| Objective:(1)Construct lentiviral vector harboring RNAi sequence targeting dynamin-related protein 1(DRP1)gene and screening cell line of stable expression shRNA-DRP1.(2)Investigate the effects and mechanisms of DRP1 gene silencing on proliferation,migration,invasion,apoptosis and cell cycle of human gastric carcinoma cell lines BGC-823 and MKN-45 in vitro and in vivo.(3)Detect the differential expression of DRP1 in gastric adenocarcinoma and explore its relationship with clinicopathologic characteristics and prognosis in gastric adenocarcinoma.Methods:(1)In order to screen the highest cell line in DRP1mRNA expression,we used qPCR to test the difference in DRP1mRNA expression among normal gastric mucosal epithelial cell GES-1,human gastric carcinoma cell lines MKN-28,SGC-7901,AGS,BGC-823 and MKN-45.Design three pairs of shRNA sequences(KD1,KD2,KD3)targeting DRP1 gene,synthesis two chains of complementary single stranded DNA encoding shRNA sequence,single stranded DNA annealed to form double stranded DNA(dsDNA)and connected to enzyme-digested lentiviral vector.The products were transformed into competent Escherichia coli(E.coli)DH5a,positive clones were screened by PCR and identified by DNA sequencing in order to check whether the insertion sequence were correct.The obtained recombinant plasmid were packaged by co-transfecting 293T cells with the help of other two plasmids,transfection efficiency was assessed by observing the expression of green fluorescence protein GFP under the inverted fluorescence microscope.Lentivirus titre was determined by dilution method of counting method.shRNA-DRP1 lentiviral vector infected MKN-45 and qPCR was carried out to detect DRP1 expression on the level of mRNA to screen out the shRNA sequence with the highest efficiency.(3)The previously tested shRNA-DRP1 lentiviral vector was transfected BGC-823 and MKN-45,set NC(transfectedempty vector)and CON(no treatment)groups as control.MTT assay,clone formation assay,scratch assay,transwell invision assay and flow cytometry(FCM)were applied to detect the changes in proliferation,migration,invasion,apoptosis and cell cycle,respectively.To further investigate the mechanisms involved in DPR1 promotes cell proliferation,western blot was used to detect the expression of DRP1,Cleaved caspase-3,p-cdk1(Tyr15),Cyclin B1 and autophagy marker LC3.Finally,to evaluate the effect of DRP1 on growth of gastric cancer cells in vivo,MKN-45carringshRNA-DRP1lentiviralvectorwas subcutaneous inoculation into the nude mouse to develop an subcutaneous xenotransplanted tumor model,volumes of planted subcutaneous tumors were measured at8 d,11 d,14 d,18 d and 21d after post inoculation and then make tumor growth curves,nude mouse were executed at 21d,then tumors was removed and weighed up.(3)DRP1expression was detected by immunohistochemistry technique in 291 specimens of gastric adenocarcinoma and matched 145 specimens of non-neoplastic adjacent tissues and non-neoplastic distant tissues.The related data were analyzed by Kaplan-Meier and multivariate COX regression model in order to make sure the relationship of DRP1expression with clinicopathologic features and prognosis in gastric adenocarcinoma.qPCR was used to test DRP1mRNA expression in 10 fresh gastric adenocarcinoma specimens,matched non-neoplastic adjacent tissues and non-neoplastic distant tissues.Results:(1)DRP1mRNA levels in MKN-45,BGC-823,AGS,SGC-7901,MKN-28and GES-1 were decreased gradually(P<0.001).PCR and DNA sequence assays showed that the obtained recombinant plasmid DNA sequence were consistent with DRP1 cDNA sequence in Gen Bank,this means the recombinant plasmid was constructed successfully.After the recombinant plasmid transfected 293T cells,a large amount of green fluorescence was found under the inverted fluorescence microscope,and reached its peak 72 h after transfection,indicating that the lentivirus packaged successfully.Dilution method of counting method found that the lentivirus titre in LV-shRNA-DRP1(KD1),LV-shRNA-DRP1(KD2)and LV-shRNA-DRP1(KD3)group were 1×10~9 TU/mL,4×10~9TU/m L and 8×10~8 TU/m L,respectively.The transfection efficiency of lentiviral vector harboring shRNA-DRP1 infect MKN-45 cell was greater than 80%,qPCR detection showed that DRP1mRNA level in KD group was remarkably below than that in CON and NC group,indicating that shRNA-DRP1 lentiviral vector had silenced DRP1 gene effectively,LV-DRP1-RNAI(KD3)group possess the highest silencing efficiency up to 92.7 percent.(2)MTT assay revealed that OD490 and OD490/fold on day 4 and day 5 in KD group were remarkably lower in contrast with CON and NC group(P<0.001);clone formation assay revealed that the colonies counting in KD group were remarkably lower in contrast with CON and NC group(P<0.001),suggesting that DRP1 gene silencing could suppress BGC-823 and MKN-45 proliferation.Scratch assay revealed that scratch healing rate in KD group were greatly diminished than that in NC group(P<0.01);transwell invision assay revealed that numbers of cell permeating septum in KD group were greatly reduced than that in NC group(P<0.01),suggesting that DRP1 gene silencing could remarkably inhibit BGC-823 and MKN-45 migration and invasion.FCM assay revealed that in contrast with NC group,apoptosis rate in KD group was rised significantly(P<0.01),percentage of cells at G2/M phase was increased(P<0.001)and percentage of cells at G1 and S phase were decreased significantly in KD group,indicated that DRP1gene silencing could cause apoptosis and G2/M arrest in BGC-823 and MKN-45.Western blot analysis showed DRP1 and Cyclin B1 were down-regulated(all P<0.001)and ratio of LC3-?to LC3-?decreased(all P<0.01),while,Cleaved caspase-3 and p-CDK1(Tyr15)were up-regulated after RNA interference in contrast with NC group(all P<0.001).Model of transplanted tumor on nude mouse revealed that in KD group showed a decrease both in tumor volume and weight compared with NC group(all P<0.001).(3)DRP1 was expressed in the cytoplasm presenting brown,the positive expression rate of DRP1 in non-neoplastic distant tissues,non-neoplastic adjacent tissues and gastric adenocarcinoma tissues were increased gradually(P<0.001 and P<0.05).In addition,DRP1 expression was associated with lymphatic metastasis(P<0.05)and TNM pathologic stage(P<0.05).Kaplan Meier survival analysis revealed that patients with positive DRP1 expression showed a shorter overall survival(OS)compared with those with negative DRP1 expression(P<0.01).Multivariate COX regression model revealed that age(P<0.05),the degree of differentiation(P<0.05),lymph node metastasis(P<0.01)and DRP1 positive expression(P<0.05)were the independent risk factors of gastric cancer.qPCR showed that DRP1mRNA levels in non-neoplastic distant tissues,non-neoplastic adjacent tissues and gastric adenocarcinoma tissues were increased gradually(P<0.001),Conclusions:(1)DRP1 gene silencing suppress BGC-823 and MKN-45 proliferation,migration,invasion,and inducing apoptosis by G2/M arrest,caspase-3 activation and inhibiting mitophagy.DRP1 may be a potential molecular therapeutic target for gastric adenocarcinoma.(2)DRP1 was aberrantly expressed in gastric adenocarcinoma tissues,and its positive expression rate was increased with lymph node metastasis and TNM staging progress.Additionally,aberrant expression of DRP1 is not only a poor prognostic factor but also one of independent risk factors for prognosis of patients with gastric adenocarcinoma. |
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