| Objectives: Pancreatic cancer is a highly invasive malignant tumor.Its onset is occult and its diagnosis is mostly advanced.The symptoms are not obvious,so its diagnosis is mostly advanced and characterized by poor prognosis.Surgery is the main treatment for pancreatic cancer,but most patients have no chance of surgical treatment at the time of diagnosis.Radiotherapy and chemotherapy are the main treatments for pancreatic cancer.Radiotherapy plays an important role when treating pancreatic cancer,and can reduce the local recurrence rate and increase the chance of reoperation in locally advanced cases.The main cause of radiotherapy failure is the presence of tumor cells resistant to conventional therapies.Therefore,increasing radiosensitivity is important for treatment.Radiotherapy resistance is a complex process involving multiple genes,factors and mechanisms.Cell cycle arrest,related genetic changes,changes in the tumor's microenvironment,autophagy regulation,stem cells and other factors lead to radiotherapy resistance.Research on radiotherapy sensitization has developed into the molecular and genetic level.Current research on the molecular mechanism of radiotherapy resistance and radiotherapy sensitivity is thus of considerable relevance to pancreatic cancer therapy.Methods: The first part : The parental pancreatic cancer cell lines SW1990 and PANC-1 were irradiated with X-ray ladder dose radiotherapy.The radioresistance cell lines SW1990-R and PANC-1-R were established with a total dose of 80 GY.With controlled parental cell lines,the radioresistance of pancreatic cancer was evaluated using a cloning test,survival curve and cell cycle test.High throughput sequencing was used to screen differentially expressed miRNA and RNA between parental cell lines and resistant cell lines.Gene ontology(GO)function annotation and the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were used to analyze the genes,biological processes and signaling pathways involved in radiotherapy resistance.Real-time PCR was used to detect the TOP10 significant difference expression miRNAs to identify the sequencing results.The second part:According to the sequencing results,miRNA-216 a and JAK2 may be related toradiotherapy resistance.Real-time PCR was used to detect the difference in the expression of related molecules in the JAK2/STAT3 signaling pathway between radioresistant and radiosensitive cell lines.The protein expression of molecules related to the JAK2/STAT3 signaling pathway in parental cell lines and radioresistant cell lines was detected with a western blot.Furthermore,the protein expression of molecules related the JAK2/STAT3 signaling pathway in pancreatic cancer cell lines was compared before and after irradiation.We transfected siJAK2 in pancreatic cancer radioresistant cells and silenced the expression of JAK2.We then examined the differences in cloning formation after X-ray irradiation in radioresistant cells compared to the control group,and a western blot was used to examine the expression of JAK2,STAT3,pSTAT3 and SOCS3 proteins.We used AG490 to downregulate the JAK2/STAT3 signaling pathway expression in pancreatic cancer radioresistant cells in order to observe the differences in cloning formation after X-ray irradiation,and to examine the differences in JAK2,STAT3,pSTAT3 and SOCS3 protein levels.According to the sequencing results,the miRNA-216 a mimic was transfected to observe the cloning formation after X-ray irradiation in pancreatic cancer radioresistance cells with an overexpression of miRNA-216 a.The expression of JAK2,STAT3,pSTAT3 and SOCS3 protein levels were examined with a western blots.We then used immunohistochemistry to detect JAK2 expression in tumors and the adjacent normal tissues of pancreatic cancer specimens,and to detect JAK2 expression in radioresistant pancreatic cancer and radiosensitive pancreatic cancer tissues.The TCAG database was used to analyze the difference in JAK2 expression between tumorous and normal tissues.Results: The second part:1.Radiotherapy resistant cell lines SW1990-R and PANC-1-R were successfully established.2.Compared to parental cell lines of pancreatic cancer,the radioresistant cell lines of pancreatic cancer were more resistant to X-ray irradiation,with stronger cell viability,more clones and cell cycle arrest.3.High-throughput sequencing detected 242 kinds of miRNAs with significantly different expression levels.In radiotherapy resistant cells,97 miRNAs were upregulated and 145 miRNAs were downregulated compared to parental cell lines of pancreatic cancer.4.GO enrichment and KEGG analysis showed that a variety ofmolecules and signaling pathways,including immune-related molecules and signaling pathways,were involved in radiotherapy resistance to pancreatic cancer.5.Real-time PCR was used to confirm that the expressions of miRNA-497-5p,miRNA-146b-3p,miRNA-181a-3p,miRNA-33a-3p and miRNA-32-5p were up-regulated,while the expressions of miRNA-7-5p,miRNA-30b-5p,miRNA-181a-5p,miRNA-296-5p and miRNA-216a-5p were downregulated.The second part: 1.When pancreatic cancer cells were irradiated with different X-ray doses,the western blots showed that JAK2 protein expression increased followed an increase in irradiation dose.2.Real-time PCR and western blot results both showed that JAK2 and STAT3 expressions in radioresistant cell lines were up-regulated and that the expression of SOCS3 was downregulated compared to parental cell lines of pancreatic cancer.3.After X-ray irradiation,JAK2 and pSTAT3 expressions were upregulated in pancreatic cell lines,and the expression of SOCS3 was downregulated compared to before irradiation.4.With transfected siJAK2 and silenced JAK2 expression,the number of clones of pancreatic radioresistant cells after irradiation decreased significantly.The protein expression of the JAK2/STAT3 signaling pathway was inhibited.5.Moreover,the CCK8 assay detected that 50 ug/mL AG490 could inhibit the colon formation of pancreatic cancer radioresistant cells after irradiation.Radiotherapy combined with AG490 could inhibit JAK2/STAT3 signaling pathway and increase radiosensitivity.6.The transfection of miRNA-216 a mimic could inhibit the colon formation of pancreatic cancer radiaresistant cells after irradiation targeting JAK2.The results indicated that the JAK2/STAT3 signaling pathway was inhibited.7.Finally,When observing 36 patients with pancreatic cancer in our hospital,the expression of JAK2 in cancer tissue was higher than in normal tissue.Likewise,the expression of JAK2 in cancer tissue was higher than in normal tissue according an analysis of the TCAG database,and the expression of JAK2 in radioresistant pancreatic cancer tissue was higher than in radiosensitive pancreatic cancer tissue.Conclusions: The differentially expressed miRNA and mRNAs between radioresistant and radiosensitive pancreatic cell lines were screened with high-throughput sequencing.Bioinformatics analysis was used for the results.These results offer insights when addressing the underlying mechanisms of the JAK2/STAT3 signaling pathway involved in radiotherapy resistance.The results showed that JAK2 expression was higher in pancreatic cancer tissue,and that the expression of JAK2 increased in radioresistant pancreatic cancer tissues.Furthermore,X-ray irradiation activated the JAK2/STAT3 signaling pathway.This suggested that continuous activation of the JAK2/STAT3 signaling pathway was a possible reason for pancreatic cancer radioresistance,and microRNA-216 a inhibited this pathway could increase radiosensitivity. |
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