prognosis.In vitro experiments were further conducted to investigate the effects of silencing or overexpressing Vinculin on cell viability,apoptosis and migration of GC or normal mucosal cells.In addition,we explored the possible upstream regulationary mechanism of Vinculin.Methods:Part 1 Expression of Vinculin in GC tissues and paracancerous tissues1.From October 2012 to October 2015,78 cases of GCs and 56 cases of non-tumor paraffin sections of paraffin-embedded tissues were collected in Qingdao University affiliated Municipal Hospital.In addition,12 cases of fresh surgical excision and matched non-tumor tissues were collected.The patient’s name,sex,age,family history,pathological reports,surgical records and other clinical data were collected and recorded.2.Immunohistochemistry(IHC)was used to detect the expression of Vinculin in normal gastric mucosa and GC tissue sections.Semi-quantitative scoring method was used to determine the expression of Vinculin by adding up the staining intensity and the proportion of stained cells.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect the expression of Vinculin in GC and adjacent tissues.The patient’s 3-year medical records and the survival time were collected,thereafter,the survival analysis curve was m drawn.Part2 The expression and function of Vinculin in GC cell lines in vitro.1.The expression of Vinculin in three GC cell lines BGC-823,SGC-7901 and normal gastric mucosa cells GES-1 was detected by qRT-PCR and Western blot.2.Vinculin was silenced in SGC-7901 and BGC-823 cells by transient transfection of siRNA.Cell viability,apoptotic rate and migration were detected by 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide salts,flow cytometry and scratch test,respectively.3.Western blot was used to detect the expression of Cyclin D1,p21,Bcl-2,Bax and Caspase-3,and the expression of matrix metalloproteinase(MMP)2 and 9 in GC cells after transfection with si-Vinculin.4.An overexpression plasmid of Vinculin was transfected into into normal gastric mucosal cells GES-1.The effects of overexpression of Vinculin on cell viability,apoptosis and migration were espectively examined by 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide salts,flow cytometry and scratch test.Western blot was performed to detect the expression of Cyclin D1 and p21,Bcl-2,Bax and Caspase-3,and MMP2 and MMP9.Part 3 The regulatory relationship between miRNA-590-5p and Vinculin in GC cells.1.The expression of miR-590-5p in GC tissues and adjacent tissues was detected by qRT-PCR.2.qRT-PCR was used to detect the expression of miR-590-5p in GC cell lines BGC-823,SGC-7901 and HGC-27 and normal gastric epithelial cells GES-1.3.Luciferase assay was used to verify the targeting relationship between miR-590-5p and Vinculin.4.In BGC-823 cells,miR-590-5p mimic and miR-590-5p inhibitor were transfected into BGC-823 cells to overexpress or knockdown miR-590-5p.The effects of silence or overexpression of miR-590-5p on the expression of Vinculin were detected by qRT-PCR and Western blot.5.Silencing the expression of Vinculin and miR-590-5p in BGC-823 cells was simultaneously transfected with si-RNA and miR-590-5p inhibitor.Thereafter,the cell viability and apoptosis rates of BGC-823 cells were detected by 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazole bromide method and flow cytometry,respectively.Results:Part 1 Elevated expression of Vinculin in GC and its relationship with clinical characteristics and prognosis of GC1.IHC showed that the positive staining of Vinculin protein was mainly located in the nucleus,lesser shown in cytoplasm,brown and yellow granules.In GC tissues,61.54%(48/78)showed positive expression and 38.46%(30/78)negative expression,while in normal gastric mucosa,25%(14/56)showed positive expression and 75%(42/78)negative expression,with a significant difference was(P = 0.001).mRNA and protein levels of Vinculin were significantly higher in GC tissues than those in adjacent groups.In addition,we found that Vinculin was not significantly correlated with sex(P = 0.937)age(P =0.884),size(P = 0.817),M stage(P = 0.297)and differentiation(P = 0.539)but correlated with T stage(P = 0.027)and N stage(P = 0.007).2.Survival curve analysis.The 3-year survival rate of patients with negative expression of Vinculin was 60.0% and the average survival time was 30.7 months.The3-year survival rate of patients with positive expression of Vinculin was 37.5% and the average survival time was 25.1 months.The average survival rate of patients with Vinculin positive expression was significantly shorter than that of patients with negative expression(Log-rank test,P = 0.0364).Part 2 Vinculin expression was increased in GC cells and promoted proliferation and migration of GC cells1.Vinculin was significantly increased in BGC-823 cells(P < 0.01),SGC-7901 cells(P < 0.001)and HGC-27 cells(P < 0.01),compared with GES-1 cells.2.Compared with the si-NC group,silent Vinculin significantly decreased the cell viability of SGC-7901 and BGC-823 cells at 24 hours,48 hours and 96 hours.SilencingVinculin could significantly increase apoptosis and decrease cell migration of SGC-7901 and BGC-823 cells.3.Silencing Vinculin could significantly reduce the expression of Cyclin D1,while significantly increase the expression of p21 in SGC-7901 cells.In addition,silencing Vinculin could significantly reduce the expression of Bcl-2,MMP-2 and MMP-9,and significantly increase Bax expression and the cleavage of Caspase-3.4.Overexpression of Vinculin in GES-1 cells could significantly promote the viability and migration of GES-1 cells,but have little effect on cell apoptosis.Part 3 The effect of silencing Vinculin on GC cells was regulated by miR-590-5p1.Compared with normal gastric tissues,miR-590-5p was significantly decreased in GC cells(P < 0.01).2.Compared with human gastric normal mucosal epithelial cell GES-1,the levels of miR-590-5p in BGC-823(P < 0.01),SGC-7901(P < 0.01)and HGC-27(P < 0.05)were all significantly lower.3.The results showed that the 283-289 3’ Untranslated Region(UTR)region of Vinculin was combined with miR-590-5p.Luciferase assay confirmed that Vinculin was one of target genes of miR-590-5p.miR-590-5p mimic could bind to Vinculin gene to reduce the expression of luciferase,while the mutated Vinculin gene could not been bound.4.Overexpression of miR-590-5p significantly reduced the mRNA and protein expression of Vinculin,and while inhibition of showed the opposite effects.5.Silencing of miR-590-5p could significantly reverse the inhibitory effect of silencing Vinculin on the cell viability of BGC-823(P < 0.05)and significantly reduce the promoting effect of silencing Vinculin on BGC-823 cell apoptosis(P < 0.01).Conclusion:1.Vinculin is highly expressed in GC and is correlated with clinical parameters including T stage,N stage and prognosis.2.Vinculin can significantly promote cell viability and migration.3.The role of Vinculinin gastric cancer may be regulated by miR-590-5p. |
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