| Objective:Salivary adenoid cystic carcinoma(SACC)is one of the most common salivary gland malignant tumors.It has a strong invasive and poor prognosis.Neoplastic myoepithelial cells(NMCs)in adenoid cystic carcinoma is the main cell component of the tumor.NMCs can synthesize and secrete proteoglycan(proteoglycans,PGs),the synthesis of PGs can provides a microenvironment for the growth of the tumor,and provides directly nutritional sources and energy for malignant biological behavior,such as tumor invasion and metastasis.PGs are macromolecule glycoprotein,which is a complex of polysaccharide molecules and proteins.PGs are the main component of the matrix,and the biosynthesis needs to be carried out under the catalysis of xylosyltransferase(XT).There are two types of XT known at present,that is,XT-? and XT-?.The one of XT-? is the key starting enzyme in the efficiency of PGs synthesis,which plays a vital role in the synthesis of PGs.As a subtype of XT-?,XT-? also participate in the biosynthesis of PGs,and the similarity of the two amino acids is chose to 55%.RNA interference(RNA interference,RNAi)is a gene silencing technique.Its core is to make exogenous and endogenous double stranded RNA induce specific degradation of homologous target gene mRNA in vivo,resulting in the silence of corresponding genes after transcription.In this experiment,RNAi technology was used to silence XT-? gene and XT-? gene respectively,in order to inhibit proteoglycans(PGs)produced by myoepithelial cells in SACC,and then the inhibition efficiency of PGs after two genes silenced was compared.Methods:1.Construction of recombinant adenovirus vector for XT-? gene andXT-? gene(Ad-shRNA-XT-?,Ad-shRNA-XT-?)with adenoid cystic carcinoma cells,divided into silence group(SACC-83-XT-?),silence group(SACC-83-XT-?),empty vector group(group SACC-83-HK),non transfection group(group SACC-83).2.Detection of XT-? and XT-? gene silence efficiency: Real-time PCR technique was used to detect the expression of XT-? and XT-? gene in SACC-83 cells after gene silenced at mRNA level.3.Detection of the inhibition rate of XT-? and XT-? protein: The inhibition rate of each protein was detected by Western blot.4.Determination of PGs content: After Blyscan Assay Kit was used to detect gene silenced 48 h,the changes of PGs synthesis and secretion in SACC-83-XT-? cells,SACC-83-XT-? cells,SACC-83-HK cells and SACC-83 cells were measured respectively,and the efficiency of PGs inhibition was compared statistically.Results:1.One percent of agarose gel result and the results of sequencing analyses showed that Ad-shRNA-XT-? and Ad-shRNA-XT-? were constructed successfully.Forty-eight hours after the transduction of Ad-sh RNA-XT-?,Ad-shRNA-XT-? and Ad-sh RNA-HK into SACC-83 cells,the transduction rate of green fluorescent protein(GFP)in both groups were 91.71%,93.05%,94.10%.There was no expression of GFP in SACC-83 cells without any transduction.2.Real-time PCR showed that XT-? gene was silenced by 64% in group SACC-83-XT-? and XT-? gene was silenced by 54% in group SACC-83-XT-?.There were no XT-? and XT-? gene silenced in group SACC-83-HK.The relative expression of XT-? mRNA and XT-? mRNA compared with that in group SACC-83-HK and SACC-83 was different significantly(P<0.05).The relative expression of XT-? gene compared with XT-? gene was significantly different(P<0.05).3.GeneTool from Syngene software was performed in this study.The results showed that the relative expression of XT-? protein was 0.34±0.06,0.68±0.16 and 0.72±0.17 in group SACC-83-XT-?,group SACC-83-HK and group SACC-83 respectively.The relative expression of XT-? protein was0.49±0.05,0.77±0.09 and 0.80±0.08 in group SACC-83-XT-?,group SACC-83-HK and group SACC-83 respectively.The XT-? protein was decreased by 38% in group SACC-83-XT-? and the XT-? protein was decreased by 31% in group SACC-83-XT-?.There is no decrease of protein in SACC-83-HK.4.The content of PGs(the total GAGs)in four groups were tested according to the standard curve.PGs inhibitory rate in group SACC-83-XT-? was 49.69%(68.33±4.75 ?g)compared with group SACC-83(135.81±10.15?g).PGs inhibitory rate in group SACC-83-XT-? was 36.47%(86.28±5.67 ?g)compared with group SACC-83(135.81±10.15 ?g)and PGs inhibitory rate in group SACC-83-HK was 6.79%(126.58±9.21 ?g)compared with group SACC-83(135.81±10.15 ?g).One-Way ANOVA showed that group SACC-83-XT-? and group SACC-83-XT-? were significantly different compared with group SACC-83-HK and group SACC-83(P <0.05).group SACC-83-XT-? was significantly different compared with group SACC-83-XT-?(P < 0.05).PGs were no inhibited significantly in group SACC-83-HK(P>0.05).Conclusion:1.Both XT-? gene or XT-? gene was silenced,the biosynthesis of proteoglycans in SACC-83 cells could be suppressed significantly.2.XT-? gene was silenced separately,the inhibition(49.69%)of proteoglycan synthesis was higher than that(36.47%)of XT-? gene silenced separately. |
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