Expression Of OCT-4 In Laryngeal Squamous Cell Carcinoma And Its Significance

Laryngeal carcinoma is the most common tumor in ENT, due to the growth of parts of the particularity of laryngeal carcinoma, and there are still more complications, side effects problem of surgery, radiation therapy and chemotherapy, to bring greater suffering for patients. Therefore, an urgent need to proposed the new diagnostic methods and simple effective treatment based on molecular biology has become a hot topic of current research.Although the pathogenesis of cancer has so far not clear, but it is certain that the incidence of tumor development is the multi-step, multi-stage and multi-and abnormal expression of genes involved in the process. Studies in a lot of scientific research institutions found that existing a small percentage of tumor cells that have stem cell characteristics, called tumor stem cell-like cells (CSCLC), the researchers believe that these CSCLC in tumor origin and development process plays a key role. Although the doctrine of the current dispute to a larger, but one thing clear is that the tumor cells should be the existence of the abnormal expression of certain key genes regulating self-renewal, and this is to ensure that tumor cells out of control, abnormal, monoclonal unlimited proliferation basis.Creatures have a series of signaling molecules to maintain stern cell pluripotency, the most widely current study is about OCT-4. OCT-4 gene is a member of POU transcription factor family, expressed in embryonic stem cells and adult stem cells were, involved in embryonic development of multiple regulation of multiple differentiation, and can control the unlimited proliferation of features. Study found that OCT-4, not only expressed in pluripotent embryonic stem cells and primordial germ cells, in some malignant germ cell tumors such as seminoma, embryonal carcinoma and solid tumors also detected abnormal expression of OCT-4. About the research on OCT-4 expression in the laryngeal carcinoma and its significance, so far little has been reported at home and abroad.In order to thoroughly explore the OCT-4 gene and malignant biological behavior; of laryngeal carcinoma, study of the role of OCT-4 in laryngeal carcinoma occurrence and development for diagnosis, treatment and prognosis of laryngeal carcinoma indicators and look for effective methods to inhibit the development of laryngeal carcinoma, this study first detected the expression of OCT-4 in laryngeal carcinoma tissue, adjacent normal tissue and vocal nodules tissue, and explore the relationship between the OCT-4 and malignant biological behavior of in laryngeal carcinoma; Successfully constructed eukaryotic expression vector containing the OCT-4 mRNA code cDNA and the siRNA sequence to detect the impact of OCT-4 on human laryngeal carcinoma Hep-2 cells and Survivin, p-STAT3/STAT3 expression level; detected the impact of sodium valproate (VPA) on laryngeal carcinoma Hep-2 cells OCT-4, p-STAT3/STAT3 and Survivin expression. The final experiment was adoption of xenografts in nude mice in vivo. From several aspects analyzed OCT-4 and VPA impact on the laryngeal carcinoma Hep-2 cells in an attempt to clarify the OCT-4 expression in laryngeal carcinoma occurrence and development and it significance; And to further clarify the relation of OCT-4 gene inhibition and VPA treatment and the inhibition of Hep-2 cells proliferation whether by inhibiting phosphorylation of STAT3 protein and reducing tumor Survivin expression. To further explore the laryngeal carcinoma occurrence and development mechanism and to find inhibition methods for laryngeal carcinoma occurrence and development and provide a theoretical basis for therapeutic approaches. This study is divided into the following four chapters.The first chapter:the expression of OCT-4 and Survivin in laryngeal squamous cell carcinoma and it significanceMethods:The expressions of OCT-4 and Survivin protein were detected in 77 cases of laryngeal carcinoma tissue,18 cases of adjacent normal tissues and 15 cases of vocal nodules tissue by immunohistochemical, Real-time PCR and western.ResultsThe expression of OCT-4 protein and survivin in laryngeal carcinoma tissues were higher than in adjacent normal laryngeal tissues and vocal cords polyps tissues; The expression of OCT-4 had significant relations with histological grade and lymphatic metastasis (P<0.01); The expression of Survivin had significant relations with histological grade,lymphatic metastasis and T staging (P<0.01); There were significantly positive correlations among the expression of OCT-4 and Survivin in laryngeal carcinoma tissues (P<0.05).The second chapter:the impact of OCT-4 on human laryngeal carcinoma Hep-2 cells in vitroMethods:1. Constructed the eukaryotic expression vector with OCT-4 gene mRNA full-length cDNA code sequence and with siRNA sequence, transfected into and screened Hep-2 cells for positive stable transfected cell clones.2. The Hep-2 cell proliferative activity and clones capacity among the control group and the transfected group was observed by MTT and plate cloning method.3. The apoptosis rate and cell cycle distribution of the Hep-2 cells among the control group and the transfected group respectively analyzed by flow cytometry (FCM).4. The expression of p-STAT3/STAT3 and Survivin in Hep-2 cells of the control group and the transfected group was detected by Real-time PCR and Western,Results:1. The pcDNA3.1-OCT-4-2和pSUPER-EGFP-OCT-4-c eukaryotic expression vector were successfully constructed, transfected into Hep-2 cells.2. MTT and Plate cloning results showed that:Compared with the control group, the Hep-2 cell proliferative activity and clones capacity were markedly increased in pcDNA3.1-OCT-4-2 group (P<0.01); while decreased in pSUPER-EGFP-OCT-4-c groups (P<0.01); no significant difference in pcDNA3.1 and pSUPER-EGFP group (P> 0.05).3. FCM results showed that:Compared with the control group, in pcDNA3.1-OCT-4-2 group the proportion of cells in G0/G1 phase decreased (P<0.01), while cells in S and G2/M phase increased(P<0.05); in pSUPER-EGFP-OCT-4-c group the proportion of cells in G0/G1 phase was significantly higher (P<0.01), while cells in S and G2/M phase reduced (P<0.05), at the same time compared with each groups the apoptosis rate increased; pcDNA3.1 and pSUPER-EGFP empty plasmid group had no significant difference (P>0.05)4. Real-time PCR and western results showed that:Compared with the control group, the p-STAT3 protein and Survivin mRNA and protein were markedly increased in pcDNA3.1-OCT-4-2 group(P<0.01); while decreased in pSUPER-EGFP-OCT-4-c group(P<0.01); no significant difference in pcDNA3.1 and pSUPER-EGFP groups(P> 0.05).The third chapter:the impact of VPA on the Hep-2 cells proliferation and it mechanismMethods:1. The proliferative activity of the Hep-2 cells treated with VPA was observed by MTT method.2. The apoptosis rate and cell cycle distribution of the Hep-2 cells treated with 3mmoL/L VPA at 0 (control),24,48,72 h was detected by FCM.3. The expression of OCT-4 and Survivin in Hep-2 cells after treated with 3mmoL/L VPA at 0 (control),24,48,72 h were detected by Real-time PCR and Western.Results:1. MTT results showed that:the VPA inhibited Hep-2 cell growth in a dose-and time-dependent manner.2. FCM results showed that:After 3mmoL/L VPA treatment, along with the time increasing the cells in G0/G1 phase increased gradually (P<0.01), cells in S phase decreased gradually (P<0.01), while the cells in G2/M phase no significant change (P> 0.05), the rate of apoptosis was time-dependent increase gradually.3. Real-time PCR and western results showed that:After 3mmoL/L VPA treatment, along with the time increasing OCT-4 mRNA and protein expression gradually increased (P<0.01), while p-STAT3 protein Survivin mRNA and protein expression decreased gradually (P<0.01).The forth chapter:the study on the impact of OCT-4 and VPA on the Hep-2 cell xenografts in nude mice and its mechanismMethods:1. After establishment of human laryngeal carcinoma xenografts in nude mice were divided into control group, pcDNA3.1-OCT-4-2 group, pSUPER-EGFP-OCT-4-c group and the VPA experimental group, Weekly measureed each tumor size and nude body mass, drawn tumor volume growth curves. The mice were killed and removed tumor weighed to calculate tumor inhibition rate at termination of treatment.2. The apoptosis in xenografts tissue in nude mice of each group were detected by TUNEL.3. the expression OCT-4,p-STAT3/STAT3 and Survivin in tumor tissue of each nude mice group were detected by Real-time PCR and western.Results1. The volume of the tumors in pcDNA3.1-OCT-4-2 nude mice group was significantly higher than the control group, the difference between the two groups was statistically significant (P<0.05); The tumor volume in pSUPER-EGFP-OCT-4-c group and; the VPA Group were significantly smaller than the tumor volume in control group, the difference among the three groups were statistically significant (P<0.05).2. TUNEL results showed that the apoptotic cells of nude mice tumor tissue in pSUPER-EGFP-OCT-4-c group and VPA group significantly increased than control group, the difference among the three groups was statistically significant (P<0.01).3. The protein and mRNA expression of OCT-4 gene in pcDNA3.1-OCT-4-2 group and VPA group were significantly higher than the control group, the difference among the three groups was statistically significant (P＜O.01); while in pSUPER-EGFP-OCT-4-c group was significantly lower than the control group, the difference between the two groups was statistically significant(P<0.01). The expression of p-STAT3 protein and Survivin mRNA and protein in tumor of pcDNA3.1-OCT-4-2 group was significantly higher than the control group, the difference between the two groups was statistically significant (P<0.01); while in pSUPER-EGFP-OCT-4-c group and VPA group was significantly lower than the control group, the difference among groups were statistically significant (P<0.01); each group was no obvious STAT3 protein in the change.Conclusion:1. The expression of OCT-4 protein were higher in laryngeal carcinoma tissues; The expression of OCT-4 had significant relations with histological grade and lymphatic metastasis, and have significantly positive correlations with the expression of Survivin in laryngeal carcinoma tissues; The OCT-4 gene can promote the Hep-2 cell proliferation activity and cloning capability, while inhibiting the OCT-4 expression will lead to cell proliferation activity and cloning capability inhibition, block cells in the Go/Gi phase and induce apoptosis, its mechanism were related to inhibition of p-STAT3 and Survivin expression.2. VPA can inhibit the proliferation of laryngeal carcinoma Hep-2 cells, blocking cell in the Go/G1 phase and induce apoptosis; VPA can inhibit p-STAT3 and Survivin expression to participate in tumorigenesis and development.3. Results in vivo and in vitro experiments were similar that OCT-4 can regulate p-STAT3 and Survivin expression to participate in tumor occurrence and development.