Experimental Studies On Silencing Of Stat3 By RNAi Induces Apoptosis Of Human Breast Cancer Cells In Vitro And In Vivo

Human breast carcinoma is one of the most frequent cancer in the femalemalignacies and deeply threatens woman health and life quality. Therefore itbecomes a very important region in studying the mechanism of breast cancerevolution, searching the novel effective methods of diagnosis and therapy andclarifying the theory basis of improving prognosis.With the completion of large-scale genome sequencing, thousands of geneshave been identified, and we know nothing about their function. Reverse geneticsis now the most efective way to assess the function of a gene, but so far there hasbeen no general method for reverse genetics other than gene targeting byhomologous recombination, which is slow and costly. Antisense approaches, suchas antisense oligonucleotide and ribozyme technologies, have been useful inreverse genetics, but only to a limited degree. As a new alternative to these reversegenetic approaches, RNA interference has attracted many researchers' attention. RNA interference(RNAi) is a sequence-specific posttranscriptional genesilencing mechanism, which is triggered by double-stranded RNA(dsRNA) andcauses degradation of mRNAs homologous in sequence to the dsRNA. RNAi wasinitially discovered in C.elegans. It is now known that RNAi operates inevolutionarily diverse organisms including plants, fungi and metazoans, and hasbecome a powerful tool for studying gene function in C.elegans. One of the majorobstacles in applying RNAi in mammalian cells is that dsRNAs longer than 30ntwill provoke the activation of dsRNA-activated protein kinase PKR, which causesnonspecific inhibition of gene expression. In 2001, Tuschl and his colleaguesinitially reported that chemically synthesized 21 nt siRNA(small interfering RNA)duplexes specifically suppress the expression of endogenous and heterologeousgenes in different mammalian cell lines, and no non-specific gene silencing effectswere observed. While the traditional antisense methods have consistently sufferedfrom the spectre of artifacts that arise from questionable specificity andincomplete efficacy, RNAi has been widely used to study gene function in plants,C. elegans, drosophila and mammalian cells as a result of the followingadvantages:1. Most of the genes in the genome of various organisms can be knocked down byRNAi, while this is not the case for antisense olignucleotides.2. RNAi is a potent method, requiring only a few molecules of dsRNA per cell tosilence gene expression.3. DsRNA is much more stable than ssRNA and more resistant to be degraded.4.High sequence specificity. Only one nucleotide mutation n the target mRNA willmake the dsRNA ineffective.The overexpression of oncogenes are genomic features of human cancers.Inhibition of abnormal expression of these oncogenes has been a common idea fordevelopment of new antitumor drugs. It has been identified that RNA interferencetechnology is a more powerful tool than antisense technology for silencingendogenous or exogenous genes in mammalian cells. STAT3 as acute-phaseresponse factor o f interleukin 6 was found and purified in 1994. Someresearch has been testified that STAT3 existed in different tissue and cells andsignal conduction path was related with cell's proliferation,differentiation andapoptosis. It has been demonstrated that persistent Stat3 signaling can directlycontribute to oncogenesis by stimulating cell proliferation and preventingapoptosis.Knocking-out STAT3 hinted that it played indispensable function onearly fetation and bone marrow's differentiation. Resent studies validated thatSTAT3 was closed related with central nerve system's disease, tumor ,and so on.Thereby Stat3 gene at present is considered as an oncogeneRNA interference (RNAi) has recently been used to silence gene expressionin various species. Whether RNAi-mediated silencing of STAT3 expression couldeffectively induce apoptosis of tumor cells in vivo and in vitro or not wasdetermined in this study. Firstly, the RNAi vectors expressing small interferingRNA(siRNA) was developed as recombinat vector targeting the coding sequence .Then , the RNAi-based gene therapy was discussed as a potentially efficaciousanticancer therapy. Therefore, it is reasonable that knockdown of Stat3 willinfluence the Stat3 signaling pathway and provide a new door for gene therapy ofcancer.Results:1. The expression rate of STAT3 protein in breast cancer was 66.7% and 17% inadjacent noncancerous breast tissue., There was significant difference betweenbreast cancer tissue, adjacent noncancerous tissue and normal breast cancertissue(P<0.05). The evidence to prove that STAT3 gene is involved in theoccurrence and development of breast cancer.2.Constructing plasmid of psilencer1.0-U6-stat3-siRNAAccording to genebank's STAT3 cDNA,we successfully constructed andsynthesized psilencer1.0-U6-stat3-siRNA as followings:⑴Select 21-23 sequence. CG content was nearly 50% and the best 45-55%. If CGcontent was higher than 70%, any sequence's function would be severely affected.⑵Avoid to contious three polyG. Or they would overhang and affect the outcomeof siRNA.⑶Select AA sequence in order to the resistence of siRNA.The selection siRNAsequence did not share significant sequence homology with other genes asanalyzed by BLAST search. Each template was consisted of 19 specific sense andantisense oligonucleotides. There were 9 bp(TTCAAGAGA) in the sense chainand 6 T as terminal signal in the antisense chain. pSilencer1.0 was digested withApa I, then digested with EcoR I followed by purification and ligation with theannealed oligodeoxyribonucleotides. The ligation mixture was transformed intocompetent E. coli. The recombinant plasmid was then purified from transformed E.coli and verified by Hind III digestion analysis.3.psilencer1.0-U6-stat3-siRNA prompt MCF-7 cell apoptosis in vitro⑴Transfection of plasmid was carried out by lipofectin using Sofastas describedby the manufacturer. If observed green luciferase, MCF-7 cells were sucessfullytransfected with siRNA and pEGFP plasmid.⑵We transfected STAT3-oriented siRNA-expressing plasmid, as well as controlplasmid, blank plasmid, separated into MCF-7 cells by Sofast TM. To evaluatethe ability of the siRNA encoding human Stat3 that we had synthesized to inhibitStat3 expression in breast cancer at both mRNA and protein level, we used themethod of RT-PCR and Western blot as well as immunohistochemical stainingusing antibody against Stat3. We compared the expression levels of Stat3 in thetumors in the siRNA group to that of the scramble (control) and blank plasmidgroups that had not received treatment with Stat3 siRNA. RT-PCR analysisrevealed that lipofection of STAT3-oriented siRNA-expressing plasmid intoMCF-7 cells led to remarkable suppression of STAT3 mRNA expressioncompared to transfection of the control siRNA group or blank plasmid group.Similarly, immunoblot analysis showed that the expression of STAT3 protein inMCF-7 cells transfected with theSTAT3-oriented siRNA-expressing plasmid wasstrongly inhibited. Transfection of a scrambled nucleotide control, had no impacton STAT3 expression at either the mRNA or the protein level. Taken together,these plasmid vectors expressing siRNA against STAT3 exerted a powerful andspecific knockdown effect with respect to endogenous STAT3 expression inMCF-7 cells.⑶After 72 hours, in cell lines tested, transfection with siRNA against STAT3increased the percentage of cells in the sub-G0/G1 fraction. Cells before thesub-G0/G1 population represent apoptotic cells. The percentage of apoptotic cellsafter transfection with siRNA against STAT3 were much higher than thoseobserved after transfection with blank plasmid or control siRNA ( P<0.01).Thesame outcome was with AO/EB experiment. Our data showed that RNAi directedagainst STAT3 significantly decreased the growth rate of MCF-7 cells, with a 30%decrease at different time points repeatedly in three separate experiments.4. Induction of Stat3 RNAi by siRNA suppresses growth and induces apoptosis ofhuman breast cancer cells implanted into nude mice⑴All animals were housed with controlled 12 hours light/dark cycles with freeaccess to water and standard laboratory diet. The experiment was conducted inaccordance with the Care and Use of Laboratory Animals. Successful animalmodel laid foundation on next experiment.⑵To determine whether tumor growths are suppressed by inhibition of Stat3 geneexpression by RNAi, we directly injected Stat3 siRNA into breast cancer. Tumorgrowth was judged by determination of tumor size and volume two weeks aftertreatment. In the siRNA group(A group), the tumor volume was significantlysmaller than that in theB,C and D groups (P<0.01). However, there was nodifference of tumor volume among B, C and D group. Consistent with this, tumorweight in the siRNA group was also significantly smaller than the B,C and Dgroups (P<0.05) whereas tumor weight in theB group was not different from the Cand D group.⑶Immunohistochemical analysis of Stat3 protein expression in tumors in theStat3 siRNA (Agroup), B,C and D groups two weeks after treatment. A groupshowed positive-Stat3 cells being predominantly peripherally-distributed ,whereasnecrosis and so on, apoptotic phenomenon in central region in tumors in theStat3 siRNA group . In the B,C and D groups ,STAT3 was positive but there wasno apoptosis. Apoptosis in tumor was evaluated by determination of Bax(apoptotic gene) and Bcl-2 (anti-apoptotic gene) that is a pair of opposite genebeing critical for regulating cell apoptosis. As results shown, Western blotanalysis revealed that weak expressions of Bcl-2 protein were observed in allgroups and the expression levels in the siRNA group were slightly lower than theB,C and D groups but no significance was achieved (P<0.05). In contrast, Baxprotein expression levels in the siRNA group were significantly higher than theB,C and D groups (P<0.01), indicating that inhibition of Stat3 gene expression byRNAi induced Bax associated-apoptosis.(4)RT-PCR and Western blot showed that STAT3 expression was lower than B, Cand D groups(p<0.01).In summary, we successfully constructed psilencer1.0-U6-stat3-siRNAplasmid. siRNA of STAT3 suppressed STAT3 expression in MCF-7 cells,inhibited tumor cells proliferation and increased cell apoptosis significantly. Thisstudy demonstrated that in vivo delivery of siRNA into tumor mass results ineffective inhibition of expression of gene encoding Stat3 and suppresseed growthand induced apoptosis of the breast cancer cells. STAT3's decrease and Bax'sincrease may be provide theory foundation for breast cancer's prognosis andtreatment. Furthermore, RNAi technique may be a promising tool for cancertherapy.