Effects Of RNA ABHD11-AS1 Alternative Splicing On The Growth Of Pancreatic Cancer Cells And Its Mechanism

Objective:To explore the effect of alternative splicing of lnc RNA ABHD11-AS1 on the progression of pancreatic cancer,and to study the expression of ABHD11-AS1-S and ABHD11-AS1-L in normal human pancreatic ductal epithelial cell line and pancreatic cancer cell lines,as well as their effects on proliferation,migration,invasion,lipid synthesis and transformation of saturated fatty acids to unsaturated fatty acids of pancreatic cancer cells,and make a preliminary analysis of possible interaction between the two transcripts.Methods:1.The expression level of lnc RNA ABHD11-AS1 in normal pancreatic tissues and pancreatic cancer tissues was compared and the relationship between the expression of lnc RNA ABHD11-AS1 and the prognosis of pancreatic cancer patients was analyzed on the GEPIA website to explore its clinical significance and research value.2.Semi-quantitative PCR and DNA sequencing technology were used to analyze the alternative splicing of lnc RNA ABHD11-AS1 and the expression of its two transcripts ABHD11-AS1-S and ABHD11-AS1-L in normal human pancreatic ductal epithelial cell line and pancreatic cancer cell lines.3.The overexpression plasmids pc DNA3.1-ABHD11-AS1-S and pc DNA3.1-ABHD11-AS1-L were constructed by genetic engineering techniques,respectively,and their overexpression efficiencies were identified by semi-quantitative PCR and RT-PCR.4.pc DNA3.1-ABHD11-AS1-S and pc DNA3.1-ABHD11-AS1-L were transfected into Bx PC3,Pa Tu8988 and PANC1 cells.CCK-8 assay,Transwell migration and invasion assays were used to detect the effects of ABHD11-AS1-S and ABHD11-AS1-L on proliferation,migration and invasion of pancreatic cancer cells.5.pc DNA3.1-ABHD11-AS1-S and pc DNA3.1-ABHD11-AS1-L were transfected into Bx PC3,Pa Tu8988 and PANC1 cells.Lipid metabolism related markers were detected at protein and m RNA levels by Western blot and RT-PCR.Maturity and nuclear translocation of SREBP1 were detected by Western blot and immunofluorescence assay.Results:1.It was showed through GEPIA website analysis that the expression level of lnc RNA ABHD11-AS1 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues,and the expression level of lnc RNA ABHD11-AS1 was negatively correlated with the prognosis of pancreatic cancer patients.2.Semi-quantitative PCR on c DNA of H6C7,Bx PC3,Pa Tu8988 and PANC1 cell lines revealed that lnc RNA ABHD11-AS1 had two bands with different molecular weights.Through DNA sequencing,it was found that the high molecular weight band retained introns and was named ABHD11-AS1-L,while the low molecular weight band did not retain introns and was named ABHD11-AS1-S.ABHD11-AS1-S was highly expressed in pancreatic cancer cells and almost not in normal pancreatic duct epithelial cells.The expression of ABHD11-AS1-L in normal pancreatic duct epithelium was significantly higher than that in pancreatic cancer cells.3.PCR and plasmid sequencing results showed that the recombinant plasmids pc DNA3.1-ABHD11-AS1-S and pc DNA3.1-ABHD11-AS1-L were constructed successfully and could efficiently overexpress ABHD11-AS1-S and ABHD11-AS1-L in Bx PC3,Pa Tu8988 and PANC1 cells.4.In addition to the increased expression of ABHD11-AS1-L,expression of ABHD11-AS1-S was also increased after upregulating ABHD11-AS1-L in PANC1 cells and Pa Tu8988 cells.However,expression of ABHD11-AS1-S was not increased after upregulating ABHD11-AS1-L in Bx PC3 cells.5.Expression of ABHD11-AS1-L was significantly decreased or even disappeared after upregulating ABHD11-AS1-S in Bx PC3,Pa Tu8988 and PANC1 cells.6.Compared with the control group,the proliferation was increased and the migration ability and invasion ability were enhanced after upregulating ABHD11-AS1-S in Bx PC3,Pa Tu8988 and PANC1 cells,while the proliferation was decreased and the migration ability and invasion ability were inhibited after upregulating ABHD11-AS1-L in Bx PC3,Pa Tu8988 and PANC1 cells.7.Compared with the control group,the protein and m RNA expression levels of SCD1 and FASN increased after upregulating ABHD11-AS1-S in Bx PC3,Pa Tu8988 and PANC1 cells,while the protein and m RNA expression levels of SCD1 and FASN decreased after upregulating ABHD11-AS1-L in Bx PC3,Pa Tu8988 and PANC1 cells.8.Compared with the control group,the up-regulation of ABHD11-AS1-S promoted maturity and nuclear translocation of SREBP1,and the up-regulation of ABHD11-AS1-L inhibited maturity and nuclear translocation of SREBP1.Conclusion:1.Lnc RNA ABHD11-AS1 generates two transcripts,ABHD11-AS1-S and ABHD11-AS1-L,through alternative splicing of intron retention mode.2.ABHD11-AS1-L may be transformed into ABHD11-AS1-S as material,and ABHD11-AS1-S may promote the degradation or splicing of ABHD11-AS1-L.3.ABHD11-AS1-S promotes nuclear translocation of SREBP1 to increase lipid synthesis and transformation of saturated fatty acids to monounsaturated fatty acids,thus promoting proliferation,migration and invasion of pancreatic cancer cells and promoting the progression of pancreatic cancer.ABHD11-AS1-L inhibits nuclear translocation of SREBP1 to decrease lipid synthesis and transformation of saturated fatty acids to monounsaturated fatty acids,thus inhibiting proliferation,migration and invasion of pancreatic cancer cells and inhibiting the progression of pancreatic cancer.