| Part one Screening of micro RNA related to radiation resistance in middle and low locally advanced rectal cancerObjective:To construct a patient-derived xenograft(PDX)model of human tumor in middle and low rectal cancer,and to detect the radiation response ability of tumor-bearing nude mice and 5 rectal cancer cell lines,and to screen out mi RNA positively correlated with radiation resistance in tumor-bearing nude mice,rectal cancer cell lines and radiation resistance.Methods:1. In the operation,fresh surgical tumor tissue samples were obtained from the second disease area(gastrointestinal surgery)of the fourth hospital of Hebei Medical University,the specimens sharpness separated into two equal parts:One of them was frozen in the refrigerator of-150℃,the other was inoculated into the forelimb or hind limb of nude mice to construct the patient derived xenograft(PDX).2.A single dose of 16Gy was used to irradiate the tumor site of the PDX model,and the tumor volume of the transplanted tumor was measured 10 days after radiation,and the tumors of the tumor bearing mice were sacrificed,and the tumor tissues were dissected for HE staining,so as to evaluate the reaction of the transplanted tumor to radiation.3.According to the results of the evaluation of transplanted tumor response to radiation screen to achieve a total reaction after accepting radiation tumor histologic specimens and non/low reaction of specimens,3patients were preserved at their corresponding low temperature-150℃refrigerator samples of micro RNAs chip detection,screening differentially expressed a fold change value>2.0 micro RNAs,and through the q RT-PCR test to screen out differences of micro RNAs in colorectal cancer tissues.4.Human rectal adenocarcinoma cell lines HRC-99,HR-8348,SW1463,SW837 and RCM-1 were sub cultured.These five kinds of cells were respectively planted in a certain number of plates.After the cells were attached to the wall,they were irradiated with single doses of 0Gy,2Gy,4 Gy,6Gy and 8Gy respectively.The clone formation experiment was conducted under the interference of radiation,and then the cell lines resistant to radiation were screened.5.According to the screening results of mi RNA chips and the results of tissue validation,q RT-PCR was used to detect the expression levels of mi RNAs that may cause increased radiation resistance of rectal cancer in the above five rectal cancer cell lines,and to screen mi RNAs that may be related to the radiotherapy resistance of rectal cancer tissues.Results:1. The success rate of PDX model construction was found to be 76.79%(43/56)after 10 days of rectal cancer tissue inoculation.A total of 55 tumors were formed in the shoulder/buttock of 43 tumor-bearing nude mice.2. On the basis of Wheeler on rectal cancer after radiotherapy histologic retreat classification(rectal cancer regression grade,RCRG)transplantation tumor response to radiation standard evaluation,the result found RCRG1specimens in 11 cases,RCRG2 specimens from 26 cases,RCRG3 level 6cases of specimen.3. mi RNA chip detection results showed that there were a total of 14 mi RNA with fold change value of>2.0,among which 4 were up-regulated in tumor tissues with radiation-resistance,including mi RNA-552-3p,mi RNA-96-5p,mi RNA-182-5p,and mi RNA-183-5p,respectively.The 10down-regulated expressions were mi RNA-3138,mi RNA-4800-5p,mi RNA-6510-5p,mi RNA-7847-3p,mi RNA-345-3p,mi RNA-1236-5p,mi RNA-6775-5p,mi RNA-1273g-3p,mi RNA-145-5p,mi RNA-381-3p,Further q RT-PCR validation in rectal cancer tissues showed that the validation results of mi RNA-552-3p,mi RNA-96-5p,mi RNA-182-5p,mi RNA-183-5p,mi RNA-3138,mi RNA-381-3p,mi RNA-1236-5p,and mi RNA-145-5p showed the same trend as those obtained by mi RNA microarray screening.4.The results of the cloning formation experiment under radiation interference showed that the resistance of 5 rectal cancer cell lines to radiation was HR-8348>HRC-99>RCM-1>SW837>SW1463.5.q RT-PCR results of four mi RNAs positively correlated with radiation resistance in five rectal cancer cell lines showed that only the expression level of mi RNA-96-5p was consistent with the trend of radiation resistance in these five rectal cancer cell lines.Summary:The expressions of mi RNA-552-3p,mi RNA-96-5p,mi RNA-182-5p and mi RNA-183-5p in rectal cancer tissues were positively correlated with the resistance to preoperative chemoradiotherapy,and the changes in the expression levels of mi RNA-96-5p in 5 rectal cancer cell lines were consistent with the resistance trend of cells to radiation,which was the key indicator of our follow-up study.Part two Target gene screening for mi RNA-96-5p regulation ofObjective:To detect the effects of miexpression changes on the radiation resistance and cell function of thecell line in rectal cancer,screen and verify its regulated target genes,and study the effects of its expression level differences on the Wnt signaling pathway.Methods:1.HR-8348 cell lines with stable down-regulation of mi RNA-96-5p expression were constructed by lentivirus packaging technology and cell transfection technology,and cloning and formation experiments under radiation interference were conducted after passage culture to further detect the influence of mi RNA-96-5p expression changes on the radiation resistance of HR-8348 cell lines in rectal cancer.2.The effects of changes in mi RNA-96-5p expression on the proliferation,migration and invasion of HR-8348 cells in rectal cancer were detected by cell function assay.3. Through bioinformatics software Target Scan mi RDB,mi RTarbase,Tarbase prediction could be mi RNA-96-5p regulating target genes,and screened in the four bioinformatics software can predict the target genes,after using the Pubmed medical literature retrieval service system to read the literature associated with the gene,then select the target genes associated with the biological behavior of tumors.4. q RT-PCR and Westren blot were used to detect the expression of the selected target genes in HR-8348 cell lines before and after the down-regulation of mi RNA-96-5p,and then possible target genes that mi RNA-96-5p regulated the radio-resistant changes of the HR-8348 cell lines in rectal cancer were screened out,and the dual-luciferase assay was used to verify whether mi RNA-96-5p directly regulated the expression of this gene.5.According to the prediction results of the differential mi RNA enrichment pathway KEGG obtained from the mi RNA chip results,the expression of relevant genes on the classical Wnt signaling pathway,which is closely related to radiation resistance,confirmed by previous research results,was analyzed by Westren blotting.Results:1.The titer transfection results of lentiviral vector showed that the virus titer of LV10N-has-mi R-96-5p-NC and lv10n-has-mi R-96-5p-inhibitor was8×10~8TU/ml and 9×10~9TU/ml,respectively.The relative expression of mi RNA-96-5p detected in HR-8348 cells after the lentiviral plasmid transfection showed that the inhibitor group was significantly lower than the untreated group.The difference was statistically significant(5.746±0.451 vs0.0036±0.00095,P<0.01).The results of clone formation experiments under radiation interference showed that the resistance of lv10n-has-mi R-96-5p-inhibitor/HR-8348 group to radiation was reduced compared with that of the blank group(P<0.001).2.Cell function test showed that the proliferation,migration and invasion abilities of lv10n-has-mi R-96-5p-inhibitor/HR-8348 were significantly lower than those of the blank group.3.A total of 3,428 genes that may be regulated by mi RNA-96-5p were predicted by the biological information software Target Scan,mi RDB,mi RTarbase and Tarbase,of which 10 target genes were predicted in the four biological information software,namely CAV1,DAB2,DDIT3,EDEM1,FOXO3,CPC3,MBD4,PDCD4,SLC25A25 and ZEB1.Pubmed medical literature retrieval service system was used to search the literature related to the above gene studies,and it was found that there were a total of 7 genes previously reported to be related to the biological behavior of tumors,namely CAV1,DAB2,DDIT3,FOXO3,GPC3,MBD4 and PDCD4 genes.4. Respectively the q RT-PCR technology and Westren blot technique for the possible target gene expression level in transfection of HR-8348 rectal cancer were detected before and after,in this study we found through the above two methods in the mi R-96-5-p-inhibitor expression level than idling/HR-8348 cells and not transfection group(NC)group were obviously up-regulated genes as the purpose of further study.The results of q RT-PCR showed no statistical differences in the expressions of CAV1,DDIT3,and PDCD4inthemi RNA-96-5p-nc/HR-8348group,mi RNA-96-5p-NC/HR-8348 group,or in the untransfected HR-8348 cells(P>0.05).The expression levels of DAB2 and MBD4 in the inhibitor group were significantly lower than those in the NC group and the untransfected group(P<0.05).The expression levels of FOXO3 and GPC3 in inhibitor group were significantly higher than those in NC group and non-transfected group(P<0.05).The Westren blot results showed no statistical differences in the expression of CAV1,DDIT3,PDCD4 and MBD4 in the three groups(P>0.05).The expression of DAB2 in the inhibitor group was significantly lower than that in the NC group and the non-transfected group(P<0.05).FOXO3 expression in inhibitor and NC groups did not see obvious difference,but not significantly higher transfection group(P<0.05),GPC3 expression in the transfection group and not transfection group were significantly increased in the NC group(P<0.05),a comprehensive analysis of the results found in the above seven genes only GPC3 meet in q RT-PCR and Westren blot test of two kinds of methods of expression in inhibitor group was obviously raised the standards,Therefore,in this study,we regarded GPC3 gene as a target gene regulated by mi RNA-96-5p in HR-8348 cells.5. In order to verify whether GPC3 gene is directly regulated by mi RNA-96-5p in HR-8348 cell line of rectal cancer,we conducted dual-luciferase detection and found that the mrna3’utr terminal of GPC3 is a direct target of mi RNA-95-5p,and mi RNA-95-5p can down-regulate its expression in HR-8348 cells of rectal cancer by directly targeting the GPC3mrna3’utr terminal.6.The Westren blot detection of colorectal cancer before and after transfection HR-8348 classic Wnt signaling pathways in cells on the expression of related genes,discovered that the hub for the pathway molecules beta catenin in total of three groups of cells expressing quantity difference is not obvious,but in its cytoplasm expression quantity of inhibitor group was obviously lower NC group and untreated group,group inhibitor beta-catenin whole-cell expression of the ratio of the amount of cytoplasm and significantly reduced;On degradation of beta-catenin complex GSK3 beta key molecules and its molecular p-GSK3 phosphorylation beta testing,found in the three groups of cells in the expression of GSK3 beta difference is not obvious,but p-GSK3 beta in inhibitor significantly lower than the amount of expression in the NC group and untreated group,inhibitor of p-GSK3 beta and GSK3 beta expression of the ratio of the amount of reduced obviously,tips on mi R-95-5-p inhibit phosphorylation of GSK3 beta levels;Further detection of the expression of CD44 and c-Myc in the downstream genes of the classical Wnt signaling pathway revealed that the expression of these two genes in the inhibitor group was significantly down-regulated compared with the expression in the NC group and the untreated group,suggesting that the down-regulation of mi R-96-5p in the HR-8348 cells of rectal cancer inhibited the activity of the classical Wnt signaling pathway.Summary:High expression of mi RNA-96-5p is an important factor leading to high radiation resistance of HR-8348 cells in rectal cancer.GPC3gene is a target gene directly regulated by it.Inhibition of mi RNA-96-5p can reduce the activity of the classical Wnt signaling pathway.Part three Screening of preoperative concurrent chemoradiotherapy advanced rectal cancerObjective:Preoperative concurrent chemoradiotherapy(CCRT),as the standard treatment for locally advanced rectal cancer(LARC),has been widely used in clinical practice,and its efficacy affects the prognosis of patients and the choice of follow-up treatment.Currently,for LRAC patients who are sensitive to preoperative CCRT,subsequent treatment options include TME resection at 8-10 weeks after CCRT treatment and a"wait and watch"strategy.However,those patients with low sensitivity or resistance to CCRT,if they receive this treatment method,not only will not benefit,but also will cause difficulties in operation and postoperative recovery due to side effects and complications of radiation.In addition,since patients still need to wait nearly 8-10 weeks for surgical treatment after the completion of preoperative CCRT,it may delay the patient’s condition and affect the prognosis.Screen to before treatment in clinical practice as well as in patients with preoperative CCRT sensitivity high in patients with high preoperative CCRT resist sex’t individualized treatment in order to better,we in this study of preoperative patients with may affect’t CCRT reactivity of multiple factors,screening for clinical doctors to CCRT reaction ability of patients before treatment to forecast and judgment,and provide reference for clinical practice.Methods:79 patients with LARC were enrolled according to the inclusion criteria.The dependent variables were complete pathological response(PCR)and no/low response(N/LR)after CCRT.According to the general clinical characteristics of patients(age,gender,pathological type,clinical T Stage(c T),clinical N stage(c N),carcinoembryonic antigen(CEA)),expression level of tumor stem cell marker CD44v6,image volume parameters of primary tumor focus(the tumor maximum longitudinal length(TML),the tumor maximum transverse diameter(TMD),approximate tumor volume(ATV),real tumor volume(RTV),tumor surface area inner the intestine(TSAI),tumor surface area(TSAO),tumor total surface area(TSA),tumor compactness(TC)),etc 15 factors that may affect the response of patients with LARC to CCRT were used as independent variables and multivariate regression analysis.In the general clinical features in patients with(age,gender,pathological type,c T,c N,CEA,cancer stem cell markers of CD44v6 expression level,Image size parameters of primary tumor lesions(TML,TMD,ATV,RTV,TSAI,TSAO,TSA,TC),for preoperative patients,and so on 15 may affect CCRT reactive factors as independent variables,multiple factors regression analysis.Results:When taking the p CR status of patients after receiving preoperative CCRT as the dependent variable,we found that the p CR status of LARC patients was positively correlated with TC,and negatively correlated with ATV and RTV.When we took the N/LR status of patients after receiving preoperative CCRT as the dependent variable,we found that the N/LR status of LARC patients was positively correlated with the expression levels of RTV,TSA,and CD44v6,and negatively correlated with TC.In addition,we,according to the results obtained by multiple factors regression analysis respectively constructed to predict patients for preoperative CCRT p CR and the results of preoperative CCRT for N/LR condition forecasting model,and on the predictive value of positive forecasting factor and forecasting model are analyzed,and found in p CR models,predictive model Z1,Z2,ATV,RTV prediction model Z,the sensitivity of the TC were 70.00%,65.00%,85.00%,85.00 and 85.00%respectively,The specificity was 93.22%,94.92%,47.46%,42.37%,89.83%,respectively.The positive predictive value was 77.78%,81.27%,35.57%,33.48%,74.73%,and the negative predictive value was90.16%,88.89%,90.27%,89.22%,94.61%,respectively.In N/LR model,the sensitivity of prediction model Z3,prediction model Z4,RTV,TC and TSA were 95.80%,79.17%,62.50%,95.83%and 62.5%,respectively,and the specificity was 70.90%,74.55%,83.64%,47.27%and 96.36%,respectively.The positive predictive values were 58.96%,57.58%,62.51%,44.23%and88.23%,respectively.The negative predictive values were 97.48%,89.13%,83.64%,96.29%and 85.48%,respectively.Summary:In patients with locally advanced rectal cancer of preoperative radiation and chemotherapy reactivity is predictable at the same time,its sensitive patients for ATV,RTV,TC value can be used as an independent predictor of patients to resist the CD44v6 expression level,RTV,TC,TSA values can be used as an independent predictor of clinical doctors can incorporate these influence factor of regression model on the reactivity of preoperative CCRT patients judgment.Conclusions:1.The expression of mi RNA-552-3P,mi RNA-96-5p,mi RNA-182-5p and mi RNA-183-5p in rectal cancer was positively correlated with the resistance of neoadjuvant radiotherapy and chemotherapy;2.Down regulating the expression of mi RNA-96-5p in HR-8348 cells can reduce the radiation resistance of the cell line;3.After down regulating the expression of mi RNA-96-5p,the proliferation,migration and invasion of HR-8348 cells were inhibited,suggesting that mi RNA-96-5p is related to the invasion and metastasis of rectal adenocarcinoma.4.GPC3 gene is a target gene directly regulated by mi RNA-96-5p in HR-8348 cells.5.In HR-8348 cells,mi RNA-96-5p regulates the classical Wnt signal transduction pathway,which is related to the response of rectal cancer patients to radiation.6. The expression of ATV,RTV,TC,TSA and CD44v6 are important for predicting the response of patients with locally advanced rectal cancer to CCRT. |
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