The Expresssion Of MALAT1Target Gene AKAP9in Colorectal Cancer And Its Regulation Mechanism

Colorectal cancer (CRC) is one of the most common malignant tumors, the morbidity and mortality are second to gastric cancer, esophageal cancer and primary liver cancer.The operation is the main treatment, but the result is poor, especially for the latter period.Metastasis is the main cause of the death of CRC patients.Many patients have occurred mirco-metastasis before the operation,and metastasis is the direct cause of recurrence.therefore, the key to improve the survival rate and cure rate in patients with colorectal cancer,is that clarifying the molecular mechanism in the invasion and metastasis of colorectal cancer.Our preliminary researches confirmed that the non-coding RNA MALAT1played an important role in colorectal cancer metastasis by regulating metastasis-associated target genes.AKAP9(A-Kinase anchoring protein9) is one of the important target gene regulated by MALAT1.AKAP9is a member of AKAPS family,and its protein located in the centrosome, Golgi apparatus and gene located in chromosome7q21-q22. The overall length of AKAP9is169.81Kb. the1-8exons of encoding gene of AKAP9gene can produce multiple isomers by alternative splicing, including AKAP450, AKAP350, AKAP120and Yotiao, etc. the function of yotiao is the most active in the AKAPs family.Yotiao can activate PKA and promote the expression of Actin.The study of the relationship of AKAP9and morbidity of colorectal cancer is still in early stages. There is no literature reported regarding AKAP9and metastasis of colorectal cancer.It is not clear that the mechanism of the regulation of MALAT1to AKAP9and the role of AKAP9in tumor metastasis.Some studies have shown that MALAT1regulates alternative splicing of associated genes by regulating SR proteins in cells. So we proposed that MALAT1activates AKAP9-PKA-EMT signaling pathway and promotes invasion and metastasis of colorectal cancer through regulating the SR proteins.The main content of this research is as the following:1, Detecting the expression of AKAP9in different transfer ability colorectal cancer cell lines and cancer tissue samples.2, Using RNA-FISH and RNA-IP experimental techniques to analyse the regulation of MALAT1to AKAP9gene by regulating SR proteins after overexpression/knockdown MALAT1in colorectal cancer cells.This thesis mainly focuses on the expression of AKAP9in different transfer ability colorectal cancer cell lines and cancer tissue samples and the regulation of MALAT1to SR protein. PART â… .Expression of AKAP9in colorectal cancer cellsThe expression of AKAP9gene in eight colorectal carcinoma cell lines was detected by real-time PCR and Western blot.At the same time the expression of AKAP9in colorectal carcinoma cell was examined by cell immunohistochemisty.Real-time PCR and Western blot detected expression of AKAP9in eight kinds of colorectal cancer cell lines. The results showed that all of the cell lines expressed AKAP9, LOVO,SW620had the stronger expression, SW480/M5, which was a human CRC cell subline with unique liver metastatic potential, established in our laboratory and used for analysis also had strong expression. The expression of AKAP9gene in SW480, HCT116, H and DLD1cell lines were lower to medium levels.The expression of AKAP9protein in colorectal cancer cells was detected by cell immunohistochemical. The results shows that the AKAP9protein expression of positive signal located within the cytoplasm, HCT116, SW480, DLD1had the negative expression, LOVO, HT29, LSI74T, SW620, M5had Positive expression. PART II.AKAP9expression in the tissue of colorectal cancer and its clinical significance54cases of fresh colorectal cancer tissues and cut edge normal mucosa tissue (10cm away from canceration edge) from general surgery of Nanfang Hospital were collected. The samples were put in liquid nitrogen immediately after surgical resection, In54cases of paired fresh CRC tissues, the expression of AKAP9in CRC tissue was significantly higher than in normal tissues (P<0.05), By the further study, there are statistically differences between the cancer tissues without metastasis and tissues with metastasis (P=0.007)Paraffin-embedded CRC tissue samples from113patients were obtained from the January2000and December2007at Nanfang Hospital, Southern Medical University. All of the enrolled patients have full-detailed clinicopathological information and follow-up results.Immunohistochemical detected the expression of AKAP9protein in primary colorectal carcinoma and corresponding normal colorectal mucosa tissues, found their correlation with metastasis of colorectal cancer. result indicated AKAP9expression was higher in113CRC tissues than in adjacent normal tissues (P<0.05), Its expression was positively correlated with patients ages and tumor depth of invasion (P=0.026; P=0.000). PART â…¢. The mechanism of MALAT1regulation SR protein and AKAP9in colorectal cancer cellsMALAT1stable overexpression cell lines were setted up and the interference of MALAT1stable cell lines was setted by using vector pEGFP C1-MALAT1/FGF and pGPU6carrier/GFP/NeoshRNA.The expression of MALAT1and SR protein were detected by RNA-FISH in colorectal cancer cells, Results showed that the MALAT1mRNA are located in the nucleus, and the exprssion is higher and display a strong co-localization with SR proteins compared with MALAT1SW480-RNAi cells.The quantitative expression of MALAT1and SR protein were detected the in colorectal cancer cells by RNA-IP. Results showed that the mRNA expression of MALAT1combined with SR proteins have statistically differences in MALAT1SW480-NC-SRSF1-IP group, the MALAT1-SW480RNAi-SRSF1-IP group, the MALAT1-SW480-RNAa-SRSF1-IP group (F=93.472, P<0.001). MALAT1and AKAP9co-expression were detected by Real-time PCR and Western blot, Western blot results and real-time PCR results are consistent. MALAT1and AKAP9had higher expression in MALATl-SW480-RMAa cells and had lower expression in MALAT1-SW480-RMAi cells.Conclusion:AKAP9gene is expressed in colorectal cancer cell lines and cancer tissue. The Expression of AKAP9is related with the metastasis and invasion of colorectal cancer. MALAT1regulate the expression of AKAP9thruogh the regulateion of SR protein.