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Study on the biological role in colon cancer cell line and its mechanism MicroRNA-222

Author: CongXueJing
Tutor: BaiChunMei
School: Beijing Union Medical College
Course: Clinical
Keywords: miR-222 colorectal cancer biological effect target gene
CLC: R735.35
Type: PhD thesis
Year: 2012
Downloads: 54
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Colorectal cancer (CRC) is the third most frequently occurring cancer and the fourth most common causes of cancer-related death worldwide. Although the treatment methods of surgery, chemotherapy, radiotherapy and targeted therapy have developed rapidly in recent years, the metastasis rate and fatality rate are still high. The5-year survival rate of stage IV patients is as low as8.1%. Therefore, further knowledge about the molecular mechanisms of CRC progression, especially those of metastasis is of great importance.MicroRNAs (miRNAs) are noncoding RNAs of approximate22nt in length that function as post-transcriptional regulators by base-pairing with the complementary sites in the3’ untranslated region(3’UTR) of the mRNA, inducing mRNA cleavage or translational repression. miRNAs exist in various normal tissues and are involved in biological processes, such as proliferation, differentiation and apoptosis, etc. miRNAs can function as either oncogenes or tumor suppressor genes depending on the function of target genes and are responsible for the initiation and progression of tumors, CRC included.Higher expression levels of miR-222were detected in CRC tumor tissues, by studying the miRNA expression profiles in the tumor and tumor-adjacent tissues of6advanced CRC patients in the Oncology Dept. of Peking Union Medical College Hospital. The result is consistent with two previous researches. We suspected that miR-222might play a role in the progression of CRC, however, the molecular mechanisms have not been elucidated yet.In this study, the human colorectal cancer cell lines HCT8and HCT116were transfected with synthesized miR-222mimics or miR-222inhibitor to upregulate or downregulate the miR-222expression respectively. The transfection efficiency was confirmed by real-time PCR. Changes in the cell proliferation, cell cycle, apoptosis, migration and chemosensitivity were assessed after the miR-222over-expression and down-regulation. The regulation mechanisms of miR-222were preliminarily explored.Section1, the biological behavior effects of miR-222on HCT8and HCT116. No statistical difference was shown in cell proliferation rate in either the miR-222over-expression or the miR-222inhibition group, compared with the respective control groups (P>0.05)(cell number detected by MTS). The effect of miR-222on HCT8and HCT116cell cycle was evaluated by flow cytometry. No statistical difference was shown in cell percentage in G0/G1, S, G2/M phase in either the miR-222over-expression or the miR-222inhibition group, compared with the respective control groups (P>0.05). Annexin V/PI staining was applied to distinguish cells of early apoptosis and late apoptosis. In either of the two groups, few cells were detected in early or late apoptosis and there was no statistical difference in cell percentage, compared with the respective control groups (P>0.05). Cell migration ability was evaluated by transwell and expressed as the average cell number on transwell membrane in each view. Significantly stronger migration ability was shown in the HCT8of miR-222over-expression group (96.315±13.14vs mimics control43.18±8.06, P<0.001). Correspondingly, significantly weaker migration ability was shown in the miR-222inhibition group (9.31±3.62vs inhibitor control46.56±6.81, P<0.001). To sum up, it was possible that miR-222significantly enhanced the capability of migration in HCT8cell lines, while no effects of miR-222were observed on the HCT8and HCT116cell lines’ proliferation, cell cycle or apoptosis.Section2, the effects of miR-222on the chemosensitivity of HCT8.5-fluorouracil and oxaliplatin was added to the HCT8cell lines transfected with miR-222mimics, miR-222inhibitor and the respective controls in different concentrations in day0,2and5. Inhibition rate was calculated based on the MTS results. No statistical difference was shown in cell inhibition rate in either the miR-222over-expression or miR-222inhibition group, compared with the respective control groups in each drug concentration and each time point (all P>0.05). The results indicated that miR-222was less likely to effect on the sensitivity of HCT8cell line to5-fluorouracil and oxaliplatin.Section3, we predicted the miR-222target genes on promoting migration. Candidate genes were selected from TargetScan and Pictar. We found that at least one conserved sequence in the3’UTR of TIMP2, RECK, MIA3, STK24, PLXNC1is perfectly complementary to the seed sequence of miR-222. And they all play a suppression role in tumor migration and are potential targets for miR-222.Conclusively, in this study, miR-222had no influence on HCT8and HCT116cell proliferation, cell cycle, apoptosis, or chemosensitivity. Notably, miR-222facilitated the migration ability of HCT8cell lines possibly via the inhibition of TIMP2, RECK, MIA3, STK24, PLXNC1by base-pairing with the complementary sites in the3’ untranslated region(3’UTR) of these mRNAs.Our study demonstrates, for the first time, that miR-222is possible to function as an oncomiR in CRC by promoting cell migration.5genes were selected as the potential targets for miR-222. Our study is a beginning of the study on molecular mechanisms for miR-222in CRC.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms > Colon tumor
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