Dissertation
Dissertation > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms

Study on the Regulation Machanism of Non-Coding RNA Gene MALAT1 in Colorectal Carcinoma Metastasis

Author YangMinHui
Tutor LiZuGuo
School Southern Medical University,
Course Pathology and Pathophysiology
Keywords MALAT 1 lncRNA Colorectal carcinoma Metastasis target gene RNA interference RNA activation realtime PCR RNA in situ hybridization proteomics bioinformatics prediction APAK-9
CLC R735.3
Type Master's thesis
Year 2010
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Completion of Human Genome Project and flourishing of tRNAscriptomic and proteomics have promoted the prosperous development of the RNomics. noncoding RNA has gradually got wide attention and recognition for it’s important role in people’s life process. Currently, human genomic researchs focus on annotation or functional partitioning of open reading frame(ORF). In eukaryotic cells, only a small fraction of the genes can encode amino acid sequences of proteins, However, the rest of sequences are unknown function sequence play extremely important roles in regulating gene expression. non-coding RNAs account for 98 percent of human genome, considered play key roles in cell biological function. non-coding RNA participate in transcription regulation, DNA duplication, chromosome pairing and chromosome aggregation. The non-coding sequences of the human genome may participate in DNA transcription regulation, copy, chromosome pairing and chromosome aggregation, etc. The latest figures show that many functional RNA molecules may hid between protein coding areas or into introns, its function should be further clarified.In our previous work, we utilized gene expression profile chip to identify differences in gene expression between colorectal cancer cells that either high or low subsequently metastasize. In total,289 independent upregulating genes were obtained in high subsequently metastasize. These upregulating genes included MALAT 1 (metastasis associated lung adenocarcinoma transcriptl). MALAT 1 is one of lncRNA (long noncoding RNA). It locates at chromosome 11q13. MALAT 1, 8.7kb at length and lack of clear open reading frame, is highly expressed in the colorectal cancer tissue compared with the counterpart resection margin of specimens. Therefore, we hypothesized that MALAT 1 are related to the regulation of the metastasis of colorectal carcinoma.To confirm our hypothesis, in situ hybridization and realtime-PCR were used to examine the expressions of MALAT 1 in 9 colorectal carcinoma cell lines and colorectal cancer tissue. After that, RNAi (RNA interfere)and RNAa (RNA activation) were performed to explore MALAT 1 functions in proliferation and invasion of colorectal carcinoma cells. At last, we used bio-informatics analytical methodology and proteomic techniques on colorectal carcinoma cells with different metastatic potentials to explore the differentially expressed proteins and to identify the gene targets of MALAT 1.PART I The expression of MALAT 1 in colorectal cancer tissues and cell linesRT-PCR, realtime-PCR and in situ hybridization were used to examine the expressions of MALAT 1 in 9 colorectal carcinoma cell lines and colorectal cancer tissue. The result of RT-PCR showed that higher MALAT 1 expressions in cell lines with high metastatic potential compared with low metastatic potential cells, and the difference was significant (t=-8.514, P<0.001). The expressions of MALAT 1 in COLO-205, LoVo and SW620 cells were higher than SW480/M5, HT29 and HCT116 cells. The difference was significant (P<0.05). Simultaneously, the expressions of MALAT 1 in Caco-2 and LS174 T cells were lowest in all. The result of realtime-PCR showed that higher MALAT 1 expressions in cell lines with high metastatic potential compared with low metastatic potential cells, and the difference was significant (t=-7.189, P<0.001). The expressions of MALAT 1 in COLO-205, LoVo, SW620 and SW480/M5 cells were higher than others, and the difference was significant (P<0.05). The result of in situ hybridization showed that higher MALAT 1 expressions in cell lines with high metastatic potential compared with low metastatic potential cells, and the difference was significant (χ2=8.586, P<0.05). The expressions of MALAT 1 in LoVo and SW620 cells were higher than others, and the difference was significant (P<0.05). The results of RT-PCR, realtime-PCR were well consistent with the results of in in situ hybridization.Reluts of RT-PCR showed that MALAT 1 expression were different in human colorectal normal tissue and malignant tumor. The expression of MALAT 1 in tumor tissues were significantly higher than normal tissue (F=99.784, P<0.001).The highest expression of MALAT 1 was in metastatic carcinoma of lymph node, and the difference was significant (P<0.01). Reluts of realtime-PCR showed that MALAT 1 expression were different in human colorectal normal tissue and malignant tumor. The expression of MALAT 1 in tumor tissues were significantly higher than normal tissue (F=182.138, P<0.001).The highest expression of MALAT 1 was in metastatic carcinoma of lymph node, and the difference was significant (P<0.01). The results of RT-PCR were well consistent with the results of realtime-PCR.PART II RNAa and RNAi of MALAT 1 in colorectal cancer cells, investigate the influence of MALAT 1 expression in Cell biologyThe specific RNA interference and RNA activation fragments of MALAT 1 gene were produced and trnasfected into SW620 cell line, with 60%-70% of the transfective efficiency detected by fluorescence microscope. The results of real time PCR showed that the RNA activation efficiency of MALAT 1 was 0.271±0.024 folds (F=177.972, P<0.001), and the RNA interference efficiency of MALAT 1 was 2.687±0.114 folds(F=1701.135,P<0.001). MTT method was carried out to observe the cell growth in vitro after MALAT 1 gene activation and silencing. Compared to SW620 cell line, RNA activation vector transfected cell had higher proliferative rate in the time-dependent manner (F=1153.007, P<0.001). The changes of cell invasive abilities after MALAT 1 gene activation were detected by invasive chamber experiment in vitro, and the results showed that compared to SW620 cell, the invasive abilities of RNA activation vector transfected cell increased significantly (F =1153.007, P<0.001). The changes of cell proliferative activity after MALAT 1 gene activation were detected by the plating efficiency of cell clone experiment in vitro, and the results showed that the invasive abilities of RNA activation vector transfected cell increased significantly (F=313.421, P<0.001) compared with SW620 cell. On the contrary, the changes of cell growth activity after MALAT 1 gene interference were detected by the MTT experiment in vitro and the results showed that compared to SW620 cell, the invasive abilities of RNA interference vector transfected cell decreased significantly (F=1701.135, P<0.001). Compared to SW620 cell, the invasive abilities of RNA interference vector transfected cell decreased significantly (t=44.472, P<0.001), and the proliferative activity of RNA interference vector transfected cell decreased significantly (F=212.160, P<0.001).Moreover, the MALAT 1/fgf (MALAT 1-functional gene fragment) recombinant vector were produced transfected into SW620 cell line. However, after transfected, we found that the expression of MALAT 1/fgf was increased(F=200.093, P<0.001), but there were no significant change of expression of the rest sequence in MALAT 1 (F=1.674, P=0.264). No significant changes of growth (F=0.892, P=0.452), proliferative (F=0.307, P=0.820) and invasive abilities in SW620 cell were found (F=4.257,P=0.071).In this study, we achieve for the first time RNA activation in vitro of lncRNA. After RNA activation of MALAT 1 in SW620 cell, we found that the growth, proliferative and invasive abilities were increase significantly compared with non transfected cells. In contrast, after RNA interference of MALAT 1 in SW620 cell, we found that the above mantioned abilities were decrease significantly compared with non transfected cells. Simultaneously, after transfected MALAT 1/fgf expression vector, and no significant changes of growth, proliferative and invasive abilities in SW620 cell were found. So we hypothesized that only when sequences of MALAT 1 completed can biological characteristics of human colorectal carcinoma be changed.PARTⅢBioinformatics and proteomics identification of MALAT-1 target genesThe protein 2D maps of SW620 cells and SW620-MALAT 1-dsRNAl vector transfected cells were analyzed by ImageMaster5.0, and we found 15 differentially expressed proteins, including 6 proteins successfully identified. Compared with SW620 cell, SW620-MALAT 1-dsRNA1 vector transfected cell had 3 up-regulated proteins and 3 down-regulated ones.There is little difference between the protein spots maps of HCT 116 and HCT-MALAT 1/fgf-5235 cells, we found that HCT 116 cells and MALAT 1/fgf-5235 vector transfected one. In this study, we found that 2 down-regulated protein spots exists in HCT 116-MALAT 1-RNAi vector transfected cells.Through bioinformatics analysis, we obtained 47 potential target genes of MALAT 1. We found that most of proteins are associated with tumor malignant transformation and metastasis behaviors involving tumor cell growth, movement, adhesions and apoptosis process. To illustrate the target genes of MALAT 1 regulated, we took these potential target genes combine with proteomics analysis. At last, we found that APAK-9 is one of MALAT-1 target genes.

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