The Experimental Study On Co-silencing Of XT-Ⅰ Gene And XT-Ⅱ Gene In Salivary Adenoid Cystic Carcinoma

Objectives:Salivary adenoid cystic carcinoma (SACC) is one of the most commonsalivary malignant tumors with the tendency of invasion, metastasis, andrecurrence. More ideal therapeutic measures for this neoplasm of poorprognosis are desired yet.Proteoglycans (PGs) secreted from neoplastic myoepithelial cells (NMCs)in SACC contribute to extracellular matrix (ECM). Abundant PGs maketypical cribriform structures full with proteoglycans and provide nutrition andmacromolecules microenvironment for the biological behaviors of SACC,including surviving, proliferation, differentiation, invasion, metastasis, andrecurrence. Serving as an indispensible material basis of morphologicalcharateristics and biological behaviors, PGs are believed to be involved intumorigenesis and development of SACC.As the initiative and rate-limited enzyme of PGs biosynthesis in vivo,human xylosyltransferases (XT) catalysis the rate-limited step in thebiosynthesis. Accordingly, the activity of XT represents the biosynthetic rateof PGs literally. XT-I and XT-II, two isoforms of XT, with unequal expressionin various tissues, play an identical catalytic role.RNA interference (RNAi) is recently regarded as the most efficient genesilencing method for its high specificity and high performance as well inblocking particular target gene expression. Adenoviral vector is generallytaken as a common medium to deliver exogenous gene to intra-cellularcircumstance. In addition, an adenoviral vector expressing multiple targetgenes may maximize availability of a vetor.In present study, to silence XT-I and XT-II simultaneously, an interferingplasmid encoding two shRNAs was designed, and then the corresponding recombinant adenoviral vector rAd5-shRNA-WJ7+WJ4was constructed.After SACC-83cell infection by the recombinant adenoviral vector, XT-I andXT-II mRNA level and PGs content were detected to assess silencingeffectiveness. SACC-83tumor-burdened mice were established prior tointra-tumor injection of recombinant adenoviral vector and followingobservation for apoptosis. These experiments were undertaken to investigatethe regulation of XT-I and XT-II on PGs in SACC and the effect ofco-silencing on apoptosis, so as to provide more evidence for biotherapy ofsalivary myoepithelial tumors.Methods:1Construction of two recombinant interfering plasmids respectively targetinghuman XT-I gene and human XT-II geneThe interfering sites and primer sequences of plasmids namedshRNA-WJ4and shRNA-WJ7were designed and synthesized to suppresshuman XT-I gene and human XT-II gene, which was followed by respectiveconjunction to linearized vectors of pGenesil-1.2and pGenesil-1.1. Throughplasmid amplification and digestive identification, the obtaining ofrecombinant plasmids, pGenesil-1.2-shRNA-WJ4andpGenesil-1.1-shRNA-WJ7, were determined.2Construction of recombinant co-silencing plasmid simultaneously targetinghuman XT-I gene and human XT-II geneFollowing double digestion of both plasmids above, fragments wererecovered and connected. Plasmid amplification, digestive identification, andsequencing were performed to verify the recombinant co-silencing plasmid,pGenesil-1.1+2-shRNA-WJ7+WJ4.3Preparation of recombinant adenoviral plasmidThe expressing box shRNA-WJ7+WJ4was transferred from plasmidpGenesil to adenoviral expressing vector pGSadeno by LR homologousrecombination in vitro, plasmid amplification and digestive identification wasoperated then.4Enclosing of recombinant adenoviral vector HEK293cells were tranfected by the connector of linearized adenoviralDNA with recombinant adenoviral plasmid fragments, and co-silencingrecombinant adenoviral vector rAd5-shRNA-WJ7+WJ4was obtained andamplified.5Synthesis of recombinant adenoviral vector rAd5-shRNA-HK as negativecontrol6Infection of SACC-83cells by recombinant adenoviral vectorsSACC-83cells were cultivated and divided into3groups: groupSACC-83-WJ7+WJ4(silencing group), group SACC-83-HK (blank vectorgroup), and group SACC-83(non-infection group). The best MOI (100) andthe moment of the best fluorescence (48h after infection) were determined，and infection rate was calculated.7Detection of XT-I and XT-I mRNA level (48h after infection)Total RNA extraction by TRIzol was followed by determination of purity,concentration, and integrity. After RT-PCR and Real-Time PCR in turn, therelative quantity of XT-I and XT-I mRNA in all groups and then silencing ratewere acquired.8Determination of PGs content in culture media of all groups (48h afterinfection)Based on the standard curve of concentration-optical density usingstandard substance, PGs content was determined by bio-staining andinhibitory rate was measured.9Establishment of tumor-burdened mice and groupingThirty-two four-week-old female BALB/c-nu nude mice,14~15g, wereraised under SPF condition.250μl cell suspension containing2.5×106SACC-83cells was injected subcutaneously at the left back of each animal. Atthe62nd day after injection of cell suspension, twenty-four tumor-burdenedanimals with mass of long diameter more than1cm were internalized anddivided, in accordance with the random number table, into3groups of8miceeach: group SACC-83-WJ7+WJ4(silencing group), group SACC-83-HK(blank vector group), and group SACC-83(non-infection group). 10Intra-tumor injection of recombinant adenoviral vectorsThe sixty-second day after inoculation of tumor cells，the first intra-tumorinjection of recombinant adenoviral vectors was performed, includingco-silencing adenoviral vector rAd5-shRNA-WJ7+WJ4for groupSACC-83-WJ7+WJ4(silencing group), negative control adenoviral vectorrAd5-shRNA-HK for group SACC-83-HK (blank vector group), and PBS forgroup SACC-83(non-infection group).4×109PFU/200μl was injected foreach animal. Such injection was taken once a week for5times totally.11Sample collectionIn the fifth week after the first intra-tumor injection,3groups oftumor-burdened mice were killed. Each mass was separated and measured forthe volume. Following instant quartering dissection of fresh neoplastic tissue,a quarter was preserved at80C, the others were used in the experimentsbelow.12Sample preparation for flow cytometry (FCM) and detection of apoptosisby FCM70%Ethanol-fixed specimens were sheared and rubbed to obtain singlecell suspension, which underwent propidium iodide (PI) staining subsequently.apoptosis of three groups were detected by FCM.13Preparation and observation of formalin-fixed/paraffin-embeddedspecimensFormalin-fixed/paraffin-embedded specimens were prepared by ordinaryprocedures, sections were stained with hematoxylin and eosin (HE), and thenexamined microscopically to evaluate morphological changes.14Preparation and observation of ultrathin section for transmission electronmicroscopySpecimens less than1mm3were successively fixed in4%glutaraldehydeand1%osmic acid, embedded in epoxy resin, sectioned into ultrathin slices,stained by uranium acetate and lead citrate respectively30min, and finallyexamined by transmission electron microscope (TEM) to evaluate cell1apoptosis. Results：1Designed siRNA sequences respectively targeting XT-I, XT-II, and negativecontrolshRNA-WJ4CAGGCAGCCCATCAAACCTshRNA-WJ7CGTCTCCTCAAGGCCGTTTATshRNA-HK GACTTCATAAGGCGCATGC2Conbinant plasmids were proved correct by enzyme digestion, agarose gelelectrophoresis, and sequencing.3Infection efficiency of SACC-83cells by recombinant adenovirus48h after recombinant adenovirus infection, according to expression ofgreen fluorescent taken as successful infection, the infection rate of groupSACC-83-WJ7+WJ4(silencing group) and group SACC-83-HK (blank vectorgroup) was98.7%and99.1%respectively.4Detection of XT-I and XT-II silencing effect by Real-Time PCR48h after infection, the relative expression of XT-I mRNA and XT-IImRNA in group SACC-83-WJ7+WJ4(silencing group) considerablydecreased compared with those in group SACC-83-HK (blank vector group)and group SACC-83(non-infection group), no difference was found betweengroup SACC-83-HK (blank vector group) and group SACC-83(non-infectiongroup). Silencing rate of XT-I and XT-II in group SACC-83-WJ7+WJ4(silencing group) was97.3%and88.0%respectively.5Effect of SACC-83cell XT-I and XT-II gene co-silencing on PGs synthesisand secretion48h after infection, PGs content of culture media from groupSACC-83-WJ7+WJ4(silencing group) decreased compared with those fromgroup SACC-83-HK (blank vector group) and group SACC-83(non-infectiongroup), with the inhibitory rate of68.61%. PGs from group SACC-83-HK wasnot inhibited.6Final volumes of transplantation tumors from tumor-burdened miceFive weeks after the first injection of adenovirus, the final volumes oftransplanted tumors from three groups of tumor-burdened mice showed no statistical difference.7Determination of apoptosis rate of transplantation tumor cell by flowcytometry (FCM)Five weeks after the first injection of adenovirus, apoptotic rates oftransplanted tumor cells from three groups of tumor-burdened mice showed nostatistical difference.8Morphological observation of nude mice transplantation tumor by opticalmicroscopicFive weeks after the first injection of adenovirus, among all groups,neither remarkable morphological differences nor significant apoptosis werefound in HE-stained formalin-fixed/paraffin-embedded specimens oftransplantation tumors form all groups.9Evaluation of transplantation tumor cell apoptosis by transmission electronmicroscope (TEM)Five weeks after the first injection of adenovirus, apoptosis is easy to findin group SACC-83-WJ7+WJ4(silencing group), but relatively rare in theother two groups.Conclusions：1Recombinant adenovirus rAd5-shRNA-WJ7+WJ4can effectively induceXT-I and XT-II gene silencing and inhibit PGs synthesis and secretion inSACC-83cells.2Five weeks after intra-tumor injection to silence XT-I and XT-II gene, cellapoptosis arose in group SACC-83-WJ7+WJ4(silencing group), but withoutstatistical significance.