The Effect Of Serine/Arginine Protein Kinase1 (SRPK1) On The Invasion,Metastasis And Angiogenesis Of Human Colorectal Cancer

Background and Purpose:Colorectal cancer(CRC)is one of the most common malignant tumors in the world.It has high incidence and mortality around the world.The recurrence and metastasis of CRC are the major factors leading to poor prognosis and survival in patients with this type of tumor.Serine/arginine(SR)proteins is a kind of the serine/arginine-rich(SR)domain family of splicing factors,which plays an important role in the regulation of m RNA selective splicing.Serine/arginine protein kinase 1(SRPK1)is a protein kinase that can phosphorylate the SR proteins,which are essential for splice-site selection,especially the modulation for RNA metabolism,localization,and translation.It has been reported that high expression of SRPK1 has been found in numerous human cancers,but its expression and mechanism in CRC is still rarely reported.In this study,the expression of SRPK1 was first explored in paraffin specimens of postoperative CRC tissues,so as to determine the clinicopathological features of patients and their relationship with the prognosis of CRC patients.Then SRPK1 m RNA and protein expression were detected in fresh CRC tissues and two CRC cell strains.Finally,by overexpressing or interfering with the expression of SRPK1,the effects of SRPK1 expression in CRC cells on malignant behaviors such as CRC cell proliferation,migration and invasion and angiogenesis were examined.Methods:1.Immunohistochemistry was used to detect the expression of SRPK1 protein in the paraffin specimens of 85 CRC patients after surgery,and its relationship with clinicopathological features was analyzed.The survival data of patients was followed up to analyze the effects of SRPK1 expression on prognosis,such as postoperative recurrence and survival of patients.Real-time quantitative PCR and Western blot were used to detect the expressions of SRPK1 m RNA and protein in fresh tissues of 15 CRC patients after surgery.2.q RT-PCR and Western blot were used to detect the endogenous expressions of SRPK1 in two strains of CRC cells(Caco2 and HT-29).SRPK1 si RNA(1~4)and control group(si NC)were transfected into corresponding CRC cell strains by the liposome method.The best SRPK1 si RNA was selected by q RT-PCR and Western blot.CCK-8 method and clone formation assay were used to detect the cell proliferation capability of each group.Transwell chamber test to detect the cell migration and invasion capability of each group.Flow cytometric(FCM)analysis after Annexin V-FITC/PI double staining was used to detect the cell apoptosis capability of each group.Meanwhile,the overexpressed SRPK1 plasmid was transfected into the CRC cell strain,and the expression of SRPK1 was detected by Western blot and CCK-8,so as to further demonstrate the effect of SRPK1 on tumor cell proliferation.3.SRPK1 si RNA and control group(si NC)were transfected into the CRC cell strain HT-29 using the liposome method.Forty eight hours later,the supernatant of cells was extracted,and the supernatant of different groups of cells were co-cultured with human umbilical vein endothelial cells(HUVEC).The expression of VEGF in the transfected HT-29 supernatant was detected by ELISA.CCK-8 method was used to detect the proliferation capability of HUVEC in each treatment group.Scratch test was used to detect the migration capability of HUVEC in each treatment group.The lumen formation test was used to detect the lumen-forming capability of HUVEC in each treatment group.Western Blot was used to detect the protein expressions of angiogenic growth factors Tie2 and Ang2 in HUVEC of each treatment group.Results:1.Immunohistochemical staining showed that SRPK1 was mainly expressed in the cytoplasm and cell membrane of CRC cells.Among the 85 CRC specimens,44 had high expression of SRPK1 protein,with an expression rate of 51.76%.SRPK1 expression was low or not stained in normal colon tissues.In 15 pairs of fresh CRC and adjacent normal tissues,the expression levels of SRPK1 m RNA and protein in cancer tissues were significantly higher than those in adjacent normal tissues(P <0.05).2.Statistically,the positive expression levels of SRPK1 in patients with CRC TNM stage III~IV and lymph node metastasis were significantly higher than those in patients of stage I~II and lymph node metastasis(P<0.05).Moreover,with the depth of tumor infiltration,SRPK1 expression in tumor deeply penetrating in the intrinsic muscular layer(T3+T4)and beyond was significantly higher than that in tumor tissues(T1+T2)that did not penetrate the muscular layer(P<0.05).Kaplan-Meier analysis showed that high expression of SRPK1 was significantly associated with a decrease in the overall survival of CRC patients.COX univariate analysis showed five pathological indicators,including TNM staging,invasion depth,presence of lymph node metastasis,and presence of distant metastasis,were correlated with the postoperative survival time of CRC patients(P<0.05).The Cox proportional hazard model was used to conduct a multivariate analysis on these four possible factors in univariate analysis,and results showed that SRPK1 and TNM staging were independent indicators of CRC prognosis(P <0.05).4.Compared with normal intestinal mucosal epithelial cells NCM460,cell strains Caco2 and HT-29 had higher expressions of both SRPK1 m RNA and protein(P<0.05).SRPK1 si RNA with the best inhibitory effect was selected by Western blot and q RT-PCR for subsequent experiments.After SRPK1 expression was inhibited,the growth and proliferation of CRC cells were also inhibited.On the other hand,after SRPK1 was overexpressed,the proliferation capability of tumor cells was promoted.In addition,both CRC cell strains that knocked down SRPK1 expression showed decreased migration and invasion capabilities,and apoptosis was significantly increased(P <0.05).5.ELISA test showed that the expression of VEGF in the supernatant of HT-29 cells transfected with SRPK1 si RNA was significantly decreased.Compare with contrl group,the supernatant of HT-29 cells with down-regulated SRPK1 expression was co-cultured with HUVEC,and the proliferation,migration,and angiogenesis capabilities of the cells were significantly inhibited.The protein expressions of pro-angiogenic factors Tie2 and Ang2 were significantly reduced.Conclusions:1.High expression of SRPK1 was associated with TNM staging,lymph node metastasis,tumor infiltration of the CRC tissues.The expression of SRPK1 and TNM stage were independent factors affecting the survival of colorectal cancer patients2.Down-regulated expression of SRPK1 could affect the CRC cells' biological behaviors,inducing proliferation,migration,apoptosis and invasion.3.Down-regulated expression of SRPK1 could affect the HUVEC' biological behaviors,inducing cell proliferation,migration,and lumen formation.