Hsp70敲低与敲除抑制小鼠结直肠癌细胞MC38恶性表型及其在人源SW1116细胞中的验证OA

In order to investigate the role of heat shock protein 70(HSP70)in the malignant progression of colorectal cancer,this study utilized mouse colorectal cancer MC38 cells as the primary model.Small interfering RNAs(siRNAs,siRNA1,2,3)for transient knockdown and single guide RNAs(sgRNAs,sgRNA1,2,3)for CRISPR/Cas9-mediated stable knockout were designed and synthesized based on the murine Hsp70 and human HSP70 gene sequences.Transient knockdown and stable knockout of Hsp70 were achieved via liposomal transfection;stable Hsp70-knockout MC38 cell lines were established through puromycin selection of sgRNA-transfected cells.Experimental groups included the Control,siRNA negative control(si-NC),knockdown(siRNA1,siRNA2,siRNA3),and knockout(sgRNA1,sgRNA2,sgRNA3)groups.Knockdown and knockout efficiencies were validated at both mRNA and protein levels using real-time quantitative PCR(RT-qPCR)and Western-blot.The most effective siRNA(siRNA group)and sgRNA(sgRNA group)were selected for subsequent functional assays.The effects of Hsp70/HSP70 loss on cell proliferation,migration,and invasion were comprehensively evaluated using Cell Counting Kit-8(CCK-8)assay,crystal violet staining,colony formation assay,and Transwell assay.To validate the translational relevance of key findings,parallel experiments were conducted in human SW1116 cells.The results indicated that in MC38 cells,all siRNA groups(siRNA1,siRNA2,siRNA3)exhibited significantly reduced Hsp70 mRNA and protein expression levels(P<0.001),while Hsp70 mRNA and protein were completely undetectable in the knockout groups.siRNA3 showed the most pronounced inhibitory effect on Hsp70 protein expression,so it was selected,along with sgRNA3,for subsequent experiments.In CCK-8 assays at 24,48,and 72 h,no significant changes in OD450 values were observed in the si-NC group compared to the Control group(P>0.05).In contrast,OD450 values were significantly decreased in the siRNA group compared to the si-NC group,and sgRNA group compared to the Control group(P<0.01,P<0.001).In the crystal violet staining,compared to si-NC and Control groups,OD595 values in both siRNA and sgRNA groups were significantly reduced(P<0.001).In the colony formation assay,compared to the Control group,MC38 cell colonies in the sgRNA group were significantly decreased(P<0.01).In the migration and invasion experiment,compared to si-NC and Control groups,numbers of migrated and invaded cells in both siRNA and sgRNA groups were significantly reduced(P<0.01).Parallel experiments in SW1116 cells yielded comparable results.These findings indicate that both knockdown and knockout of Hsp70/HSP70 significantly suppress cell viability,short-term proliferation,migration,and invasion in MC38 and SW1116 cells,suggesting that HSP70 was a critical factor maintaining the malignant phenotype of colorectal cancer cells.