| Objective1. To selectively construct of three specific adenovirus expression vector ofRNA interference for CXCR4and negative control. Specific silencing CXCR4ingastric cancer cell line SGC7901and test the efficiency.2. To investigate the effect of RNAi on proliferation and invasion of gastriccancer line SGC7901by specific inhibiting CXC chemokine receptor type4expression in gastric cancer line SGC7901.Method1. Retrieved CXCR4gene sequence in the GENBANK, designed four shorthairpin RNA (shRNA) and synthetized shRNA after homology search withouterror. Constructed recombinant adenovirus vector shpAd-hCXCR4-eGFP. Therecombinant adenovirus plasmid was ultra pure extracted by getting rid ofendotoxin. HEK293A cell was transfected by means of liposome Lipofectamine2000, through the eGFP gene green flourescent expression to detecttransfection situation and packaged virus, virus titer was ermined by end-pointdilution method. With the optimal multiplicity of infection (MOI)to transfectgastric cancer cell SGC7901, RT-PCR and Western-blot were used to detect thehighest silence efficiency group.2. With optimal MOI to transfect experiment group, negative control groupand set up blank control group, RT-PCR and Western-blot were used to detect CXCR4mRNA and protein expression in gastric cancer cells after CXCR4genewas silenced by recombinant adenovirus vector. MTT method drew SGC-7901cell growth curve, and detected the inhibition effect of recombinant adenovirusvector on proliferation of gastric carcinoma cell SGC-7901; Transwell chamberwas used to detect cancer cell migration and invasion ability, obtain theinfluence on gastric cancer cell SGC-7901invasion of recombinant adenovirusvector.Result1.Gene sequencing, PCR detection and enzyme digestion showed thatrecombinant adenovirus expression vector shpAd-hCXCR4-eGFP wasconstructed successfully.2.Inverted fluorescence microscope found that inside HEK293A cells oftransfection of recombinant adenovirus expression vector and its positive,negative control had a large number of green fluorescent expressed andobvious cytopathic effect (cytopathic effect, CPE). Adenovirus was packagedsuccessfully,the test resultof virus titers was4x109PFU/ml. The best MOI=100,the optimal transfection volume of adenovirus vector was12.5L.3. After the shpAd-hCXCR4-eGFP was transfected into gastric cancer cellSCG-7901, results of RT-PCR and Western blot detection showed that: inCXCR4-shRNA1group, CXCR4mRNA and protein expression weresignificantly lower than the other two groups with significant difference (P <0.05); the difference between three groups of experimental group and thecontrol group, the negative control group was significant (P <0.01); there wasno significant difference between the blank group and negative control group (P>0.05). CXCR4-shRNA1group silence efficiency was the highest,about78.08%.4.After the CXCR4-shRNA1was transfected into gastric cancer cellSGC-7901, via MTT method to draw growth curve, the results show that: the blank group and the control group cells grew rapidly, the difference between thattwo groups had no significant (P>0.05), the experimental group aftertransfected with adenovirus vector, the degree of cell proliferation decreasedsignificantly, and compared to the former two groups, there was significantdifference between them(P <0.05).5. Cell invasion experiment results show that: in the SDF-1Chemotaxis24h,the transmembrane cell number in blank group, negative control group and theexperimental group were264.7+15.7,270.5+16.9,113.8+9.2, comparedshRNA-CXCR4adenovirus vectors to blank group and the control group, thetransmembrane cell number significantly decreased, difference between themwas significant (P <0.05). And the transmembrane cell number in blank groupand control group didn’t obviously decrease, no significant difference betweenthat two groups(P>0.05).Conclusion1.CXCR4-shRNA adenovirus expression vector was constructedsuccessfully and the highest scilence efficiency group was screened out.2.CXCR4-shRNA recombinant adenovirus vector was transfected intogastric cancer cell SGC-7901successfully and it demonstrated thatrecombinant adenovirus vector CXCR4-shRNA can inhibit CXCR4gene mRNAand protein expression in gastric cancer cell strain SCG-7901.3.Recombinant adenovirus vector CXCR4-shRNA can significantly inhibitthe proliferation of gastric cancer cell SGC-7901.4.Recombinant adenovirus vector CXCR4-shRNA can reduce SGC-7901gastric cancer cell’s invasion force. |
