| Pancreatic Cancer (PC) accounted for8%-10%of digestive tract malignant tumor is one of the digestive tract tumors with extremely high malignant degree. Recently, the incidence rate on a global scale of PC was getting higher year by year. Due to pancreatic cancer, there were nearly about276000people lost their lives on earth in2011.Although the treatment has been improved, owing to the lack of typical clinical symptoms in the early period and its fast progress, easy early invasion and metastasis distantly, and short effective tumor markers and detection means, and so on, lead to difficult early diagnosis of pancreatic cancer, when many cases were diagnosed clinically, most of patients with pancreatic cancer had lost the only chance for radical surgery. Moreover, the prognosis of patients with pancreatic cancer was very poor, because it’s not sensitive to radiation and chemotherapy; the overall survival rate of two years was less than20%. There were a large number of documents reported both at home and abroad that many microRNAs (miRNA) took part in the regulation of pancreatic cancer mechanism or acted as tumor suppressor genes and ontogenesis.MiRNA is a set of non-coding endogenous single stranded molecule small RNA protein which was first discovered in c. elegant embryos by Lee and his companies. So far, miRBase database have collected nearly200species, twenty thousand kinds of miRNA including animals, plants, and predicted that miRNA may control about50%of human genes. Micrornas regulate gene expression mainly through combining with the sequence of specific target genes, inhibiting synthesis of target protein. So miRNA can be served as a targeted-therapy for malignant tumor.Recent studies have shown that more than100kinds of micrornas existed abnormal expression in pancreatic cancer, in the blood circulation of patients with pancreatic cancer, pancreatic juice, feces (feces, urine, sputum), organization of medium, abnormal expression of miRNAs can be detected.In blood circulation, Li and his co-workers found that miR-210a and miR-200a significant rise in serum of patients with pancreatic cancer through testing. In pancreatic juice, Habbe and his team workers found that miR-155and miR-21presented down-regulated expression in healthy subjects, on the contrary, obviously up-regulated expression in pancreatic cancer patients. In faeces, link found that miR-216a and other four miRNAs expressional level in patients with pancreatic cancer was lower than normal control group. By examining stool specimens in normal control group and pancreatic cancer patients. In the organization, Zhang and his team workers found eight micrornas (miR-196a etc) expression up-regulated in most pancreatic cancer tissue. Those micrornas with abnormal expression in pancreatic cancer could be used as tumor markers.Currently, standarded chemotherapical scheme for pancreatic cancer is based on gemcitabine, but which is short for improving clinical symptoms and prolonging survival rate. Growing number of studies demonstrate that the miRNA can serve as targeted therapy for pancreatic cancer. Park and collaborators proved that miR-21down-regulated expression could make pancreatic cancer cells activity low at cellular level by transfecting antisense oligonucleotide into miR-21.Ali also found that restraining miR-21can enhance pancreatic cancer cell chemosensitivity for gemcitabin. Mercatelli and his workers found that regulated and controlled RNAi miR221/222expressional level as the target of tumor therapy through the study of intervening nude mice transplanted tumor of prostate cancer in using inhibitor, which offered variety of possible targeting selection for pancreatic cancer targeted-therapy.Studies have shown that miR-216a down-regulated expression in pancreatic cancer, lung cancer, liver cancer, breast cancer, prostate cancer and other malignant tumors. Our previous study found that statistical difference existed in the expression of70miRNAs by through comparing miRNA expression in pancreatic cancer with the normal pancreatic tissue, miR-216a expressed significantly lower among patients with pancreatic cancer than the normal. As a result, miR-216a may be a potential biomarker. We further forecasted target gene possibly for miR-216a, and proved that JAK2could act as targeting gene for miR-216a in pancreatic cancer on the cell level by using the agonist and inhibitors, miR-216a could make expression of JAK2of targeting gene down, then promoted apoptosis of pancreatic cancer cells. Therefore, it maybe a new treatment for pancreatic cancer to silence gene,antisense oligonucleotide blocking or micrornas modification for miR-216a expression, Although the clinical application was been not very satisfied, testing results of animal excited us, to be sure, miR-216a has a great potential value as a marker in early diagnosis and treatment for pancreatic cancer.Purposes:Based on current research status of miR-216a and its possible role in pancreatic cancer development, this study aims at establishing the animal model of nude mouse of pancreatic cancer, further verifying miR-216a playing possible role in the pathogenesis of pancreatic cancer through from whole animal level,and lay a theoretical foundation for further clearing the molecular mechanism of pancreatic cancer and anti-tumor effect, thus providing a new basis and methods for early diagnosis of pancreatic cancer, molecular targeted-therapy.Methods:1:To establish the model of subcutaneous transplanted tumors derived from human pancreatic cancer PANC-1cells in the nude mice, total of18nude mice in number2:When the accumulated volume of nude mice subcutaneous transplanted tumors of pancreatic cancer were grown to not less than50mm3(V=ab2/2, calculation of tumor volume, a as long diameter and b as short diameter), those mice were randomly divided into three groups:solvent control group (PBS), negative control group (miR-216a agomir NC) and drug intervention group (miR-216a agomir).when nude mice were grouped later, respectively tumors were injected with corresponding reagent on the first day, fourth day, seventh day, tenth day. We observed growth rate and the volume change of tumors in vivo of nude mice in each group, used vernier caliper to measure the longest diameter of tumor and the shortest path for drawing the tumor growth curve.3:Nude mice were given drugs were euthanized by cervical dislocation method after13days, removed tumors, one part of the organization was fixed in neutral buffered formalin,another part was used for measuring content of miR-216in subcutaneous transplanted tumor tissue derived from human pancreatic cancer PANC-1cells in the nude mice each of groups by PCR (real-time quantitative immunofluorescence)to observe the tumor proliferation; In situ end labeling technique (TUNEL) to detect transplanted tumor tissue cell apoptosis. the rest of tissue was frozen in the liquid nitrogen, and then transferred to-80℃refrigerator for related testing analysis, used real-time quantitative PCR to detect the content of miR-216a in the tumor, Western blot method to detect JAK2protein expression of transplanted tumor tissue.4:SPSS13.0statistical software was used for statistical analysis,, mean plus or minus standard deviation (x plus or minus s)showed all the data, data were analyzed by single factor analysis of variance or inspection statistical analysis, P<0.05indicated that the difference was statistically significant.Results:1:The establishment of the model of subcutaneous transplantation tumor derived from human pancreatic cancer PANC-1cells in the nude mouse.Nude mice had an general condition during tumorigenicity, and then nude mice were subcutaneously been injected with pancreatic cancer cells, when tumor volume in all the groups grew up to more than50mm3after two weeks, which marked the establishment of the model of subcutaneous transplantation tumor derived from human pancreatic cancer PANC-1cells in the nude mouse successfully。2:Drew the tumor growth-curveAccording to the experimental results, we drew tumor growth curve, and found that tumor growth of three groups were not completely inhibited, but compared with solvent control group and the negative control group, the rate of tumor growth of subcutaneously transplanted tumor in nude mice of intervention group were been slowed, the volume of transplanted tumor growth had been narrowed considerably.The significant difference in the average growth rate (p=0.008) existed between intervention group (162.28%plus or minus42.6%) and solvent control group (362.86%plus or minus131.54%), also which existed between Intervention group (162.28%plus or minus42.6%) and the negative control group (371.87%plus or minus141.14%)(p=0.006); but no obvious difference was found between Solvent control group and negative control group (p=0.893),The real growth in volume of there groups were respectively intervention group (89.76plus or minus15.82mm3) and solvent control group (209.77plus or minus61.26mm3) and negative control group (208.50plus or minus765mm3), compared with negative control group and solvent control group for intervention group.difference existed statistically significant (P value were0.011and0.032, less than0.05); it was no difference appeared between negative control group and solvent control group (p value is1.000greater than0.05). Dead nude mice were not found among the drug groups.3:Measuring content of miR-216a in subcutaneous transplanted tumor tissue derived from human pancreatic cancer PANC-1cells in the nude mice each of groups by PCR (real-time quantitative immunofluorescence)Compared solvent control group (1.01plus or minus0.12) and negative control group (1.97plus or minus0.29) with Intervention group (5029.43plus or minus5029.43), there were significant differences (P=0.000, respectively) in the miR-216a relative expression, and the significant difference(P=0.003)also existed between solvent control group and negative control group, which was likely to miR-216a segment not specially modified contained in negative control group when nude mice were given by medication. From the results we can preliminary get judgment; content of miR-216in intervention group gets greatly improved after drug intervention given.4:Western blot testing expression levels of JAK2protein in the each transplanted tumor tissueThe average relative grey value of JAK2in three groups were respectively0.102plus or minus0.798(Intervention group),0.739plus or minus0.211(solvent control group),0.833plus or minus0.329(negative control group).The differences of The average relative grey value of JAK2in Intervention group were very significant compared with solvent control group(p=0.001) and negative control group(p=0.006), there was no significant difference (p=0.909, greater than0.05) between solvent control group and negative control group, the expressional level of JAK2protein in intervention group was down, further validated that rise of miR-216a could inhibit or block the expression of JAK2in vivo, the result was consistent with our previous experimental result in vitro.5:Expression of Ki-67and PANC-1in the transplantation tumor tissue of nude mouseThe average optical density of Ki-67in the intervention group and solvent control group, negative control group were respectively0.2963plus or minus0.3048^0.4612plus or minus0.4687、0.4722plus or minus0.4662.the results could be obtained by using LSD test of single factor variance that there were very significant difference (p=0.000,0.000)comparing Intervention group with solvent control group and negative control group, but no significant difference (p=0.645, greater than0.05)existed between solvent control group and negative control group; we could further infer that The average optical density of Ki-67in the intervention group was extremely reduced after being given drug, so we thought that miR-216a could affect expressional level of Ki-67by regulating cell proliferation.The average optical density of PCNA in the intervention group and solvent control group, negative control group were respectively were0.3502plus or minus0.0879,0.4358plus or minus0.0270,0.4460plus or minus0.0214; we knew that p=0.014through nonparametric test of multiple independent samples, there were significant differences among three groups, according to the average rank,we further inferred that average optical density value in intervention group was the lowest, followed by the solvent control group, the highest for the negative control group, Therefore, the expression of PCNA in nude mice transplanted tumor tissues after injection of the miRNA-216a agomir were been obviously decreased. This suggested that the effect on pancreatic cancer cell proliferation PANC-1nude mouse for intervention group could depend on inhibiting the expression of PCNA and then slowed tumor cell growth.6:TUNEL experimentThe optical density value of Intervention group and solvent control group and negative control group were respectively0.7048plus or minus0.02481,0.2247plus or minus0.04316,0.2140plus or minus0.04853. We Further accept parameter test, the result:p=0.003, show that there are significant difference between there groups, according to the average rank,we can think that the optical density value of Intervention group was the highest, followed by solvent control way, negative control group.TUNEL experiment told us that the intervention group given miR-216a agomir make cancer cell apoptosis rate up, further verifying miR-216a in the process of development of pancreatic cancer as a tumor suppressor.Conclusion:MiR-216a agomir could promote the expression of miR-216a.enhancing expression of miR-216a could inhibit the growth in of subcutaneous transplanted tumors derived from human pancreatic cancer PANC-1cells in the nude mice; down regulated the level of JAK2gene expression in the subcutaneous transplanted tumors; thus could restrain JAK2protein expression, beyond that,the expression of Ki-67and PCNA in the transplanted tumor were been down regulated, and induced cell apoptosis in the transplantation tumors. In all, miR-216a has potential clinical prospect of application serving as early marker for pancreatic cancer tumor and target for gene therapy... |
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