Knockdown Of STAT3 Expression Using SiRNA Inhibits The Growth Of Colorectal Cancer Cell Lines

The colorectal carcinoma is one of the most lethal cancers in humans. The majority of patients died of local recurrence despite aggressive medical intervention including surgery,radiotherapy,chemotherapy. In recent years,gene therapy of tumors has become the most noticeable research field, furthermore,the identification of new molecular targets may lead to more effective treatments for colorectal cancer. STATs,signal transducers and activator of transcription ,are latent cytoplasmic transcription factors that function as intracellular effectors of cytokine and growth factor signaling pathways. STAT proteins were originally defined in the context of normal cell signaling,where STATs have been implicated in control of cell proliferation,differentiation,and apoptosis. stat3,a member of STATs family,have been shown to play a key role in promoting proliferation , differentiation ,anti-apoptosis or cell cycle progression. it transmit signals from cell surface receptors directly to the nucleus by binding directly to high affinity DNA binding sites or by associating with other transcription factors. There is evidence to implicate that stat3 is constitutively activated and overexpressed in a variety of tumours cell lines including leukemias,multiple myeloma,head and neck cancer,breast cancer,lung cancer.. This suggests that stat3 represents a promising molecular target for novel tumor therapy. Recent studies have indicated that inhibition of STAT3 activity in human tumor cells induces apoptosis and /or growth arrest in vitro. In human head and neck squamous carcinoma cells,interrupting stat3 signaling by antisense olignucleotides or decoy oligunucleotide abrogate transforming growth factor-αinduced oncogenic growth of these cells. blocking stat3 signal by antisense induce human colorectal cancer cells to apoptosis also; stat3βis a dominant-negative stat3 variant,which is a truncated form of stat3 that contains the dimerization and DNA-binding domain but lacks the transactivation domain. As a consequence,stat3βcan bind DNA but cannot transactivate gene expression,thus blocking stat3 signaling in a trans-dominant negative fashion in most cases. Blocking stat3 signaling by stat3βin human myeloma cells down-regulates IL-6-induced expression of the antiapoptotic gene,Bcl-XL,resulting in a dramatic sensitization of cells to Fas-mediated apoptosis in vitro. Blocking stat3 activity by dominant-negative stat3βin breast cancer cells leads to apoptosis also. similar to multiple myeloma,disrupting stat3 signaling in head and neck cancer and colorectal cancer cells inhibit Bcl-XL expression and induces apoptosis. knockdown of stat3 expression by siRNA induces apoptosis in astrocytoma cells .these findings raise the possibility that targeting stat3 may result in antitumor responses in vivo in a wide variety of human cancers. RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing triggered by double- stranded RNAs(dsRNAs) homologous to the silenced gene. this phenomenon was first observed from studies in Caenorhabditis elegans and Drosophila melanogaster and was subsequently found in other organisms. The use of RNAi in mammalian studies has only recently been established by introducing short interference duplex RNA (siRNA). In order to determine the role of stat3 in colorectal cancer directly,In this study,we use DNA-vector-based RNAi approach for establishing a long-term siRNA strategy to blocking stat3 expression in colorectal cancer in vitro and vivo and to examine consequences of the loss-of-function.1 Plasmids construction we use DNA vector-based RNAi approach to produce siRNA in vivo .first,we design two pairs double stranded siRNA oligonucleotide against stat3 ( accession numbers for the human stat3 NM003150 genebank) that contained a sense strand of 19 nucleotide equences followed by a short spacer(TTCAAGAGA),the reverse complement of the sense strand,and five thymidines as an RNA polymerase III transcriptional stop signal. oligos were annealed in the anneal buffer (100mM K-acetate,30mM HEPES-KOH pH 7.4,and 2 mM Mg-acetate,Incubate the mixture at 900C for 3min and then at 370C for 1hr and cloned into the ApaI–EcoR I site of pSiencer1.0-U6/3.1-H1 vector which can express hairpin siRNAs under the control of the U6/H1 promoter .2 Cell culture and transfectionsthe human colorectal cancer cell lines HCT8 were cultured in IMDM (GIBCO;INC)supplemented with 10%FCS. lipofectamine 2000 (Invitrogen) was used as the transfection reagent following manufactures directions using 6ug of pSilencer3.1-H1-stat3 and 9-18 ul lipofectamine per 10cm dish,cells were cultured for 5-20 hrs then changed to fresh medium with 10%FCS. and lysed 24-72hrs after transfection3 RT-PCR and Western blot analysis were used to detectthe stat3-mRNA and stat3 protein level , the results showed the pSilencer3.1-H1-siRNA-stat3 could significantly inhibited the mRNA and protein level of STAT3 expression.4 In vitro,the recombinant of siRNA-stat3 could suppress the growth of HCT8 cells in time-dependent,by inducing G1-phase arrest and cell apoptosis, Which brings promise future for colorectal cancer treatment.5 in vivo,the recombinant of siRNA-stat3 could suppress the growth of HCT8 in nude mice. Taken together,we drew a conclusion that the recombinant pSilencer3.1-H1-siRNA-stat3 could inhibited the expression of STAT3 in human colorectal cancer,importantly,direct inhibition of stat3 signaling was accompanied by growth inhibition and induction of apoptosis in colorectal cancer cells in vitro and vivo.