| Background and ObjectiveColorectal cancer (CRC) is one of the gastrointestinal malignant tumors, the morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer. The incidence of CRC in our country has obvious rising trend. In recent years, with new technology application on diagnosis and treatment and update of chemotherapy drugs, the prognosis of patients with CRC has greatly improved, but overall survival of patients has not been significantly improved, its main reason is that many patients have occurred mirco-metastasis before the operation. Metastasis is the main cause of the death of CRC patients. Since metastasis of tumor is a complex process, understanding the key mechanisms and molecules involved in the complex process of tumor invasion and metastasis contribute to the development of effective therapeutics for treating CRC patients. With the development of molecular biology techniques, the research on etiology and pathogenesis of tumor has made important progress, but the research on tumor metastasis has not substantive breakthrough. Focusing on and strengthen the basic research on metastasis mechanisms, illustrating molecular mechanisms of tumors metastasis, are a frontier subject in the research of tumors, and have important scientific significance for improving survival rates and cure rate of patients with malignant tumors.With the project of human genome being completed, the non-coding RNA play an important role in the process of life. The regulatory function of long chain non-coding RNA gene has aroused researchers’s great interesting. The non-coding RNA may form a RNA regulation network, which has not yet been fully known, regulate the expression of genetic information in the senior eukaryotic cells, and play an important role in important life activities, such as gene transcription and its processing, cell differentiation and individual development, genetic and epigenetic. The previous study has confirmed that the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), is upregulated in colorectal cancer tissues and can promote cells proliferation, migration, invasion in vitro by regulating its target gene AKAP9 expression in colorectal cancer. The expression of AKAP9 is upregulated in colorectal cancer tissues, and closely relate to infiltration depth and metastasis of colorectal cancer. However, whether or not AKAP9 associate with the proliferation, invasion and metastasis of colorectal cancer, and what’s molecular pathways is involved in promoting metastasis of colorectal cancer. There is no related research that has reported at home and abroad. We are not yet know that the molecular mechanism of the noncoding RNA MALAT1 regulating AKAP9 gene, and the biological function of AKAP9 gene and molecular mechanism of AKAP9 regulating colorectal cancer metastasis.Cdc42-interacting protein 4 is a protein that in humans is encoded by the TRIP10 gene CIP4. CIP4 is required to coordinate membrane tubulation with reorganization of the actin cytoskeleton during endocytosis. CIP4 can bind to lipids such as phosphatidylinositol 4,5-bisphosphate and phosphatidylserine and promote membrane invagination and the formation of tubules. Moreover, CIP4 is required for the formation of podosomes and actin-rich adhesion structures specific to monocyte-derived cells. Many researches have reported that expression of CIP4 protein is abnormal in many tumors, CIP4 protein can promote proliferation, invasion and metastasis of breast cancer, lung adenocarcinoma, and osteogenesis sarcoma tumor. In addition, CIP4 protein is closely related with EMT (epithelial mesenchymal transition) process. Previous research has found that AKAP350, which is an alternate splicing isoform of AKAP9 gene, can interact with CIP4 at the Golgi apparatus. Some studies have reported that CIP4 can promote TGF-β1-induced EMT in renal proximal tubular epithelial cells. Moreover, knock-down of CIP4 strongly increases the formation of tubular E-cadherin vesicles at adherens junctions. Thus, we hypothesized that AKAP9 may interact with CIP4 protein and induce EMT by regulating CIP4 protein expression, and then promote invasion and metastasis of CRC.Therefore, we hypothesized that MALAT1-AKAP9-CIP4 molecular signal axis may participate in regulating the proliferation, invasion and metastasis of colorectal cancer. But, there is no research reported that MALAT1 is how to regulate its target gene AKAP9 expression and what’s function of AKAP9-CIP4 molecular signal on proliferation, invasion and metastasis of CRC. Therefore, this study aim to explore MALAT1-AKAP9-CIP4 molecular signal axis how to regulate proliferation, invasion and metastasis of colorectal cancer, to provide reliable experimental basis for explore the mechanism of colorectal cancer growth and metastasis.Methods1. The mechanism research of how MALAT1 regulates AKAP9 expression in CRC cells(1) RNA immunoprecipitation (RNA-IP), Co-immunoprecipitation (Co-IP) and Immunofluorescent localization were employed to detect the interaction between MALAT1 and SRSF1 protein, the interaction between MALAT1 and SRPK1 protein, and the interaction between SRSF1 and SRPK1 protein.(2) Western blotting and Quantitative reverse transcription-PCR (qRT-PCR) were employed to detect the SRSF1 expression in Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells. Western blotting, Immunofluorescent and The nucleus plasma separation technology were used to detect the phosphorylated SRSF1 protein in Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells, and intracellular location of phosphorylated SRSF1 protein.(3) Western blotting, Quantitative reverse transcription-PCR (qRT-PCR) and Immunofluorescent were used to detect the SRPK1 expression in Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells. According to the concentration gradient, the RNAa-MALAT1-SW480 cell was treated with SRPK1 protein activity inhibitor SRPIN340. Western blotting was carried out to detect the SRPK1,AKAP9 and phosphorylated SRSF1 protein expression.(4) SRPK1 gene express plasmid was transfected to RNAi-MALAT1-SW480 cell, and Western blotting and The nucleus plasma separation technology were used to detect the SRPK1, AKAP9 and phosphorylated SRSF1 protein expression. The CCK-8 cell proliferation assays, Wound Healing assays, Transwell migration and invasion assays were carried out to detect cell proliferation, migration and invasion in vitro after transfection of SRPK1 gene express plasmid in RNAi-MALAT1-SW480 cell.(5) Knocking down SRPK1 expression in RNAa-MALAT1-SW480 cell, and Western blotting and The nucleus plasma separation technology were used to detect the SRPK1, AKAP9 and phosphorylated SRSF1 protein expression. The CCK-8 cell proliferation assays, Wound Healing assays, Transwell migration and invasion assays were carried out to detect cell proliferation, migration and invasion in vitro after knocking down SRPK1 expression in RNAa-MALAT1-SW480 cell.2. To explore the function of AKAP9 on CRC growth and metastasis in vitro and in vivo(1) RNA interference (RNAi) was targeted on mRNA or promoter region of AKAP9 gene. RNAi cDNA sequence was cloned into the GV115 lentiviral expression vector according to the manufacturer’s instruction (Shanghai Genechem Co). Pseudovirus particles were subsequently used to infect CRC cells. Lovo and HT29 cell lines stably suppressing AKAP9 were generated.(2) The CCK-8 cell proliferation assays, Plate colony formation assays, Wound Healing assays, Transwell migration and Invasion assays were carried out to detect cell proliferation, migration and invasion in vitro after stably suppressing AK.AP9 expression in Lovo and HT29 cell lines.(3) Xenograft tumours were generated by subcutaneous injection to assess the effect of knocking down AKAP9 on tumour growth in vivo. A tail vein xenograft model, in which tumour cells were injected into tail vein, was employed to evaluate the effect of knocking down AKAP9 on the metastasis of CRC cells in vivo.3. The verification of the interaction between AKAP9 and CIP4, and the regulatory effects of AKAP9 on CIP4 expression(1)Co-immunoprecipitation (Co-IP) and Immunofluorescence co-localization were employed to detect the interaction between AKAP9 and CIP4 protein.(2) Western blotting, Quantitative reverse transcription-PCR (qRT-PCR) and Immunofluorescent were used to detect the CIP4 expression in Lovo and HT29 cells with stably suppressing AKAP9 expression.4. The verification of CIP4 expression in CRC cell lines and CRC tissues(1) Western blotting was used to detect the CIP4 expression in normal human fetal colonic mucosa cell line (FHC) and colorectal cancer cell lines including HT29, Lovo, M5, SW620, SW480, LS174T, HCT116 and DLD1.(2) Immunohistochemical was employed to detect the expression of CIP4 and AKAP9 in 38 paired paraffin-embedded CRC tissue samples.5. To analysis the effect of AKAP9-CIP4 on EMT of CRC cell lines(1) Western blotting and Immunofluorescent were used to detect the EMT related protein (E-cadherin、N-cadherin Vimentin) expression in Lovo and HT29 cells with stably suppressing AKAP9 expression.(2) Knocking down CIP4 expression and over-expressing CIP4 expression in Lovo and HT29 cells, and Western blotting was used to detect CIP4, E-cadherin, N-cadherin and Vimentin expression.(3) Lovo and HT29 cells were treated with 5ng/ml TGF-β1 for 48 hours, and Western blotting was used to detect AKAP9, CIP4, E-cadherin, N-cadherin and Vimentin expression.(4) Verifying hypothesis that AKAP9 is required for TGF-β1-induced EMT and CIP4 rescue AKAP9 function in Lovo and HT29 cells via detecting AKAP9, CIP4, E-cadherin, N-cadherin and Vimentin expression by Western blotting.6. To explore the effect of AKAP9-CIP4 on biological function of CRC cell linesCIP4 gene express plasmid was transfected to Lovo and HT29 cells with stably suppressing AK.AP9 expression. And CCK-8 cell proliferation assays, Wound Healing assays, Transwell migration and Invasion assays were carried out to detect cell proliferation, migration and invasion in vitro.7. Statistical analysisSPSS 16.0 software was used for statistical analysis. Quantitative values of all experiments are expressed as the mean ± standard deviation (SD). Relative quantification value(2"△△Ct) of qRT-PCR in cells were analysed by One-way ANOVA, with the SNK, LSD or Dunnett’s T3 tests for multiple comparisons. It also analysed through two-tailed independent-samples t-Test. The data of colony formation assay, Transwell migration and invasion assay were analysed through One-way ANOVA and two-tailed independent-samples t-Test. The data of CCK8 assay was analysed by Factorial design analysis of variance. Correlation analysis between CIP4 and AKAP9 expression in CRC tissues, Normal distributed data choose Pearson correlation coefficient, non-normal distributed data choose Spearman correlation coefficient. Differences were considered significant if P<0.05.Results1.Long non-coding RNA MALAT1 modulates AKAP9 expression and cells biological function via promoting SRPK1-catalyzed SRSF1 phosphorylation in colorectal cancer cells.(1) MALAT1, SRPK1 and SRSF1, interacted with each other and formed a complex.Reverse transcription-PCR (RT-PCR) results in RNA-IP experiment showed that SRPK1 and SRSF1 protein interacted mRNA of MALAT1 in SW480-SRPK1-IP and SW480-SRSF1-IP groups are obviously higher than that in corresponding negative control groups. The total RNAs extracted from each group were reversely transcripted into cDNAs, and Quantitative reverse transcription-PCR (qRT-PCR) results showed that the mRNA of MALAT1 interacted with SRPK1 and SRSF1 protein in SW480-SRPK1-IP and SW480-SRSF1-IP groups are obviously higher than that in corresponding negative control groups (P<0.05, P<0.001). Co-IP experiment results showed that SRPK1 interacts with SRSF1 protein in colorectal cancer SW480 cells. The above experimental results showed that MALAT1, SRPK1 protein and SRSF1 protein interact with each other and form a complex.(2) MALAT1 promoted SRSF1 phosphorylation in nucleus of CRC cell lines.Western blotting and Quantitative reverse transcription-PCR (qRT-PCR) results showed that SRSF1 protein expression in Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells has no statistical differences (F=2.373, P>0.05; F=0.335, P>0.05). However, the nucleus protein of Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells were extracted by the nucleus plasma separation technology, and Western blotting was used to detect phosphorylated SRSF1 protein expression. Compared with Scranmble-SW480 group, the phosphorylated SRSF1 protein expression in RNAa-MALAT1-SW480 group was significantly higher than that in the control group (Scranmble-SW480) (F=71960, P<0.001), however, the phosphorylated SRSF1 protein expression in RNAi-MALAT1-SW480 group was significantly lower than that in the control group (Scranmble-SW480) (F=71960, P<0.001) The immunofluorescence results revealed that phosphorylated SRSF1 protein is enrichment in the nucleus, and MALAT1 activated increases SRSF1 phosphorylation. On the contrary, knockdown of MALAT1 expression decreases SRSF1 phosphorylation in SW480 cells. Thus, MALAT1 had no effect on SRSF1 expression, but MALAT1 positively regulated SRSF1 phosphorylation in nucleus of CRC SW480 cell lines.(3) MALAT1 regulated SRPK1 expression in CRC cell lines, and inhibition of SRPK1 activity inhibited phosphorylated SRSF1 and AKAP9 protein expression in RNAa-MALAT1-SW480 cells.Western blotting and Quantitative reverse transcription-PCR (qRT-PCR) showed that SRPK1 expression in Scranmble-SW480, RNAa-MALAT1-SW480 and RNAi-MALAT1-SW480 cells have statistical differences (F=18.561, P<0.01; F=226.34, P<0.001). The SRPK1 expression in RNAa-MALAT1-SW480 group was significantly higher than that in the control group (Scranmble-SW480), however, the SRPK1 expression in RNAi-MALAT1-SW480 group was significantly lower than that in the control group (Scranmble-SW480). The immunofluorescence results yielded the similar effect. Thus, MALAT1 positively regulated SRPK1 expression in CRC cell lines.The RNAa-MALAT1-SW480 cells were treated with SRPK1 inhibitor SRPIN340 by concentration gradient. The inhibitor were used at 0uM,10 uM, 40 uM and 50 uM for 48 hours respectively, western blotting analysis revealed that SRPK1 inhibitor SRPIN340 can inhibit phosphorylation of SRSF1 and AKAP9 protein induced by MALAT1 in RNAa-MALAT1-SW480 cells with a dose-dependent, compared with control-treated cells (Blank and dimethylsulphoxide 0.02%) (F=0.658.307, P<0.001; F=59.675, P<0.001). However, SRPK1 inhibitor SRPIN340 had no effect on SRPK1 protein expression (F=0.120, P>0.05). Thus, these results showed that SRPIN340 can inhibit SRPK1 activity, but has no effect on SRPK1 protein expression, moreover, SRPIN340 can inhibit phosphorylation of SRSF1 and AKAP9 protein expression.(4) Over-expression of SRPK1 expression restored AKAP9 expression and cell biological function by restoring the level of SRSF1 phosphorylation in RNAi-MALAT1-SW480 cells.The SRPK1 expression plasmids and empty vectors were transduced into RNAi-MALAT1-SW480 cells generating sub-cell lines RNAi-MALAT1/SRPK1 and RNAi-MALAT1/Vector-SW480 cells. Western blotting was used to detect SRPK1, AKAP9 and phosphorylated SRSF1 protein expression, and the results revealed that SRPK1, AKAP9 and phosphorylated SRSF1 protein expression are significantly decreased in RNAi-MALAT1/Vector-SW480 group (F=184.199, P<0.001; F=852.730, P<0.001), compared with Scramble-SW480 group. However, over-expression of SRPK1 expression restored AKAP9 and phosphorylated SRSF1 protein expression in RNAi-MALAT1/SRPK1-SW480 group (F=184.199, P<0.001; F=852.730, P<0.001), compared with RNAi-MALAT1/Vector-SW480 group.Moreover, The nucleus plasma separation technology and Western blotting were conducted to detect the phosphorylated SRSF1 protein expression in nucleus and cytoplasm of Scramble-SW480, RNAi-MALAT1/Vector-SW480 and RNAi-MALAT1/SRPK1-SW480 cells. The results showed that phosphorylated SRSF1 protein expression restored by overexpression SRPK1 is not obvious in cells cytoplasm proteins, but is enriched in cells nucleus proteins. Compared with nucleus protein of Scramble-SW480 group, phosphorylated SRSF1 protein expression was significantly decreased in nucleus protein of RNAi-MALAT1/Vector-SW480 group (F=473.610, P<0.001). However, over-expression of SRPK1 expression restored phosphorylated SRSF1 protein expression in nucleus protein of RNAi-MALAT1/SRPK1-SW480 group (F=473.610, P<0.001), compared with nucleus protein of RNAi-MALAT1/Vector-SW480 group. The restored level of SRSF1 phosphorylation in nucleus restored alternative splicing of AKAP9 Pre-mRNA, thus restoring SRPK1 protein expression in RNAi-MALAT1-SW480 cells restored AKAP9 protein expression.In addition, CCK8 assays showed that cell proliferation ability is significantly lower in RNAi-MALAT1/Vector-SW480 group than that in Scramble-SW480 group (F=155.933, P<0.001), however, over-expression of SRPK1 expression restored proliferation ability in RNAi-MALAT1/SRPK1-SW480 group (F=155.933, P<0.001), compared with RNAi-MALAT1/Vector-SW480 group. Wound healing and Transwell migration assays showed that migration abilities are significantly lower in RNAi-MALAT1/Vector-SW480 group than that i Scramble-SW480 group(F=2645,P＜0.001;F=2020,P＜0.001)，however, over-expression of SRPK1 expression restored migration abilities in RNAi-MALAT1/SRPK1-SW480 group (F= 2645, P<0.001; F=2020, P<0.001) compared with RNAi-MALAT1/Vector-SW480 group. Transwell invasion assay showed that invasion ability is significantly lower in RNAi-MALAT1/Vector-SW480 group than that in Scramble-SW480 group (F= 612.122, P<0.001), however, over-expression of SRPK1 expression restored invasion ability in RNAi-MALAT1/SRPK1-SW480 group(F= 612.122, P<0.001), compared with RNAi-MALAT1/Vector-SW480 group.(5) Down-regulation of SRPK1 expression attenuated AKAP9 expression and cells biological function by reducing the level of SRSF1 phosphorylation in RNAa-MALAT1-SW480 cells.Two siRNAs were used to knock down SRPK1 expression, and two siRNAs and control were transduced into RNAa-MALAT1-SW480 cells generating sub-cell lines RNAa-MALAT1/siCtrl, RNAa-MALAT1/siSRPK1-1 and RNAi-MALAT1 /siSRPK1-2-SW480 cells. Western blotting was used to detect SRPK1, AKAP9 and phosphorylated SRSF1 protein expression, and the results revealed that SRPK1, AKAP9 and phosphorylated SRSF1 protein expression are significantly increased in RNAa-MALAT1/siCtrl-SW480 group (F= 804.517, P<0.001; F=4417, P<0.001) , compared with Scramble-SW480 group. However, knockdown of SRPK1 expression attenuated AKAP9 and phosphorylated SRSF1 protein expression in RNAa-MALAT1/siSRPK1-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 groups (F=804.517, P<0.001; F=4417, P<0.001), compared with RNAa-MALAT1/siCtrl-SW480 group.Moreover, The nucleus plasma separation technology and Western blotting were conducted to detect the phosphorylated SRSF1 protein expression in nucleus and cytoplasm of Scramble-SW480, RNAa-MALAT1/siCtrl-SW480 RNAa-MALAT1/siSRPK1-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 cells. The results showed that phosphorylated SRSF1 protein expression is not obvious in cells cytoplasm proteins, but is enriched in cells nucleus proteins. Compared with nucleus protein of Scramble-SW480 group, phosphorylated SRSF1 protein expression was significantly increased in nucleus protein of RNAa-MALAT1/siCtrl-SW480 group (F=286.811, P<0.001). However, knockdown of SRPK1 expression attenuated phosphorylated SRSF1 protein expression in nucleus protein of RNAa-MALAT1/siSRPK1-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 group (F=286.811, P<0.001), compared with nucleus protein of RNAi-MALAT1/siCtrl-SW480 group. The reduced level of SRSF1 phosphorylation in nucleus attenuated alternative splicing of AKAP9 Pre-mRNA, thus down-regulation of SRPK1 protein expression in RNAa-MALAT1-SW480 cells attenuated AKAP9 protein expression.In addition, CCK8 assays showed that cell proliferation ability is significantly higher in RNAa-MALAT1/siCtrl-SW480 group than that in Scramble-SW480 group (F=381.239, P<0.001), however, knockdown of SRPK1 expression attenuated proliferation ability in RNAa-MALAT1/siSRPKl-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 groups (F=381.239, P<0.001), compared with RNAa-MALAT1/siCtrl-SW480 group. Wound healing and Transwell migration assays showed that migration abilities are significantly higher in RNAa-MALAT1/siCtrl-SW480 group than that in Scramble-SW480 group (F=200.39, P<0.001; F=128.877, P<0.001), however, knockdown of SRPK1 expression attenuated migration abilities in RNAa-MALAT1/siSRPK1-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 groups (F=200.39, P<0.001; F=128.877, P<0.001), compared with RNAa-MALAT1/siCtrl-SW480 group. Transwell invasion assay showed that invasion ability is significantly higher in RNAa-MALAT1/siCtrl-SW480 group than that in Scramble-SW480 group (F=396.765, P<0.001), however, knockdown of SRPK1 expression attenuated invasion ability in RNAa-MALAT1/siSRPK1-1-SW480 and RNAa-MALAT1/siSRPK1-2-SW480 groups (F=396.765, P<0.001), compared with RNAa-MALAT1/siCtrl-SW480 group.The above results indicated that MALAT1 modulates AKAP9 expression via regulating SRPK1-catalyzed SRSF1 phosphorylation, consequently, regulates proliferation, migration and invasion abilities of CRC cell lines.2. AKAP9 promotes CRC growth and invasion in vitro and in vivo(1) The Lovo and HT29 cells with high AKAP9 expression were selected to knock down AKAP9 expression. The sliencing AKAP9 expression lentiviruses were stably transduced into Lovo and HT29 cells generating sub-cell lines Lovo/shAKAP9 and HT29/shAKAP9, their controls were infected with lentiviruses containing empty vectors. Western blotting and Quantitative reverse transcription-PCR (qRT-PCR) showed that the protein and mRNA level of AKAP9 expression were significantly knocked down in Lovo/shAKAP9 and HT29/shAKAP9 cells (F=13.446, P<0.01, F=9.529, P<0.05; F=8.46, P<0.01, F=2.723, P<0.05), compared with Lovo/shCtrl and HT29/shCtrl cells. The immunofluorescence assay yielded the similar effect.(2) CCK-8 assay showed that the down-regulation of AKAP9 inhibited cell proliferation of Lovo and HT29 cells compared with their control cells (Lovo/shCtrl and HT29/shCtrl) (P<0.01; P<0.001). The plate colony formation assays yielded the similar effect (P<0.01; P<0.001)(3) Wound healing assay showed that down-regulation of AKAP9 markedly repressed the migration healing ability of Lovo and HT29 cells compared with their control cells (Lovo/shCtrl and HT29/shCtrl) (P<0.001; P<0.05). Transwell migration assay showed that down-regulation of AKAP9 markedly repressed the migration ability of Lovo and HT29 cells compared with their control cells (Lovo/shCtrl and HT29/shCtrl) (P<0.001; P<0.001). Transwell invasion assay showed that down-regulation of AKAP9 markedly repressed the invasion ability of Lovo and HT29 cells compared with their control cells (Lovo/shCtrl and HT29/shCtrl) (P<0.001; P<0.001)(4) Subcutaneous tumor growth in the Lovo/shAKAP9 and HT29/shAKAP9 groups was slower than that in the Lovo/shCtrl and HT29/shCtrl groups, and the tumor size in Lovo/shAKAP9 and HT29/shAKAP9 groups was smaller than that in the Lovo/shCtrl and HT29/shCtrl groups (P<0.001; P<0.001). Immunostaining confirmed that the AKAP9 expression and the cell proliferation index Ki-67 were down-regulated (P<0.01, P<0.01; P<0.01, P<0.01)(5) Tail vein xenograft model showed that no mice in Lovo/shAKAP9 and HT29/shAKAP9 groups have lung metastatic lesions compared with Lovo/shCtrl and HT29/shCtrl groups, however,5/6 of mice in the Lovo/shCtrl group and 4/6 of mice in the HT29/shCtrl group have lung metastatic lesions. No metastatic nodules are discovered in the other organs in any group. The number of mice with lung metastatic lesions in the two groups were significant differences (P= 0.003; P= 0.014)3. AKAP9 interacted with CIP4 and regulated CIP4 expression in CRC cell lines.(1) Co-IP assay showed that AKAP9 protein can specific interact with CIP4 protein in Lovo and HT29 cells; similarly, immunofluorescence co-localization analysis revealed co-localization of AKAP9 and CIP4 proteins in Lovo and HT29 cells.(2) Western blotting and Quantitative reverse transcription-PCR (qRT-PCR) showed that the protein and mRNA level of AKAP9 and CIP4 expression are significantly knocked down in Lovo/shAKAP9 and HT29/shAKAP9 cells (F=3.829, P<0.01,F=2.84, P< 0.001; F=7.248, P<0.001, F=6.285, P<0.01; F=5.170, P <0.001, F=13.077, P<0.01; F=8.780, P<0.01, F=13.914, P<0.01), compared with Lovo/shCtrl and HT29/shCtrl cells. The immunofluorescence assay yielded the similar effect.The above results showed that AKAP9 can interact with CIP4 and regulate CIP4 expression in CRC cell lines.4. CIP4 was up-regulated in CRC cell lines and CRC tissues.(1) We examined CIP4 expression in HT29, Lovo, M5, SW620, SW480, LS174T, HCT116 and DLD1 CRC cell lines and normal human fetal colonic mucosa cell line (FHC) by Western blotting. Relative CIP4 expression was significant difference between FHC cell and the CRC cell lines (F=4741, P<0.001). The Dunnett’s T3 multiple comparison indicated the expression of CIP4 in CRC cell lines was higher than that in the FHC cell. Meanwhile, The expression of CIP4 was positively correlated with AKAP9 expression in the most of CRC cell lines.(2) Immunohistochemical showed that CIP4 and AKAP9 are significantly increased in 30 of 38 CRC tissues specimens, and the average expression level of CIP4 and AKAP9 are significantly higher in 38 CRC tissues than that in 38 normal counterparts (P<0.05; P<0.05). Moreover, CIP4 and AKAP9 expression in 38 cases of colorectal cancer tissue samples have positive correlation (r=0.337; P =0.038).5. AKAP9-CIP4 was involved in TGF-β1-induced EMT.(1) Western blotting and immunofluorescence assays were used to detect the EMT related proteins (E-cadherin, N-cadherin, Vimentin) expression in Lovo/shAKAP9 and HT29/shAKAP9 cells, and Western blotting results revealed that E-cadherin expression is significantly up-regulated in Lovo/shAKAP9 and HT29/shAKAP9 cells (F=2.571, P<0.001; F=0.348, P<0.01), and N-cadherin and Vimentin expression are down-regulated in Lovo/shAKAP9 and HT29/shAKAP9 cells (F=4.121, P<0.01,F=3.118, P<0.01; F=2.671, P<0.001, F=5.984, P< 0.05), compared with Lovo/shCtrl and HT29/shCtrl cells. The immunofluorescence assay yielded the similar effect.(2) Lovo and HT29 cells were transfected with two siRNAs to knock down CIP4 protein expression, and the proteins were preformed by Western blotting to detect CIP4, E-cadherin, N-cadherin, Vimentin protein expression. The results showed that CIP4, N-cadherin and Vimentin protein expression are significantly decreased in Lovo and HT29 cells with knocking down CIP4 expression (Lovo/siCIP4-1,Lovo/siCIP4-2, HT29/siCIP4-1, HT29/siCIP4-2) (F=455.855, P <0.001, F=6572, P<0.001; F=892.652, P<0.001; F=276.189, P<0.001), however, the E-cadherin protein expression is significantly increased in Lovo/siCIP4-1, Lovo/siCIP4-2 and HT29/siCIP4-1, HT29/siCIP4-2 groups (F=3658, P<0.001; F=155.015, P<0.001), compared with Lovo/siCtrl and HT29/siCtrl groups. Meanwhile, Lovo and HT29 cells were transfected with CIP4 expressing plasmid to over-express CIP4 protein expression, and the cell proteins were preformed by Western blotting to detect CIP4, E-cadherin, N-cadherin, Vimentin protein expression. The results showed that CIP4, N-cadherin and Vimentin protein expression are significantly increased in Lovo and HT29 cells with over-expressing CIP4 expression (Lovo/CIP4 and HT29/CIP4) (F=0.175,P<0.001, F=4.377,P< 0.001; F=6.472, P<0.01; F=0.022, P<0.001), however, the E-cadherin protein expression is significantly decreased in Lovo/CIP4 and HT29/CIP4 groups (F=7.654, P<0.01; F=3.519, P<0.001), compared with Lovo/Vector and HT29/Vector groups.(3) Lovo and HT29 cells were treated with 5ng/ml TGF-β1 for 48 hours, and the cell proteins were preformed by Western blotting. The results showed that the AKAP9, CIP4 and N-cadherin protein expression are significantly increased in Lovo and HT29 cells treated with 5ng/ml TGF-β1 (Lovo/TGF-β1 and HT29/TGF-β1) (F=4.565, P<0.01,F=10.799, P<0.05; F=4.37, P<0.001, F=6.397, P<0.05; F=0.506, P<0.05; F=0.032, P<0.001), however, the E-cadherin protein expression is significantly decreased in Lovo/TGF-β1 and HT29/TGF-β1 groups (F=9.616, P< 0.01; F=13.085, P<0.01), compared with Lovo/Control and HT29/Control groups.(4) Western blotting assay showed that:compared with the naked Lovo and HT29 cells, TGF-β1 stimulates the cells and induces AKAP9 and CIP4 expression, moreover, induces EMT of the cells. Compared with Lovo and HT29 cells treated with TGF-β1 and shCtrl, AKAP9 and CIP4 expression are down-regulated in Lovo and HT29 cells treated with TGF-β1 and shAKAP9, and EMT of cells is inhibited. However, compared with Lovo and HT29 cells treated with TGF-β1 and shAKAP9, CIP4 expression is restored in Lovo and HT29 cells treated with TGF-β1, shAKAP9 and CIP4, and EMT of cells is restored (F=846.921, P<0.001, F=151.105, P< 0.001; F=1746,P<0.001, F=627.425, P<0.001; F=297.011, P<0.001, F=7027, P <0.001; F=289.121, P<0.001, F=397.951, P<0.001; F=330.430, P<0.001, F=278700,P<0.001)The above results showed that AKAP9-CIP4 is involved in TGF-β1-induced EMT.6. CIP4 was required for AKAP9-induced proliferation, migration and invasion ability of CRC cell lines.(1) CCK-8 assay showed that the proliferative ability of cells is significantly lower in Lovo/shAKAP9 and HT29/shAKAP9 groups than that in Lovo/shCtrl and HT29/shCtrl groups, however, the proliferative ability of cells is significantly restored in Lovo/shAKAP9/CIP4 and HT29/shAKAP9/CIP4 groups, compared with Lovo/shAKAP9 and HT29/shAKAP9 groups (P<0.001, P<0.001).(2) Wound healing assay showed that the migration healing ability of cells is significantly lower in Lovo/shAKAP9 and HT29/shAKAP9 groups than that in Lovo/shCtrl and HT29/shCtrl groups, however, the migration healing ability of cells is significantly restored in Lovo/shAKAP9/CIP4 and HT29/shAKAP9/CIP4 groups, compared with Lovo/shAKAP9 and HT29/shAKAP9 groups (P<0.01, P<0.001). Transwell migration assay showed that the migration ability of cells is significantly lower in Lovo/shAKAP9 and HT29/shAKAP9 groups than that in Lovo/shCtrl and HT29/shCtrl groups, however, the migration ability of cells is significantly restored in Lovo/shAKAP9/CIP4 and HT29/shAKAP9/CIP4 groups, compared with Lovo/shAKAP9 and HT29/shAKAP9 groups (P<0.001, P<0.001).(3) Transwell invasion assay showed that the invasion ability of cells is significantly lower in Lovo/shAKAP9 and HT29/shAKAP9 groups than that in Lovo/shCtrl and HT29/shCtrl groups, however, the invasion ability of cells is significantly restored in Lovo/shAKAP9/CIP4 and HT29/shAKAP9/CIP4 groups, compared with Lovo/shAKAP9 and HT29/shAKAP9 groups (P<0.001, P<0.001).The above results showed that AKAP9 regulates EMT and invasion and metastasis abilities of CRC cell lines by regulating CIP4 expression.Conclusion1. Long non-coding RNA MALAT1 modulates AKAP9 expression via promoting SRPK.1-catalyzed SRSF1 phosphorylation, consequently, regulates growth, migration and invasion ability of colorectal cancer cells.2. AKAP9 promotes tumour growth, invasion and metastasis in vitro and in vivo.3. AKAP9 can interact with CIP4, and regulate CIP4 expression in colorectal cancer cells.4.CIP4 expression is up-regulated in colorectal cancer cells and tissues, and there is a positively correlation between CIP4 and AKAP9 expression in colorectal cancer cells and tissues.5.AKAP9-CIP4 was involved in TGF-β1-induced EMT.6.CIP4 was required for AKAP9-induced growth, migration and invasion of colorectal cancer cells. |
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