Experimental Studies On Biological Effect Of SiRNA Recombinant Expression Vector Targeting Livin Gene To Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (CSCC), as the most commonly occurring cancer in order of incidence in skin cancer, is one of the common skin cancer, and its mechanisms remain unknown up to date. Although it has made considerable progress in Mohs surgery (Mohs micrographic surgery),chemotherapy, radiotherapy, biotherapy, immunotherapy, etc, incidence of CSCC is still increasing, for some special CSCC cases which are not suitable to adopt Mohs micrographic surgery are becoming more and more, and the CSCC is an invasive carcinoma with high degree of malignancy, drug resistance, high incidence of lymph node metastasis, distant metastasis and late spread through the blood, which can lead to the CSCC patients relapse after treatment and even death.A lot of factors lead to the occurrence and development of CSCC, including the external environmental factors, genetic factors, and internal factors such as oncogene mutation, tumor suppressor gene inactivation, abnormal apoptosis and signal transduction pathway, This indicates that the occurrence and development of CSCC is a process with multifactor, polygene and multi-stage, and its important mechanism is cell proliferation and apoptosis imbalance.Livin, a in inhibitor of apoptosis proteins (IAPs) family, has strong inhibitor of apoptosis by direct binding to cysteine aspartate-specific protease (Caspase), so as to inhibits apoptosis and promotes the growth of cancer cells. Studies have shown that Livin was over-expression in many tumor cells including CSCC, while Caspase-3 and second mitochondria-derived activator of caspases (Smac), a factors of pro-apoptotic function, are often in low expression or even lack of. Livin and Caspase-3, Smac, factors of two opposite effects, are negatively related. RNA interference (RNAi) to silence expression of Livin can promote tumor cell apoptosis and inhibit tumor cell proliferation, and increasing the sensitivity of tumor cells to chemotherapeutic, which showing that it is related to the occurrence, development and prognosis of tumor in the over-expression of Livin.Concerning inhibiting target gene expression, RNA interference (RNAi) technology can be efficiently and specifically, and it is a powerful tool for the study of endogenous gene function and signal transduction pathways. In this study, CSCC cell lines with silencing of Livin were established by RNAi silencing Livin gene. Through a series of in vivo and in vitro experimental study, the changes of cell growth, proliferation, apoptosis, cell cycle, migration, cloning capability was observed after Livin gene expression being silenced. It was simultaneously detected the changes in the level of expression of caspase-3 and Smac, also the resistance reversal of the chemotherapy drug bleomycin (BLM) in the treating CSCC and its possible mechanism. These studies provided new targets and methods for therapy of CSCC. This study included four parts as follows. Part 1 Expression of Livin and Smac in cutaneous squamous cell carcinomaObjectiveTo detect the expression of Livin and Smac in human cutaneous squamous cell carcinoma tissues, human cutaneous squamous cell carcinoma cell line A431, normal skin tissue and human keratinocyte cell line HaCaT. To investigate the expression and significance of Livin and Smac, and analyze the correlation of expression of Livin and Smac, and clinical and pathological features in cutaneous squamous cell carcinoma tissues.Methods1. The pathologically-confirmed human cutaneous squamous cell carcinoma tissue specimens of 50 cases selected from Plastic Surgery from January 2009 to August 2011, and 20 cases of normal skin tissue specimens is also.2. Cultured human cutaneous squamous cell carcinoma cell line A431, human skin keratinocyte cell line HaCaT.3. Livin and Smac protein expression was detected in CSCC and normal skin specimens by SP immunohistochemical technique, the expression of these two proteins were comprehensive determined by the percentage of positive cells and the intensity of staining, and their correlation was analyzed.4. Western blot and Real-time-quantitive-RT-PCR were used to detect the gene expression of Livin and Smac in human cutaneous squamous cell carcinoma cell line A431 and human keratinocyte cell line HaCaT.5.To study correlation of Livin or Smac and tumor pathological characters.Results1. Livin and Smac were found in a brown pattern in the cytoplasm. The positive expression rate of Livin protein was 70.0%(35/50) in 50 CSCC tissues but in none of the normal skin tissues.The protein expression in CSCC was higher than those in normal skin tissue. The positive of Livin protein expression had significant differences between cutaneous squamous cell carcinoma and normal skin tissues (P<0.05). The positive expression rate of Smac was 46.0%(23/50) in 50 CSCC tissues and that of Smac was 100%, Smac protein showed lower expression in CSCC than normal skin tissues (P< 0.05).2. Both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.842±0.191 and 0.828±0.230 in human cutaneous squamous cell carcinoma cell line A431,but that of human keratinocyte cell line HaCaT were 0.249±0.023 and 0.175±0.020. The positive expression of Livin mRNA and Livin protein in human cutaneous squamous cell carcinoma cell line A431 were higher than those in human keratinocyte cell line HaCaT. The positive of Livin mRNA and Livin protein expression had significant differences between human cutaneous squamous cell carcinoma cell line A431 and human keratinocyte cell line HaCaT (P<0.05). Both Smac mRNA and Smac protein correspondence expressions were consistently detected in 0.328±0.038 and 0.231±0.032 in human cutaneous squamous cell carcinoma cell line A431,but that of human keratinocyte cell line HaCaT were 0.824±0.183 and 0.808±0.215.Smac mRNA and Smac protein showed lower expression in human cutaneous squamous cell carcinoma cell line A431 than human keratinocyte cell line HaCaT (P<0.05).3. The expression of Livin and Smac had not relationship with age, sex, the sizes of CSCC(P>0.05).The expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC with a statistically significant difference (P<0.05).The positive expression rate of Livin protein was increased in CSCC from highly differentiated to poorly differentiated,and the positive expression rate of Smac was decreased in CSCC from highly differentiated to poorly differentiated.The expression of Livin protein was negatively related to that of Smac protein in CSCC.Summary1.The expression of Livin protein in CSCC was high, while the positive expression of Smac protein in CSCC was lower, and the expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC.2. There were negatively related between Livin and Smac.Part 2 Construction and identification of Livin gene siRNA eukaryotic expression vectorsObjectiveThe Livin-siRNA eukaryotic expression vectors of human Livin gene was constructed and identified, so as to investigate the inhibitory effect of gene silencing mediated by Livin-siRNA and stable expression in targeting to cells, in order to provide stable transfection vectors for the study of the Livin gene function.Methods1. The two siRNA target sequences for Livin gene were designed, so as to synthesize hairpin-shaped single-stranded DNA, and to form double-stranded DNA after annealing.2. The double-stranded DNA with pSilencer2.1/U6 vector was connected, the recombinants were transformed and selected, and the inserted fragments were identified by sequence and enzyme cut. Two siRNA target sequences for Livin gene were obtained.3. The cultured cells were randomly allocated into silence Livin group 1 (transfection of pSilencer2.1/U6-si438), silence Livin group 2 (transfection of pSilencer2.1/U6-si475), empty vector group (transfection of pSilencer2.1/U6) and blank group (untransfected the A431 cells).4. We transfected Livin siRNA vectors into CSCC A431 cell line by shRNA-LipofectamineTM 2000.5. After G418 selection stably transfected cell lines were obtained. The mRNA and protein expression level of Livin was detected in the CSCC A431 cells transfected by Real-time-quantitative-RT-PCR and Western blot. Results1. According to human Livin known sequence(No.NM-022161,No.NM-139317)of GenBank, RNAi explorer of GeneLinkTM Company and siDESIGN center of Dharmacon Company were used to scan, two siRNA target sequences with 19bp were obtained by BLAST homological analysis confirmed,438-456 (GTGGTT-CCCCAGCTGTCAG),475-493 (GGAAGAGACTTTGTCCACA). We synthesized the two siRNA target sequences for Livin gene for two hairpin-shaped single-stranded DNA and at both ends with BamHⅠand HindⅢwithin the enzyme residues (named Livin438 and Livin475),and double-stranded DNA were formed after annealing(named si438 and si475).2.After the double-chain DNA (si438 and si475) was connected to pSilencer2.1/ U6 vector, the recombined plasmid were transformed and selected, and the inserted fragments were identified by DNA sequence analysis and after enzyme cut.Two siRNA target sequences for Livin gene (pSilencer2.1/U6-si438 and pSilencer2.1/ U6-si475) were obtained and were in consistency with the design.3. The stable transfected cell lines were obtained by G418 selection in silence Livin group 1 (transfection of pSilencer2.1/U6-si438), silence Livin group 2 (transfection of pSilencer2.1/U6-si475), empty vector group (transfection of pSilencer 2.1/U6).4. Both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.826±0.205 and 0.813±0.189 in empty vector groups, and that of blank group were 0.852±0.197 and 0.832±0.202.The Livin mRNA and Livin protein expression level did be not changed significantly between blank group and empty vector groups no a statistically significant difference (P>0.05). However, both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.253±0.031 and 0.233±0.024 in silence Livin group 1,and that of silence Livin group 2 were 0.132±0.031 and 0.128±0.031. The Livin mRNA and Livin protein expression level in silence Livin group 1 and silence Livin group 2 were significantly reduced in comparison with the two control groups with a statistically significant difference (P<0.05), the more than 70% the Livin gene silencing effect were achieved, and silence Livin group 2 showed the lowest Livin mRNA and Livin protein relative expression.Summary1. Two human Livin gene RNAi vector were successfully constructed. Optimal RNAi vector (pSilencer2.1/U6-si475) and two CSCC A431 cell lines of silencing of Livin were established successfully.2. Livin gene of target cells can be silenced by RNAi technology, The more than 70% the Livin gene silencing effect were achieved after the CSCC A431 cell were transfected into, and the expression of target sequences for Livin gene is inhibited in the transcription and protein levels.Part 3 Effects on biological behavior of cutaneous squamous cell carcinoma A431 cell lines silenced by reconstructed single siRNA vector targeting Livin genesObjectiveTo investigate the effect on biological behavior of cutaneous squamous cell carcinoma A431 cell lines silenced by reconstructed single siRNA vector targeting Livin genes and to study its possible mechanism, so as to provide the basis of scientific experiments for further clinical trials.Methods1.In vitro experiments are grouped into Livin group (stably transfected pSilencer2.1/U6-siLivin and silent Livin gene expression of A431 cells), empty vector group (stably transfected pSilencer2.1/U6 of A431 cells),blank group (untransfected the A431 cells).2.The mRNA and protein expression level of Smac were detected in the cells in each group by Real-Time-quantitative-PCR and Western blot, and activities of caspase-3 were detected by ELISA.3.The cell proliferation and resistance were detected by MTT. 4. The cell cycle and apoptosis were detected by flow cytometry.5. The ability to metastasis were detected by transwell hole.6. The clonogenic capacity were detected by Colony formation assay.Results1. Compared with the mRNA and protein expression level of Smac in blank group and empty vector group, the mRNA and protein expression level of Smac in silence Livin group was significantly increased with a statistically significant difference (P<0.05), and the activities of caspase-3 was increased significantly.2. MTT technique revealed that compared with the OD value in blank group and empty vector group, the OD value in silence Livin group was decreased significantly with a statistically significant difference (P<0.05), and compared with the cell apoptosis rate in blank group and empty vector group, the cell apoptosis rate in silence Livin group joint BLM treatment group was significantly increased (P<0.05).3. Compared with the rate of apoptosis in blank group and empty vector group, the rate of apoptosis in silence Livin group was increased significantly with a statistically significant difference (P<0.05), and compared with the cells G0/G1 phase percentage in blank group and empty vector group, the cells G0/G1 phase percentage in silence Livin group was significantly increased (P<0.05),and the cells S and G2/M phase percentage in silence Livin group was significantly decreased (P<0.05).4. Compared with the average numbers of the cell passed through matrigel in blank group and empty vector group, the average numbers of the cell passed through matrigel in silence Livin group was decreased significantly with a statistically significant difference (P<0.05), and cell colony formation rate was significantly lower(P<0.05).Summary1. Through upregulating the expressions of Smac and activities of caspase-3, Livin gene is silenced by pSilencer2.1/U6-siLivin wich can inhibit proliferation and ability to metastasis and induce apoptosis in A431 cells.2. After Livin gene is silenced by pSilenccr2.1/U6-siLivin, the sensitivity of A431 cells to BLM chemotherapy were enhanced, and the resistance of A431 cells to BLM could be reversed.Part 4 Effects of silencing Livin gene by RNA interference on xenograft growth of cutaneous squamous cell carcinoma A431 cells in nude mouseObjectiveTo investigate livin gene expression and growth inhibition of silencing Livin gene by RNA interference on xenograft of cutaneous squamous cell carcinoma A431 cells in nude mouse, so as to provide the basis for further clinical trials.Methods1. The nude mice tumor xenograft model was establish by injecting the human CSCC A431 cell lines into the left armpit subcutaneous tissue of the nude mice. All the nude mice were randomly divided into three groups in the eighth day after inoculation, which are the blank group, the empty vector group, the treatment group.2. The xenograft was injected normal saline (the blank group), empty pSilencer2.1/U6 vectors (the empty vector group), pSilencer2.1/U6-siLivin vectors (the treatment group). The tumor growth was observed and detected at the same time, and the curve of tumor growth was then described. The nude mice were putted to death and photoed on the 33th days, and tumors were measured, stripped and weighed.3. Pathological changes of tumor was observed with HE staining.4. Real-time-quantitative-RT-PCR were used to detect mRNA level of Livin and Smac in tumor tissues of nude mouse, Western blot were used to detect protein expression level of Livin, Smac and Caspase-3.Results1. Implanted tumors were observed apparently 8 days after the nude mice inoculated with A431 cells. The growth of the tumor in treatment group were significantly slower than in the empty vector group and the blank group,and tumor volume was significantly lower also.2. The average tumor weight in the treatment group were 0.58±0.03g, which were significantly lower than those of the empty vector group and the blank group (1.24±0.05g and 1.27±0.08g, P<0.05).3. The tumor growth was inhibited, while the necrosis and apoptosis were evident in the treatment group.4. Compared with the mRNA and protein expression level of Livin in blank group and the empty vector group, the mRNA and protein expression level of Livin in the treatment group was decreased significantly with a statistically significant difference (P<0.05), and compared with the mRNA and protein expression level of Smac in blank group and empty vector group, the mRNA and protein expression level of Smac in the treatment group was significantly increased (P<0.05), and the protein expression level of Caspase-3 increased significantly also.Summary1. The growth of A431 cell xenograft in nude mice were inhibited by silencing Livin gene.2. The protein expression level of Smac and Caspase-3 were increased significantly by silencing Livin gene, and experimental results was consistency in vitro and in vivo. 1. The expression of Livin protein in CSCC was high, while the positive expression of Smac protein in CSCC was lower. There were negatively related between Livin and Smac, and the expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC.2. Two human Livin gene RNAi vector were successfully constructed. Optimal RNAi vector (pSilencer2.1/U6-si475) and two CSCC A431 cell lines of silencing of Livin were established successfully.3. Through upregulating the expressions of Smac and activities of caspase-3, Livin gene is silenced by pSilencer2.1/U6-siLivin wich can inhibit proliferation and ability to metastasis and induce apoptosis in A431 cells.4. After Livin gene is silenced by pSilencer2.1/U6-siLivin, the sensitivity of A431 cells to BLM chemotherapy were enhanced, and the resistance of A431 cells to BLM could be reversed.5. Livin gene is silenced wich can inhibit proliferation and induce apoptosis in A431 cells, and experimental results was consistency in vitro and in vivo.