Effects Of Long-noncoding RNA Mi-Lnc70 On Mouse Pancreatic Cancer Cell Min6

Long noncoding RNAs(lncRNAs)regulate the biological behaviors of tumor cells,such as apoptosis,proliferation,cell cycle and metastasis.The expression level of LncRNA in different tumor tissues is significantly different and has cell specificity.LncRNAs are expected to become new tumor markers or therapeutic targets.Mi-Lnc70 is an LncRNA that specifically expressed in mouse pancreas.Compared with mouse fibroblasts,Mi-Lnc70 is highly expressed in mouse pancreatic cancer cells.In this study,three LNA Gapmers were designed and constructed according to the DNA sequence of mouse Mi-Lnc70.Mi-Lnc70knockdown was performed on mouse pancreatic cancer cell line Min6 in vitro.It was found that the knockdown efficiency of Gapmers#1 is the highest.Through testing the combination of transfection time,cell density and concentration of Gapmer,the optimized transfection condition is confirmed,which is transfection at 72h,cell density at 9�10~5 or 1.5�10~6 and Gapmers at 75nM.The knockdown efficiency reach 50%~80%.Compared with the blank and negative control,the activity of cells after Mi-Lnc70 knockdown was decreased.The migration,invasion and healing ability of the Mi-Lnc70 knockdown cells was weakened by transwell and cell scratch analyses.The migration ability was decreased to 56%,and the invasion ability was decreased to 32.5%.Meanwhile,the cell cycle was blocked at G2/M phase,and the apoptosis was increased.The results showed that the Mi-Lnc70 knockdown could effectively inhibit the malignant phenotype of mouse pancreatic cancer cells.At the same time,the effects of Mi-Lnc70 knockdown on the islet characteristics of Min6 were also examined in this study.Real-Time PCR results showed that the expression of Insulin1,Insulin2,Somatostatin,Pax4,MafB,Isl-1,NeurD1,Pax6,Pdx1,Nkx6.1 and Glucagon,as well as miRNA(miR-124a,miR-15a-3p,miR-15b,miR-23b-3p,miR-30d-5p,miR-16-1-5p,miR-195-5p,miR-7 and miR-375),which are involved in the gene expression of pancreatic organogenesis,were down regulated with Mi-Lnc70 knockdown.The expression levels of Mi-Lnc77,Mi-Lnc78,Mi-Lnc85and Mi-Lnc87 were increased,while the expression levels of Mi-Lnc71,Mi-Lnc72,Mi-Lnc75,Mi-Lnc76 and Mi-Lnc80 were significantly decreased.IF analyses showed that the knockdown of Mi-Lnc70 weakened the immunofluorescence of C peptide,insulin,glugagon and somatostatin,as well as the immunofluorescence of the key?cell specific transcription regulator PDX1.Western blot also confirmed that the expression of PDX1,insulin and glucagon protein was down regulated in Mi-Lnc70knockdown cells.The secretion of insulin and glucagon in the supernatant of Mi-Lnc70 knockdown cells was detected by ELISA,which showed no significant difference in the level of insulin secretion,while the level of glucagon secretion increased.In conclusion,the knockdown of mouse long noncoding RNA Mi-Lnc70weakened the migration,invasion and healing ability of Min6 cells,and also reduced the expression of insulin related transcription factors and pancreatic organogenesis genes.It plays an important role in islet development and cell carcinogenesis.The study of its mechanism may provide a reference for further understanding the pathogenesis of pancreatic cancer.