Using CRISPR-Cas9 Gene Editing Technology To Study The Effects Of LIAS Gene On Cell Proliferation Of PANC-1 Pancreatic Cancer And Its Clinicopathological Significance

BackgroundPancreatic ductal adenocarcinoma is one of the digestive tract tumors with a high degree of malignancy,characterized by insidious onset,rapid progression,and high mortality.Due to its high frequency silencing genes and unique tumor microenvironment,pancreatic cancer has high recurrence rate and metastasis rate after surgery.At the same time,it shows broad tolerance to chemoradiation and is insensitive to immunotherapy.Therefore,compared with other common malignant tumors,the prognosis of pancreatic ductal adenocarcinoma has not been significantly improved,with a 5-year survival rate of less than 10%.In order to overcome the limitations of traditional cancer treatment,gene targeted therapy may bring a new therapeutic strategy for pancreatic cancer."Oncogene addiction" refers to the apparent dependence of the occurrence and development of tumors on certain overactive genes,oncogenic proteins,or signaling pathways.When these genes,proteins,or signaling pathways are inactivated,tumor growth is also severely affected.This theory provides a biological and theoretical basis for molecular targeted therapy.Based on the above theory,our research group applied proteomics technology to identify 17 human central proteome members as short-lived proteins.The study found that the rapid downregulation of these proteins may be related to acute cell apoptosis induced by addiction oncogenes.Lipoic acid synthase(LIAS)is one of these proteins.LIAS belongs to the biotin and lipoic acid synthase family and is involved in the synthesis of antioxidant lipoic acid.Relevant studies have shown that LIAS can affect the progress of diseases such as diabetes,atherosclerosis and neonatal epilepsy by regulating oxidative stress and mitochondrial function.At the same time,The mutation of LIAS can indirectly regulate HIF-1,which may have an impact on tumor proliferation and angiogenesis.In addition,researchers found that the expression of LIAS is closely related to the infiltration of immune cells in atherosclerosis,pulmonary fibrosis and other diseases.This provides research clues and ideas for its immune escape in the tumor microenvironment.Finally,LIAS is a key upstream regulator of cuproptosis,a newly discovered regulatory cell death mode.Cuproptosis is just one of the potential strategies to overcome the resistance of cancer cells to apoptosis.In conclusion,LIAS has potential research value for cancer proliferation,angiogenesis,and immune escape.However,there is no study on pancreatic cancer by LIAS.Therefore,LIAS is expected to become a potential target for the treatment of pancreatic cancer,providing theoretical support and experimental basis for its future research in the treatment and prognosis of pancreatic cancer.PurposesThis project aims to study the expression and clinicopathological significance of LIAS in pancreatic cancer.Using CRISPR-Cas9 gene editing technology constructs pancreatic cancer cell line with stable knockout of LIAS gene and explore its effect on the proliferation of pancreatic cancer cells.These studies will preliminarily determine whether LIAS can be used for gene-targeted therapy in pancreatic ductal adenocarcinoma or not.Methods(1)The expression of LIAS in pancreatic and adjacent tissues was first analyzed by using the GEPIA 2.0 database.Then TCGA database was applied to statistical analyze the expression of LIAS in different clinicopathological stages and grades.Finally,the TISIDB website was used to statistically analyze the relationship between LIAS expression and immune cell infiltration in pancreatic cancer.(2)The expression of LIAS was detected in normal tissues and pancreatic ductal adenocarcinoma tissues with different degrees of differentiation by immunofluorescence.And the relationship between the expression level of LIAS and different clinicopathological features such as tumor tissue differentiation,recurrent organs,age and sex were statistically analyzed.(3)Using CRISPR-Cas9 gene editing technology,we have constructed PANC-1 pancreatic cancer cell line with LIAS gene knocked out.(4)The changes in the proliferation ability of PANC-1 pancreatic cancer cells with targeted knockout of LIAS gene were explored in vitro by using the cell proliferation assay(CCK 8)and cell clone formation assay.Results(1)GEPIA 2.0 database analysis showed that the expression of LIAS in pancreatic cancer tissues was higher than that in normal tissues.According to TCGA database analysis,the expression of LIAS was lower in the local stage of T3 than that of T1(P=0.04).The expression of LIAS was not significantly different from lymph node involvement(phase N)and distant metastasis(phase M).The expression of LIAS in moderately differentiated(G2 grade)pancreatic cancer was lower than that in highly differentiated(G1 grade)pancreatic cancer(P=0.012).The expression of LIAS in clinical stage II was significantly lower than that in clinical stage I(P=0.0062).TISIDB website analysis showed that the expression of LIAS was closely related to the infiltration of immune cells in pancreatic cancer.(2)Immunofluorescence analysis of clinical samples showed that the expression of LIAS in pancreatic cancer tissues was higher than that in adjacent tissues,and the expression of LIAS in highly differentiated pancreatic cancer tissues was higher than that in moderately and poorly differentiated pancreatic cancer tissues.Through statistical analysis of the relationship between the expression of LIAS and different clinicopathological characteristics of pancreatic cancer,it was found that the higher the clinical stage of pancreatic cancer,the lower the expression of LIAS,with a statistically significant difference(P=0.0037).The expression of LIAS was correlated with whether the organ was involved(P=0.001),but no significant correlation with age,sex or infiltration(P>0.05).(3)Using CRISPR-Cas 9 gene editing technique,the proliferation capacity of PANC-1 was significantly inhibited compared with that of PANC-1 cells in vitro(P<0.05).ConclusionsThis study reveals the expression of LIAS in pancreatic cancer in different clinicopathological stages.Meanwhile,the expression of LIAS was closely correlated with the immune cell infiltration.Targeted knockdown of the LIAS gene suppresses the proliferation of pancreatic cancer cells in vitro.Therefore,we speculate that LIAS may be the potential target for gene-targeted therapy in pancreatic cancer.