The Clinical Significance And Mechanism Of Keratin 17(KRT17) Expression In Pancreatic Cancer

Objective:1.To analyze the expression of KRT17 in pancreatic cancer and its clinical significance;2.To clarify the role of KRT17 in the proliferation and apoptosis of pancreatic cancer cells;3.Explore the molecular mechanism of cell proliferation in promoting pancreatic KRT17.Methods:1.Eighteen samples of patients with pancreatic cancer（PDAC）were collected,including tumor tissues and paracancerous tissues,and the expression of KRT17 was measured by real-time quantitative PCR（qPCR）;TCGA database was also analyzed.In the pancreatic cancer dataset,analyze the expression of KRT17;download the clinical information of pancreatic cancer in the TCGA database,analyze the relationship between disease-free survival and overall survival in patients with pancreatic cancer and the expression of KRT17 genes using bioinformatics methods,and explore the significance of KRT17 expression.2.First,detect the expression levels of KRT17 in the three pancreatic cancer cell lines PANC-1,MIAPa Ca-2 and KP-3 and the pancreatic immortal cell line H6c7 were detected using qPCR and western blot;secondly,specific interference fragments for the KRT17 gene were synthesized,and inserted into the lentiviral interference vector（refered as KRT17-siRNA-1 and KRT17-siRNA-2）and the corresponding control interference vector（named as Scr-siRNA-1 and Scr-siRNA-2）;the above-mentioned interference vectors were respectively transfected into PANC-1 cells,and the knockdown efficiency of KRT17 was determined by qPCR and Western blot;then,the lentiviral interference vectors Scr-siRNA-1,Scr-siRNA-2,KRT17-siRNA-1 and KRT17-siRNA-2 were transfected into PANC-1 cells;MTT method was used to detect cell viability for 5 consecutive days,and the effect of KRT17 gene knockdown on PANC-1 cell proliferation was analyzed;PANC-1 cells transfected with Scr-siRNA-1,Scr-siRNA-2,KRT17-siRNA-1 or KRT17-siRNA-2 lentiviral vector were stained with Annexin V-APC,and analyzed apoptotic percentage of PANC-1 cells using FCM（flow cytometry）,the cells at the G₀/G₁,S and G₂/M phases was analyzed,and the KRT17gene knockdown was analyzed Reduce the effect on cell cycle;inoculate 6-well culture of PANC-1 cells infected with Scr-siRNA-1,Scr-siRNA-2,KRT17-siRNA-1 and Scr-siRNA-2 lentiviral interference vector in logarithmic growth phase Plate,culture for14 days,cells were treated with 4%paraformaldehyde,and then stained with Giemsa solution for 10-20 minutes,performed clone count for assessing the effect of KRT17gene knockdown on PANC-1 cell clone formation;Transwell experiment examined the effect of KRT17 silence on migration PANC-1 cells;Finally,PANC-1 cells（5×10⁶ cells）transfected with Scr-siRNA-1 and KRT17-siRNA-1 lentivirus were inoculated into BALB/c nude mice subcutaneously,and measured the tumor volume 5 consecutive weeks.Then,xenografts were weighed to explore the role of KRT17 silencing.3.Total RNA from PANC-1 transfected with Scr-siRNA-1 and KRT17-siRNA-1lentiviral interference vector was extracted using Trizol,qPCR and western blot analysis of proliferation and apoptosis-related signal molecules ERK1/2,Akt,Bad,The expression levels of p38 MAPK,SAPK/JNK,Caspase-3 and Caspase-7.Results:1.Bioinformatics analysis showed that tumor tissue of patients with pancreatic cancer KRT17 m RNA were significantly higher than in normal pancreatic tissue（P<0.01）;The results also show that HPA website,KRT17 protein expression in pancreatic tumor tissue than in normal tissue.Consistant with above results,the results of TCGA clinical information analysis showed that the overall survival rate（OS）and disease-free survival rate（DFSR）of patients with high KRT17 expression were poor（both P<0.05）.2.The qPCR results showed that the expression of KRT17 in pancreatic cancer cells PANC-1,MIAPa Ca-2 and KP-3 was significantly higher than that in pancreatic immortalized cells H6c7.The qPCR test results showed that,compared with the Scr-siRNA-1 group or Scr-siRNA-2 group,the expression of KRT17 m RNA in the KRT17-siRNA-1 group or KRT17-siRNA-2 group was significantly decreased（P<0.01）,and its efficiencies of KRT17 knockdown were 63.2%and 82.5%respectively.Western blot results confirmed that the KRT17 protein expression of KRT17-siRNA-1or KRT17-siRNA-2 transfected cells was significantly lower than that of Scr-siRNA-1or Scr-siRNA-2 group.MTT assay showed proliferation KRT17-siRNA-1 or KRT17-siRNA-2 group than Scr-siRNA-1 or Scr-siRNA-2 group was suppressed significantly（P<0.01）.The above results indicate that KRT17 knockdown inhibits proliferation of PANC-1 cells.The percentage of G₀/G₁ phase cells was increased obviously in the KRT17-siRNA-1 or KRT17-siRNA-2 groups compared to the SCR-siRNA-1 or SCR-siRNA-2 groups（P<0.01）,but the cellular percentage at S phase declined significantly（all P<0.01）,which suggested that KRT17 silencing arrested the cell cycle of PANC-1 cells at G₀/G₁ phase.Flow cytometry detection of early apoptosis of PANC-1 cells showed that compared with Scr-siRNA-1 or Scr-siRNA-2 group,the percentage of early apoptotic cells in KRT17-siRNA-1 or KRT17-siRNA-2 group was significant elevated（P<0.01）.After staining with Giemsa solution,compared with Scr-siRNA-1 or Scr-siRNA-2 group,the clone number in KRT17-siRNA-1 or KRT17-siRNA-2 group was significantly declined（all P<0.01）.Transwell found that KRT17 silencing significantly inhibited the invasion ability of PANC-1 cells（P<0.01）.Finally,xenograft in the KRT17-siRNA-1 group was significantly reduced compared with the Scr-siRNA-1 group（P<0.05）.3.The results from qPCR showed that the levels of down-regulated ERK1/2 and up-regulated Bad in the KRT17-siRNA-1 group were found,compared to those in the Scr-siRNA-1 group;At the same time,western blot testing verified the qPCR results.Conclusion:1.The highly expression of KRT17 in tumor tissues from patients with PDAC is positively correlated with their poor prognosis.2.KRT17 silencing significantly slowed the biological characterization of pancreatic cancer PANC-1 cells,including proliferation,clone formation and xenograft growth,at the same time,promoted its apoptosis,and resulted in cell cycle arrest at G₀/G₁ phase.3.KRT17 silence inhibits PANC-1 cell proliferation,which may be achieved by elevating ERK1/2 and decling Bad.