The Relationship Between Chaperone HSPA5 In Endoplasmic Reticulum Stress And Proliferation、Invasion Abilities Of A549 Lung Cancer Cells

Objectives To research the relationship between chaperone HSPA5 in Endoplasmic reticulum stress and proliferation,invasion abilities of A549 lung cancer cells,and to further study the specific pathways and the role of relevant components in the pathway,so as to explore the new target of drug therapy for lung cancer.Methods The expression levels of HSPA5 in cancer and adjacent tissues in 21 patients with lung cancer were studied by Western Blot to contrast the expression level of HSPA5 in lung tissues with normal tissues.The HSPA5 mRNA levels in cancer tissues and adjacent tissues in 96 lung cancer patients were tested by real-time fluorescence quantitative PCR to verify the difference of HSPA5 transcription levels.CD513B-Cas9 and CD513 B were used to construct the CRISPR-Cas9 knockout plasmid and overexpression plasmid,and the right plasmid was transfected into A549 cells.Successful HSPA5 CRISPR-Cas9 knockout and overexpression cell models tested by selecting single clone,doing Westerrn Blot and sequencing were selected for the experiment.HSPA5 overexpression plasmid was transfected into cells in the HSPA5 knockout group,so as to study the functional changes of A549 cells in the rescue expression group.Through CCK8 cell proliferation experiment and clone formation assay,we compared the proliferation abilities of the CRISPR-Cas9 knockout group,overexpression group,rescue expression group and control group.The migration ability and invasion abilities of A549 cells were researched by Transwell chambers.Results The result of the pre-experiment showed that the expression of HSPA5 in lung cancer tissues was higher than that in para-tumorous tissues.We successfully constructed A549 cell models of CRISPR-Cas9 knockout,overexpression and rescue expression of HSPA5.The CCK8 cell proliferation showed that compared with the control group,the proliferation ability of A549 cells in the CRISPR-Cas9 knockout group was reduced,however the proliferation ability ofA549 cells in the overexpression group was increased.The proliferation ability of A549 cells in the rescue group was between the knockout group and the control group.The clone formation assay showed that the clone formation number in the control group,knockout group,rescue expression group and overexpression group was about 300,210,250 and 360 respectively.The clone formation number in the overexpression group was the most,in the control group was the second,in the rescue expression group was the third,in the knockout group was the lowest.The Transwell cell migration experiment showed that the number of A549 cells passing through the small chamber in the overexpression group was the most,in the control group was the second,in the rescue expression was the third,and in the knockout group was the lowest.We counted the number of A549 cells through the small chamber:the overexpression group was about 1750,the control group was about 1500,the rescue expression group was about 900,and the knockout group was about 800.The Transwell cell invasion experiment showed that the number of A549 cells passing through the small chamber in the knockout group was 260,in the in the rescue expression was about 380,in the control group was about 410,and in the overexpression group was about 500.Conclusion After knock out the HSPA5 gene of lung cancer A549 cells,the proliferation ability,migration ability and invasion ability of cells are weakened,however the proliferation ability,migration ability and invasion ability of A549 cells are enhanced after overexpress the HSPA5 gene.The proliferation,migration and invasion abilities of A549 cells in the rescue group are between the control group and the knockout group.In conclusion,the expression of HSPA5 gene was positively correlated with proliferation,migration and invasion abilities of A549 cells.