In-vivo And In-vitro Experimental Study Of OSCC Cell Apoptosis Induced By Inhibition Of STAT3 Gene By RNA Inteference

As the most common malignant oral or perioral tumor, oral squamous cell cancer(OSCC) accounts for over 80% of the total incidence of oral tumors, making it a malignant disease deserving much attention. Despite its declining incidence in the developed world, OSCC incidence is on the raise globally. In recent years, although considerable progresses have been made in the treatment of a variety of malignant tumors, resulting in improved 5-year survivals, the treatment for OSCC has been lagged behind with particular difficulty in late-stage treatment due to the imperceptible features at early stages. Therefore greater attention should be paid to the treatment of OSCC in the effort to seek better treatment options.Routine treatment options for OSCC include surgical therapy, radiotherapy, chemotherapy, and immunotherapy, etc., all of which have substantial shortcomings in spite of the certain efficacy achieved in inhibiting the genesis and development of tumors. For instance, radio and chemotherapies would inevitably result in serious side effects, while in surgical treatment, there are difficulties in practice and eradication of tumor is impossible,and immunotherapy showed no significant efficacy. Given this, gene therapy and molecular-targeted therapy, which has newly emerged for the treatment of tumors, have won the favor of the research field with less adverse reactions and accurate efficacy. RNA interference (RNAi) technology, one of the novel gene therapies for disease diagnosis and treatment, can effectively hinder disease progression by identifying and interfering with the pathogenic target genes. Recently it has drawn increasing attention in the treatment of tumors.STATS,the coding gene of signal transduction activating factor 3, is often considered as the proto-oncogene. The downstream pathway of STAT3 signal transduction can affect the expression of many factors, including cyclinD1, Bcl-xl,surviving and c-myc, etc, that are related to the promotion of tumor progression. Besides, STAT3 plays a role in the immune escape process in tumors. Therefore STAT3 gene has been selected as the target to be subject to appropriate interference for effective gene therapy of tumors. In this study, RNAi therapy, one of the gene therapies, was applied, using the tumor-related proto-oncogene STAT3 as the treatment target. RNAi plasmid specific for the STAT3 gene locus was designed and introduced to the OSCC cells. The influence of the RNA interference effect of this plasmid on OSCC progression was firstly observed in vitro, and then tested in vivo in animal models of OSCC, in order to verify the tumor inhibition effect of RNAi with STAT3 gene.In this study, the treatment method for OSCC was explored from an entirely new perspective, in the hope to provide a new paradigm for the treatment of OSCC.Objective:To investigate the in-vitro inhibition of OSCC cells and in-vivo tumor inhibition effect in OSCC animal models by knocking out STAT3 gene using RNA interference technology.Method:(1)Immunohistochemical assay was using in detecting normal oral mucosa, atypical hyperplasia and patients with oral squamous cell carcinoma of STAT3, EGF and EGFR protein expression.(2) The STAT3 gene interfering plasmid, i.e., psilencer1.0-U6-stat3-siRNA plasmid, was constructed.(3)The expression levels of STATmRNA and the related proteins in OSCC cells were determined in different in-vitro and in-vivo study groups using downstream gene engineering technologies such as Western blot, RT-PCR, and IHC methods, so as to assess the tumor inhibiting effect of RNAi.(4)In-vitro test:The siRNA template specific for STAT3 RNAi locus was designed and synthesized in vitro. The plasmid vector was then subject to enzyme cutting for linearization and collected. The samples then underwent quantification of nucleic acids. The linearized plasmid vector was connected to the interference fragment by T4 ligase. Subsequently the competent cells were prepared and co-incubated with the recombinant plasmids for transformation of competent cells by plasmids. Extraction and identification of recombinant plasmid were also required. Transfection of cells by the recombinant plasmid was then performed for the four test groups, namely, positive recombinant plasmid group, negative recombinant plasmid control group, blank plasmid control group, and blank vehicle control group. After completion of the tests for each group, the appropriate in-vitro tests were performed for each group to determined the inhibition on the expression of STAT3-related proteins. SDS-PGE was prepared for the immunoblotting test by the process of protein electrotransfer,blockage, binding with antibody, colorization, and photographing ,etc.; the expression of STAT3mRNA was determined by extracting total RNA of the transfected cells followed by RNA quantification, cDNA collection and reverse transcription reaction, primed synthesis,polymerase chain reaction, and determination of PCR products, etc; apoptosis of the tested cells were determined using flow cytometer; colorimetric reagent was prepared, cells were subject to trypsin enzymolysis, and cell apoptosis was tested as appropriated using MTT method by the process of colorization and color comparison, etc.(5)In-vivo test:Athymic mice were inoculated with human Tca8113 tongue squamous cell resuspension culture to establish the animal model of human tongue squamous cell carcinoma in ahtymic mice. Modeling was completed when the tumor had grown to the size of 50mm3. Psilencer1.0-U6-stat3-siRNA recombinant plasmid, blank plasmid and control plasmid were then transferred into the tumor cells using electroblot. The status of tumor growth and the increase rate of tumor size were regularly monitored, and tumor tissues were collected at the end of test for weighing. mRNA expression of STAT3 gene was determined by RT-PCR method, the expression of proteins MMP2 and VEGF from STAT3 and the related genes was monitored by western blot method, and the expression of BAX and BCL-2 by IHC method.Results:(1) oral squamous cell carcinoma carcinogenesis STAT3 in protein expression in normal mucosa, dysplasia and carcinoma rates were higher, respectively, 13.3% (8 / 60), 30.0% (9 / 30), 71.2% (43/60) group had a significant difference (P <0.05), and EGF protein in normal oral mucosa, dysplasia and oral squamous cell carcinoma rates were 25.0% (15/60) , 46.7% (14/30), 76.7% (46/60), the group had a significant difference (P <0.05); EGFR protein in normal mucosa, dysplasia and carcinoma rate 15.0% (9 / 60), 36.7% (11/30), 70.0% (42/60), the group had a significant difference (P <0.05), the three were positively correlated. In oral squamous cell carcinoma of STAT3 protein expression and the histological grade cancer, the depth of invasion and lymph node metastasis (P <0.05); EGF expression and depth of cancer invasion and lymph node metastasis (P <0.05); EGFR protein expression and depth of cancer invasion and lymph node metastasis (P <0.05).(2) Psilencer1.0-U6-stat3-siRNA plasmid was found to be successfully constructed as determined by enzyme cutting.(3)The results of the in-vitro test suggest that recombinant plasmid exhibited relatively strong pro-apoptosis and proliferation inhibition effect on the tongue squamous carcinoma cells in the test group; the results of both Western blot test and reserve transcription PCR test suggest that recombinant plasmid produced inhibition on cell proliferation and promoted apoptosis. In order to verify the molecular mechanism of the pro-apoptosis and proliferation inhibition effect of recombinant plasmid, MTT test and cell cycle test by flow cytometer were performed. The results of MTT test suggest that the recombinant plasmid significantly inhibited cell proliferation, and flow cytometer test suggests that cell cycle was blocked by recombinant plasmid at the GO/G1 phase, with hypodiploid peak present before normal diploid peak. The apoptosis rate was high.(4)The results of the in-vivo test suggest that animal model of subcutaneous implantation of OSCC was successfully constructed in athymic mice; the growth rate of tumor size was significantly lower in the test group than in all the control groups(p<0.05); the ultimate tumor size was significantly smaller in the test group than in all the control groups(P<0.05); mRNA expression levels of STAT3 gene determined by RT-PCR suggest that STAT3mRNA expression level was significantly higher in the test group than in the control groups(P<0.05); expression of proteins MMP2 and VEGF from STAT3 and the related genes monitored by western blot method suggests that protein expression level was significantly lower in the test group than in the control groups (P<0.05); and expression of BAX and BCL-2 from STAT3 and the related genes monitored by IHC method suggests that expression level of the related proteins was significantly lower in the test group than in the control groups(P<0.05).Conclusions:(1)STAT3, EGF and EGFR in oral squamous cell carcinoma invasion, metastasis and mucosal epithelial carcinogenesis process plays an important role, suggesting that the detection of STAT3, EGF and EGFR is expected to become oral squamous cell carcinoma early diagnosis and prognosis of the objective indicators .(2)After successful cell transfection, siRNA gene blocking plasmid involving STAT3 effectively inhibited the activation of and information transfer by STAT3 signal transduction pathway, which further resulted in blocking of tumor cell cycle, inducing in-vitro apoptosis of OSCC cells. This indicates that STAT3siRN may be used as a therapeutic molecule for OSCC.(3)The results of the in-vivo test suggest that successful transfection of STAT3 siRNA plasmid into the tumor sites of the test models effectively inhibited the increase of tumor size. Inhibition of STAT3 expression inhibited the expression of MMP-2 and VEGF at the downstream of the signal pathway, indicating that inhibition of STAT3 signal pathway could indirectly inhibit the expression of the tumor-related genes of MMP-2 and VEGF, etc., thus inhibiting the genesis and development of tumor.