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The Preliminary Study of Hsa-miR-622Inhibits Radiotherapy Sensitivity of Patients with Advanced Rectal Cancer by Targeting RB1

Author: YuJiang
Tutor: DingYanQing
School: Southern Medical University,
Course: Pathology and Pathophysiology
Keywords: miRNA hsa-miR-622RB1 rectal cancer rediosensitivity bioinformatics
CLC: R735.37
Type: PhD thesis
Year: 2013
Downloads: 13
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Abstract


Background and purposeEpidemiological characteristics of colorectal cancer in China suggest that colorectal cancer especially low rectal cancer important disease is an important disease which affects the health. With the development of economy, the rapid change in lifestyle, diet structure, the incidence of colorectal cancer in China are increasing, the incidence is trending gradually to the level of Europe and the United States closely. But compared with western developed countries, the epidemiology of colorectal cancer in China has its own characteristics; first, the age of patients is younger, median age of onset of colorectal cancer at the age of45, than the patients in Europe and the United States about12to18years. Second, middle and low rectal cancer is more frequency detected. Rectal cancer accout for about70%of the colorectal cancer and70%of colorectal cancer locates in8cm from the anal verge. Third, large numbers of advanced patients greatly influences the treatment the effect. As a consequence, the5year survival rate has remained at a low level.Preoperative neoadjuvant radiotherapy has become the standard scheme for the treatment of advanced lower rectal cancer, worthy of further research on molecular markers for predicting the radiosensitivity. In recent years, with a few big sample randomized controlled study completion in Europe and the United States, adjuvant radiotherapy showed preoperative chemoradiotherapy has more andvantages compared with afteroderative for the treatment of advanced rectal cancer, first, the surrounding area of tumor blood supplication did not destroy, so the tumor has good blood supply and high oxygenayion, result in the exact effect of preoperative radiotherapy. Second, the tumor degradation significantly after neoadjuvant radiotherapy compared with high negative margin rate and doubled anus preserving operation rate. Third, preoperative radiotherapy will increase tumor local control rate than that after the implementation. Last but not least, the adverse reaction was significantly lower than the afteroperative postoperative. More reports, preoperative radiotherapy can improve the patient’s long-term survival rate. However, rectal cancer a kind of adenocarcinoma, the whole efficiency of radiotherapy is not as sensitive as squamous cell carcinoma and malignant lymphoma. Patients with obvious degraion, part degration and no degration each account for about1of/3, and the total efficiency is about70%, the latter benefited less from neoadjuvant radiotherapy. Apparently, patients with invalid treatment cannot benefit from preoperative radiotherapy, even only added to the toxic effects of treatment, even a delay of the treatment. Therefore, looking for molecular markers related to radiosensitivity of rectal cancer, making a pefect choice of patients should be the focused in the current study. Many studies trying to find biological markers to predict the radiosensitivity in rectal carcinoma to get abetter patients selection, including P53and P21, but it is difficult to be widely applied. At present, we are mainly depend on clinical features of tumors, including tumor size, the activity of the tumor, the degree of tumor differentiation and imaging examination. Unfortutinary, those kinds of clinical indexes are lack of correlation with tumor radiosensitivity. Therefore, the search for new molecular markers related to radiosensitivity of rectal cancer is worth looking forward to.MicroRNA is expected to become a new molecular diagnostic marker, which almost involved in the whole process of the development of tumor. While the Giuseppina Della Vittoria Scarpati et al. Selected the miRNAs to predict to sensitivity of patients to radiotherapy in38cases of advanced low rectal cancer patients before neoadjuvent therapy by microarray assay that was verified by RT-Q-PCR. Combined with clinical data, it showed that hsa-miR-622and hsa-miR-630are perfect the sensitivity to radiotherapy with both100%sensitivity and specificity. After screening and Preliminary experiments, we chose hsa-miR-622as the object of study.MethodsRelationship between the expression of hsa-miR-622and radiation sensitivity.1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. Then SW480, HT29, HCT116, LS174.T, SW837, HR8348were given radiotherapy dose of2,3,4,5Gy processing, cell proliferation after radiotherapy was observed by CCK-8and plate colony forming experiment.2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Comparison of the sensitivity of tumor-bearing nude mice.3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree.The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining.Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines. Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy.2、Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group.Characteristic analysis and a prediction of target gene of hsa-miR-6221、Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622.2、RB1gene3’UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3’UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RB13’UTR and inhibit RBI gene expression in posttranscriptional level through the dual luciferase system in293FT cell. The result was convinced in the SW837cell line.3、Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. Then we take a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor. 4、Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual.5、The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy.ResultsRelationship between the expression of hsa-miR-622and radiation sensitivity.1. Expression of hsa-miR-622in SW480, HT29, HCT116, LS174.T, SW837, HR8348cells was fluorescence quantitative PCR detection. When the conference group was disputed, there was significant difference between five cell lines (F=118.335, P<0.001). The results showed that hsa-miR-622was the highest expressed in HR8348. It was higher than that of SW480(P<0.001), HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and higher than that of HT29(P<0.001), HCT116(P<0.001), SW837(P<0.001) expression in the cells, there was significant difference. Hsa-miR-622expression in HT29higher than that in HCT116(P=0.032), SW837(P=0.018) expression in the cells, there were statistical differences. But the expression of hsa-miR-622in the left two cell lines showed no significant difference between SW837and HCT116(P=0.741). The expression of hsa-miR-622decreased in HR8348, SW480, HT29, HCT116, SW837, and LS174.T. It relatively high expressed in HR8348, SW480, and HT29. All of the cell lines were given radiotherapy of2,3,4,5Gy. The results of CCK-8and plate clone formation assay showed that SW480, HT29, HR8348are not sensitive to radiotherapy, while HCT116, Ls174.T, SW837is sensitive to radiotherapy. We found that endogenous hsa-miR-622expression correlated with the radiosensitivity of cells negatively.2. Expression of hsa-miR-622detected by fluorescence quantitative PCR tumor-bearing nude mice tumor tissue. Results showed that hsa-miR-622expression were statistically significant in subcutaneous tumors formed by five cell lines in nude mice when the conference group was disputed (F=315.155, P<0.001). Dunnett T3comparison found:HR8348cells and the expression of hsa-miR-622was the highest in the tumor tissue formed by HR8348, and it was higher than that of SW480(P=0.011), HT29(P=0.002), HCT116(P=0.004), SW837(P=0.003) expression in the cells, there was significant difference. Hsa-miR-622expression was higher in SW480cells, and it was higher than that of HT29(P=0.066), HCT116(P=0.022), SW837(P=0.016) expression in the cells. The expression of hsa-miR-622in HT29was higher than in HCT116(P=0.008), SW837(P=0.005) expression in the cells, there was statistical differences. Expression of hsa-miR-622in HCT116cell was higher than that expressed in SW837(P<0.001). Comparison of the sensitivity of tumor-bearing nude mice showed that:there was significant difference in radiosensitivity of subcutaneous tumor in nude mice formed by different cell lines (F=6.141P=0.005). Homogeneity of variance test showed there was no variance of homogeneity, LSD comparison showed that:radiosensitivity of subcutaneous tumor formed by HT29is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.024), while there was no significant difference compared with that of HR8348(P=0.829) and SW480(P=0.984). Radiosensitivity of subcutaneous tumor formed by SW480is lower than that of HCT116(P=0.004), Ls174.T (P=0.012) and SW837(P=0.036), while there was no significant difference compared with that of HR8348(P=0.845). Radiosensitivity of subcutaneous tumor formed by HR8348is lower than that of HCT116(P=0.002), Ls174.T (P=0.008) and SW837(P=0.025). There was no difference between HCT116and Ls174.T (P=0.522), Ls174.T and SW837(P=0.562), SW837and HCT116(P=0.233) in sensitivity to radiotherapy. We found that endogenous hsa-miR-622expression in the tissue of mude mice hearing human tumor correlated with the radiosensitivity of cells negatively.3.17turner tissue specimens before radiotherapy were collected status in rectal cancer patients. The expression of hsa-miR-622was detected in17cases with advanced status in rectal cancer patients before radiotherapy. After a comparison of CT and colonoscopy results of the patients before and after radiation, the tumor regression grade was classified by the standard of Mandard tumor remission degree. By contrast,5cases were classified as TRG4, the hsa-miR-622expression level was higher, and the lowest expression in3patients achieved complete remission.The effect of silence and overexpression of hsa-miR-622on colorectal cancer cells and tumor-bearing nude mice to radiosensitivity1. SW480and HCT116were selected for the transient transfection. Hsa-miR-622was interfered in SW480, while HCT116was transfected with mimics to overexpress hsa-miR-622. Efficiency of cell transfection was detected by RT-Q-PCR. The t test of the results showed that hsa-miR-622was higher expressed in SW480N.C than the expression in SW480IN (t=13.313, P=0.006), the difference was statistically significant. The expression of hsa-miR-622was lower in the HCT116N.C than in the HCT116expression (t=13.313, P=0.008), the difference was statistically significant. It indicated that hsa-miR-622expression was infected by the transient transfection. The apoptosis of the different group after radiotherapy was detected flow cytometry experiment with PI staining. The sensitivity of the cell was decreased in mimics transfected cell line compared with the control group (t=16.186, P<0.001), while the sensitivity was enhenced in the inhibitor transfected cell line compared with the control group (t=13.556, P<0.001). Then we use lentiviral to transfect cells to overexpress or silence of hsa-miR-622stablily. Lentiviral transfection efficiency was detected by RT-Q-PCR in four cell lines, single sample t test was usd to analyze the results, expression of hsa-miR-622was no difference between the SW480IN and SW480N.C (t=4.285, P=0.050), HCT116-miR622and HCT116N.C showed no significant difference (t=2.853,P=0.104); The expression of hsa-miR-622in HT29IN was higher than that of HT29N.C (t=5.387,P=0.033); The expression of hsa-miR-622in SW837miR-622was higher than that of SW837N.C (t=5.541,P=0.031). Cell proliferation assay (CCK-8and plate colony formation assay) was used to compare the varience of sensitivity of the cell to radiotherapy. Compared with the control group, the sensitivity of the interference group was enhenced, while it was decreased in the overexpression group of cell radiosensitivity.2. Cells transfected with letiviral were injected subcutaneously into athymic nude mice. The nude mice were treat with radiotherapy after the diameter of the tumor was more than1cm according to clinical therapeutic dose. The tumor size was recorded every three days by vernier caliper in both with radiotherapy group and without radiotherapy group. Tumor regression rate (radiotherapy group/without radiotherapy group), an index to evaluate the radiation sensitivity, was used to compared the control group and the interference group, control group and the overexpression group. The results showed:tumor reduction rate was more obvious in SW480IN than thatin SW480N.C (t=5.572, P=0.005), the results were statistically difference. Nude mice injected with HCT116N.C was more significantly regressed than that with HCT116miR-622(t=3.283,P=0.030), the results were statistically difference. It wais also verified in SW837(t=2.917, P=0.043).Characteristic analysis and prediction of target gene of hsa-miR-6221. Taking hsa-miR-622as the search term, we tried to predict the target genes of hsa-miR-622in microRNA.org, TargetScan. Intersection the two datebase was classified by GO classification, and further verification was made in the DAVID database. According to the annotations and score of gene in microRNA.org database, three the target gene is closely related with hsa-miR-622. According to the literature, there were important relations between tumor occurrence and development and RB1. In addition, RBI was play an important role in the regulation of cell cycle and influencing on apoptosis.2. RB1gene3’UTR expression vector and hsa-miR-622binding site mutated MutRB1gene3’UTR expression vector was constructed. We tried to verify whether hsa-miR-622can combine with RBI3’UTR and inhibit RB1gene expression in posttranscriptional level through the dual luciferase system in293FT cell.combined with the translation of the RBI level. Results in293FT cell transfected with RBI gene3’UTR plasmid analysied by single factor variance analysis showed that the difference between groups was significant (F=139.970, P<0.001), test of homogeneity of variance by Levene homogeneity of variance show there was homogeneity of variance (F=1.103, P=0.407). LSD two two comparisons showed: luciferase activity was inhibited in the group transfected with hsa-miR-622compared with blank (P<0.001) and N.C (P<0.001). There was statistically difference. It showed that hsa-miR-622can combine with RBI gene3’UTR, leading to the inhibition of luciferase activity. There was no significant difference between blank group and N.C group (P=0.838), the control sequence had no effect on luciferase activity. Luciferase activity has no difference in the group transfected with hsa-miR-622inhibitor compared with blank (P=0.231) and N.C inhibitor (P=0.168). The results of single factor analysis of variance based on the plasmid of MutRB1gene showed no significant difference between groups (F=2.397, P=0.120). The results are also verified in SW837cells.3. Edogenous expression of RBI in tissues of nude mice subcutaneous tumor was detected by western-blot. With a contrast of the relationship of the radiosensitivity and RB1expression in nude mice bearing human tumor, we found the higher expression of RB1in the tumor tissues, the more sensitive to radiotherapy of the nude mice. Using western-blot to detect the expression of RB1in tissues from the nude mice injected with stably transfected cell and analyzed its relationship with radiotherapy sensitivity. The results showed that RB1expressed lower in the tumor tissue which was formed by the cells transfected with overexpression lentiviral compared with N.C group. And the RBI expressed higher in the inhibitor group compared with N.C group.4. Paraffin specimens of the patients with advanced rectal cancer before radiotherapy were immunohistochemically stained. Relationship to the expression of RBI and radiosensitivity were compared among individual. We found that RBI expressions were all strongly positive in27patients, no statistically significant differences between individuals.5. The expression of RB1, E2F1, and E2F8in first to eighth hour was detected by western-blot in the cells that were treated with radiotherapy. The result showed that three albumin has the same trend after radiotherapy in1-8h. They were all decreased first and then increased, but dreased again at last. The relationship between them needs to be studied further Conclusion1. The relationship between differential expressions of Hsa-miR-622and radiotherapy sensitivity of colorectal cancer cell was negative correlation2. Radiosensitivity of the nude mice bearing human tumor associated negatively with hsa-miR-622expression.3. RB1is a direct target gene of hsa-miR-6224. The relationship between differential expressions of Rbl and radiotherapy sensitivity of colorectal cancer cell was positive correlation5. Radiosensitivity of the nude mice bearing human tumor associated positively with RB1expression

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms > Rectal cancer
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