| Part I The construction and identification of siRNA CLIC1 recombinant lentivirus vectors and stable siRNA CLIC1 gastric cancer cell linesObjectivesTo construct siRNA CLIC1 recombinant lentivirus vectors by using CLIC1-siRNA3,which was screened as the most effective segment in our preliminary work,and to construct stable and monoclonal siRNA CLIC1 gastric cancer cell lines.Q-PCR was used to select the siRNA CLIC1 gastric cancer cell line with the highest inhibition rate.MethodsWe selected CLIC1siRNA3 as the most effective segment,which is identified by our preliminary experiment.By using gene recombination technology,we transfected prepared double-stranded DNA into lentiviral vector GV115,and verified the recombined lentiviral vector using the method of double enzyme digestion and DNA sequencing.The recombined lentiviral vector containing CLIC1 siRNA was cotransfected with the lentiviral packaging plasmids mixture into 293T cells.Then the virus particles were collected,and virus titer was tested using hole dilution method.In addition,negative sequence virus purchased from Shanghai Ji Kai company was used as negative control.Human gastric cell lines SGC-7901and MGC-803 were infected with the virus,and then 6 groups of cell cloning clusters(SGC-7901and MGC-803,respectively)with GFP strong expression were used to establish two stable monoclonal cell lines.Fluorescence quantitative PCR(Q-PCR)was used to test the CLIC1 inhibition rate,and the cloning group with the best inhibition rate was identified.ResultsAfter sequencing,RNA interference recombinant lentiviral expression vector targeting CLIC1 was established successfully.The recombined lentiviral particles were collected by using 293T cells packaged with recombined lentiviral vector,and the virus titer was 1.2E+9 TU/mL.The infection efficiencies of recombined lentiviral particles for gastric cancer lines were all higher than 80%.Six groups of stable monoclonal cell lines with CLIC1 low expression were acquired,and the group with the best inhibition rate was identified by Q-PCR.The inhibition rate in gastric cell lines SGC-7901 and MGC-803 was 89.3%and 93.8%,respectively.ConclusionRNA interference recombinant lentiviral expression vector targeting CLIC1 was established successfully.Stable gastric cancer cell lines with CLIC1 low expression were acquired.Which provides a stable vector for further study of the relationship between gastric cancer and CLIC1 gene.Part ? Roles of siRNA-mediated inhibition of CLIC1 expression on the biological behaviors of gastric cancer cellsObjectivesTo explore the effect of silencing CLIC1 by siRNA interfence on the the biological behaviors of gastric cancer cells.MethodsProliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry.PI single-staining was used to observe cell cycle.Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.ResultsCompared with control group and negative control group,the absorbance value(24h,48h,72h,96h and 120h)of knock down group significantly lower after transfection(P<0.05).At 24h,48h,72h,96h and 120h after transfection,cell proliferation rate in SGC-7901 knock down group was 27.829%,52.663%,71.785%,28.778%and 20.450%,respectively;and in MGC-803 knock down group was 16.91%,42.169%,127.244%,101.388%and 34.118%,respectively.Compared with control group and negative control group,the apoptosis rate was significantly higher in knock down group(SGC-7901%:0.743±0.031 vs 0.587±0.025 vs 0.597±0.032,F=26.628,P<0.01;MGC-803%:4.370±0.125 vs 1.453±0.166 vs 1.510±0.079,F=506.432,P<0.01).The ratio of G0/G1 phase in SGC-7901 knock down group was significantly lower than control group and negative control group(F=172.299,P<0.01);but the ratio of G2/M phase was significantly higher(F=2665.338,P<0.01).The cell cycle in MGC-803 was the same with that in SGC-7901.In Polycarbonate membrane transwell chamber and Matrigel tests,the number of penetrating cells in SGC-7901 knock down group and MGC-803 knock down group was significantly less compared with control group and negative control group(P<0.05).ConclusionThe inhibition of CLIC1 expression effectively enhanced gastric cancer cell proliferation and increased gastric cancer cell apoptosis in vitro.Inhibiting CLIC1 gene expression results in G2/M phase arrested in cell cycle in gastric cancer cell.Inhibiting CLIC1 gene expression suppresses gastric cancer cell invation ability and migration ability in vitro.Our data suggests that the specific down-regulation of CLIC1 by RNAi is a promising gene therapy approach for the treatment of gastric carcinoma.Part ? Screening the differentially expressed protein of gastric cancer cell line SGC-7901 by inhibiting the expression of CLIC1ObjectiveTo screen the differentially expressed protein of gastric cancer cell line SGC-7901 with the inhibition of CLIC1 based on the technique of iTRAQ combined with mass spectrometric analysis.MethodsTotal protein was extracted and received enzymatic hydrolysis.iTRAQ marker,mass spectrometry and searching of database were used to screen eligible proteins.The criteria were that the ratio of 114/115 was more than 2.0 or less than 0.5.And then bioinformatic analysis was performed for the differentially expressed protein.ResultsTotally 1170 protein peptides were identified in Human database,including 54 differential proteins.In the differential proteins,36 were high expressed,and 18 were low expressed.Totally 858 protein peptides were identified in Mouse database,including 39 differential proteins.In the differential proteins,24 were high expressed,and 15 were low expressed.The differential proteins were mainly intracellular,with the main functions of conjugation and biological regulation.ConclusionDifferential proteins were identified successfully,which provided clues and basis for exploring the action mechanism of CLIC1 in gastric cancer.Part ? Roles of siRNA-mediated inhibition of CLIC1 expression on the integrin and apoptosis genes and proteins of gastric cancer cellsObjectiveTo evaluate the expression of apoptosis and integrin genes and proteins by inhibiting CLIC1 in gastric cancer cells.MethodsFluorescence quantitative PCR and Western blot were used to test the expression of apoptosis(Bcl-2,survivin,Fas)and integrin(?1,?3,?v,?1)genes and proteins.ResultsThe expression of integrin gene and protein(a3,?v,?1)were decreased significantly in two knock down groups(SGC-7901:70.924%,35.491%,55.209%and MGC-803:46.729%,53.765%,70.935%)(P<0.05).The expression of integrin al gene and protein were upregulated significantly in SGC-7901 knock down groups,but decreased significantly in MGC-803(P<0.05).The expression of Bcl-2 gene and protein were both decreased significantly in SGC-7901 and MGC-803 knock down group(mRNA:65.200%and74.725%,protein:60.023%and 54.023%)(P<0.05),but the expression of Fas gene and protein were both upregulated significantly(mRNA:97.702%and 173.826%,protein:83.091%and 76.935%)(P<0.05).The expression of survivin gene and protein did not change significantly(P>0.05).ConclusionBased on the level of gene and protein,we found that integrin ?3,?v,?1 were down-regulated in gastric cancer cells SGC-7901 and MGC-803,and integrin al was up-regulated in SGC-7901,but down-regulated in MGC-803 after silencing CLIC1 expression.Combined with the roles of integrins in tumor,we conclud that the silence of CLIC1 can affect the progression of gastric cancer by regulating the expression of integrins.By inhibiting the expression of CLIC1,the expression of Fas was up-regulated,Bcl-2 was down-regulated and Survivin had no change in gastric cancer cell line SGC-7901 and MGC-803.Combining with the experimental results of the part ?,we concluded that the expression of Fas and Bcl-2 could promote apoptosis of gastric cancer cells after silencing CLIC1 expression. |
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