| IntroductionEsophageal cancer is one of the most common gastrointestinal tumors, and its typical symptom is the dysphagia. According to the pathological type, the esophageal cancer can be divided into squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma. Among all of the malignant tumors, esophageal cancer mortality rate ranks as the sixth. Although the 5 year survival rate (10%-20%) was significantly higher than that (about 4%) in 30 years ago due to treatment of ESCC, the survival rate is still at a low level. Esophageal squamous cell carcinoma (ESCC) is the most common digestive tract invasion of malignant tumor and shows poor prognosis, the study showed that the survival rate between squamous cell carcinoma and adenocarcinoma had no obvious differences. At present, the conventional effective methods for the treatment of ESCC mainly include radiotherapy and chemotherapy, and the most commonly used surgical treatment. So far, although a variety of treatment methods have been applied to ESCC, but the five-year survival rate is still not satisfactory. Therefore, biomarkers that can predict ESCC prognosis will be of great significance in the treatment of ESCC, and can be used in high risk population screening of patients with ESCC. So, study on the molecular mechanism of ESCC will be helpful to the treatment of patients with ESCC.Transforming growth factor β(TGF-β) is a multifunctional growth factor, and mainly secreted by macrophages in the tumor microenvironment, stromal cells and tumor cells, and can be used as an important inflammatory factor that plays an important role in the process of maintaining the stability of epithelial cells. At the same time, TGF-p also plays an important role in many important cellular processes, including cell cycle regulation, cell differentiation and extracellular matrix synthesis process. One of the main roles of TGF-β is inhibition of cell proliferation; it is closely linked to the abnormal occurrence of a variety of tumors, such as colon cancer, bladder cancer, ovarian cancer, renal carcinoma, lymphoma and squamous cell carcinoma. In normal epithelial cells, TGF-β can induce cell cycle arrest. However, TGF-β may play an important role in the invasion and metastasis of esophageal squamous cell carcinoma.Serine-arginine protein kinase (SRPK) is a kind of protein kinase for specific phosphorylating RNA splicing in arginine serine domain, and plays a key role in the process of cell growth. In the human body, SRPK is mainly divided into two subtypes, namely SRPK1 and SRPK2, and these two subtypes are mainly distributed in different tissues, with specific regulation of mRNA protein and serine acid-arginine precursor, to ensure the normal function of cells, and these two subtypes also play an important role in cell cycle regulation. SRPK1 is a highly conserved protein in eukaryotes, a PKC superfamily and a kinase domain of the kinase domain was divided into two parts. Some studies indicate that SRPK1 is rich in serine/arginine protein sequence (also known as RS-domain protein) and plays an important role in phosphorylation of specific amino acid sequences. RS-domain protein functions in the pre mRNA processing, translation, shear, chromosome reconstruction, cell cycle regulation and cell structure in the reconstruction process. The SRPK1 spacer can participate in the transport from cytosol to nucleus. The research showed that the expression level of SRPK1 increased and breast cancer, lung cancer and liver cancer risk and prognosis. Studies have found that SRPK1 also plays an important role in glioma cell growth and invasion, migration. But the specific mechanism of SRPK 1 in tumor progression remains to be studied. According to the results of the current study, we think that the expression of SRPK1 gene silencing can exert anti-tumor effect in tumor progression. There are a number of signaling pathways involved in the process of proliferation, migration and apoptosis in TGF-β-mediated ESCC.However, so far, the exact role of TGF-β-SRPK1 in ESCC is still unclear. To elucidate the role and significance of SRPK1 and TGF-β in the cell proliferation and apoptosis of esophageal squamous cell carcinoma cells, we used immunohistochemistry and Western blot assay and RT-PCR method to detect the expression of SRPK1 in 120 cases of ESCC samples, and then we analyzed and summarized the expression and biological function of SRPK1 in ESCC tissues and cell lines, and then explored the development of effective anti-SRPK1 therapy in the clinical practice.Part one. The expression of SRPK1 in esophageal squamous cell carcinoma and its clinical significanceObjectiveIn this study, we used the immunohistochemistry, gene and protein methods to observe the expression of SRPK1 in esophageal carcinoma and normal esophageal tissues, and then we analyzed its relationship with the occurrence of esophageal cancer and tumor metastasis, pathological grade and clinical stage et al. SRPK1 may act as an oncogene in diagnosis and treatment of esophageal cancer. Study on the pathogenesis of esophageal cancer will provide a new target for the treatment of ESCC.Materials and methods120 cases of esophageal cancer patients were collected in this experiment from November 2008 to June 2010. After surgery, two pathologists independently confirmed the diagnosis of esophageal squamous cell carcinoma. All cases of the cancer tissues were attributed into the experimental group, the corresponding control specimen were collected at 5cm away from the margin of cancer tissues. Differential expression of SRPK1 protein was detected by immunohistochemical method in the esophageal cancer tissues and matched normal tissues, and explores the role of SRPK1 in the development of tumors. The experimental data were analyzed by SPSS 19.0 statistical software, and analysis between SRPK1 and the clinicopathological factors of esophageal carcinoma was subjected to the X2 test. We used a=0.05 as a test standard.Result1. Srpkl expression in ESCC tissuesThe immunohistochemical staining showed that, in 120 cases of ESCC,65 cases showed high SRPK1 expression, and in the 65 cases of ESCC, SRPK1 protein mainly expressed in the cytoplasm and nucleus, including 29 cases (++) and 31 (+++). However, SRPK1 proteins were rarely staining in the adjecant normal tissues. In addition, the expression level of SRPK1 mRNA was significantly increased in tumor tissues. Compared with the non metastatic ESCC tissue, SRPK1 mRNA and protein was significantly increased in metastatic ESCC tissue.2. The relationship between SRPK1 and clinicopathological featuresThere was no significant correlation between SRPK1 expression and tumor clinicopathological parameters (such as age, gender or tumor size) (p>0.05). On the contrary, a significant correlation between the expression of ESCC and metastasis of SRPK1 was found, and the difference has statistical significance (p<0.001).3. The relationship between SRPK1 expression and the prognosis of patients with ESCCKaplan-Meier survival curves showed that the expression level of SRPK1 and the total survival rate is closely related (p<0.000). In addition, the single factor analysis confirmed that patients with high expression of SRPK1 had the higher risk of death (p<0.001). At the same time, the single factor analysis showed that ESCC metastasis has a certain relationship with the total survival rate. However, there are no correlations between the prognosis and depth of invasion, age, gender. The results of multivariate analysis further showed that SRPK1 expression can be used as independent predictors of prognosis in patients with ESCC (p<0.001).ConclusionThe expression of SRPK1 in esophageal carcinoma tissues was higher than that of normal esophageal tissue. There were statistically significant differences with the lymph node metastasis. In addition, the expression level of SRPK1 and the total survival rate is closely related, and the high expression of the patients significantly increased risk of death, therefore, the expression of SRPK1 was an independent predictor of prognosis in patients with ESCC.Part two. The role of SRPK1 in TGF-β mediated cell proliferation and apoptosis in esophageal squamous cell carcinomaObjective1. To explore the expression level of SRPK1 in TGF-P mediated ESCC cell lines.2. To explore the role and significance of SRPK1 in TGF-β mediated human ESCC cells.3. To explore the biological effects of SRPK1 in TGF-β mediated human ESCC cells.Materials and methods1. Construction of SRPK1-WT and SRPK1-KD plasmid and adenovirus vectorThe establishment of human ESCC cell lines EC 109, TE8 and TE2 cells detected by related SRPK1 cell level. Wild type SRPK1 (SRPK1-WT) full-length cDNA was amplified by RT-PCR in normal esophageal epithelial cells mRNA, and SRPK1 (SRPK1-KD) activity of kinase mutants through mutation of the 109th catalytic lysine into alanin. After that, the SRPK1-WT or SRPK1-KD cDNA were transfeted into pcDNA3.1 (-) A-myc/his expression vector. And then we constructed the expression system of SRPK-WT or SRPK1-KD using adenovirus AdEasyTM XL adenovirus vector system.2. SRPK1 siRNAtransfectionIn this study, the plasmid and SRPK1 siRNA were transfected into cells by liposome 2000, Western blot confirmedvthe transfection efficiency of SRPK1 siRNA and the control siRNA. The qPCR, Western blot and RT-PCR were used to detect the expression level of SRPK1 in EC 109, TE8 and TE2 cells. After that, western blot method was used to detect the phosphorylation level of AKT and JNK to assess the potential role of TGF-P-SRPK1 signaling pathway.3. Cell proliferation assay was used to detect the survival rate of cells with SRPK1-WT or SRPK1-KD.4. The TUNEL experiment and immunoblotting were used to reflect the effects of of SRPK1 on ESCC cell apoptosis. The wound healing assay and invasion assay were used to explore the function of SRPK1 in the metastasis and invasion of EC 109 cells. 5. Flow cytometry was used to study the role of SRPK1 in regulation of EC 109 cell cycle and control factor.Results1. The expression of SRPK1 in ESCC cell linesThe expression level of SRPK1 in different cell lines was different. SRPK1 showed the highest expression level in TE2 cells. SRPK1 showed the lowest expression level in EC109 cells. SRPK1 is mainly expressed in cell membrane or cytoplasm in EC109, TE8 and TE2 cell lines.2. Effect of SRPK1 on cell proliferationThe wild type SRPK1 can enhance the proliferation of ESCC three cells, and the kinase inactivation type can inhibit cell proliferation. At the same time, SRPK1 gene silencing can significantly inhibit the proliferation of cancer cells.3. Effect of SRPK1 on cell apoptosisTUNEL assay and Western blotting showed that SRPK1 knockout cells showed the high expression of activated caspase 3 and cyclin D1, while the expression of activated caspase 3 and cyclin D1 in the siRNA control cells were not increased (p<0.01). At the same time, after si-SRPK1 treatment, EC 109 cells appeared apoptosis morphological changes such as cell shrinkage related, rounded, cell membrane vacuolization, nuclear condensation fracture etc. The same changes were observed in TE8 and TE2 cells.4. SRPK1 regulates AKT and JNK signaling pathwayCompared with the control siRNA group, in the si-SRPK1 group, the expression level of phosphorylated-AKT in three ESCC cell lines were significantly decreased (p<0.01), and the expression level of-JNK phosphorylation was significantly increased (p< 0.01).5. SRPK1 regulates the process of metastasis and invasion of ESCC cellsSRPK1 suppressed the G1 phase of the cell cycle to S phase transformation. At the same time, compared with the control group, the expression level of cyclin D1 and CDK2 in ESCC cells was inhibited. Wound healing assay showed that SRPK1 siRNA transfected EC 109 cells metastasis were decreased obviously. Similarly, in the invasion assay, compared with untransfected group, invasiveness of SRPK1 siRNA transfected EC 109 cells was inhibited (p<0.001).ConclusionsThe expression level of SRPK1 in ESCC cell lines with different differentiation level shows that SRPK1 has a role in the development of ESCC, and has a certain effect on cell signal transduction and initiation process. SRPK1 can regulate the TGF-β pathway to regulate the balance between proliferation and apoptosis, and can act as an important part of TGF-β signaling pathway. SRPK1 can also regulate migration and invasion, and act as a key regulator of transformation of G1 phase to S phase. |
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