Mechanism Of SRPK1 To Promote The Anti-apoptosis Of Colorectal Cancer Cells

Objective:Analyze the correlation between SRPK1 high expression level and colorectal cancer clinical stage and poor prognosis through detecting the expression level of SRPK1 in colorectal cell lines,surgical tissues and paraffin pathological sections;Declare the molecular mechanism of SRPK1 in enhancing anti-apoptosis ability of colorectal cell via promoting NF-?B pathway.Method:1.Using Western Blotting and Real-time PCR to analyze the translation and m RNA transcription levels of SRPK1 in six colon cancer cell lines and five pairs of colon cancer surgical tissues.Immunohistochemistry was used to detect the expression of SRPK1 in 347 paraffin pathological sections of colorecal cancer.The SPSS 19 statistical software was applied to analyze the results of immunohistochemistry.Chi-square test analyze the relationship between SRPK1 expression level and clinicopathologic grade TNM.Kaplan-Meier analysis of the correlation between the expression level of SRPK1 and the survival and prognosis of colorectal cancer patients.Statistical results with p ? 0.05 were considered as statistically significant.2.Using the SW480 and HCT-116 cells to construct SRPK1-OE and SRPK1 RNAi stable cell line and verified the expression of SRPK1 by WB and q PCR.We checked cell viability with MTT assay,tested cell anti-apoptosis ability via Annexin V/FITC and verified the expression of Bcl-2,cleaved-Caspase 3 and cleaved-PARP by WB in SRPK1-OE and SRPK1 RNAi stable cell lines respectively.3.Analyzing the transcriptional activity of NF-?B and transcription level of downstream target genes of NF-?B by luciferase assay and q PCR method in SRPK1-OE and SRPK1 RNAi stable cell lines.We used immunofluorescence assay and WB to detect the expression and nuclear translocation of NF-?B/p65 and the phosphorylation level of IKK?,I?B? as well.4.MTT assay and Annexin V/FITC were used to detect the viability and anti-apoptotic capacity in SRPK1-OE and SRPK1 RNAi stable cell lines under under the condition of10?M SRPIN340.Subcutaneous tunorigenesis in nude mice was used to detect the tumor size of mice in PBS group,adriamycingroup and SRPIN340 groups on were determined respectively.Result:1.The expression of SRPK1 in colorectal cancer cell lines SW480,HCT-116,HT-29,HCT-8,Caco2,LS174 T was significantly higher than the normal cell line NCM-460 both in translation and transcription level.At the same time,the expression level of SRPK1 in cancer tissues is also much higher compare with adjacent tissues in the 5 pairs of colorectal cancer tissues.Immunohistochemistry and SPSS results how that the expression level of SRPK1 is significantly correlated with the malignancy of colorectal cancer.2.The up regulation of SRPK1 enhanced cell viability and anti-apoptosis capacity and increased expression of Bcl-2 and active cleavage fragments of Caspase-3 and PARP.Down regulation of SRPK1 obtained an opposite result.3.The up regulation of SRPK1 raised the transcriptional activity of NF-?B and transcription level of downstream target genes of NF-?B pathway,increased the phosphorylation level of IKK?,I?B? and promoted nuclear translocation of NF-?B/p65.4,SRPIN340 can partially inhibit the anti-apoptotic ability of SRPK1,but in nude mice experiments,its inhibition in tumor formation was not obviously.Conclusions:The high expression of SRPK1 is significantly correlated with the malignancy of colorectal cancer and the poor prognosis of colorectal cancer patients;The high expression of SRPK1 could promote the anti-apopotosis ability of colorectal cancer cells;The high expression of SRPK1 could increased the phosphorylation level of IKK?,I?B? and promoted nuclear translocation of p65 to enhance NF-?B transcription activity.