The Role And Mechanism Of SNRPA1 In Colorectal Cancer Cells

Objective: Colorectal cancer(CRC)is the third most common cancer in the world and the fourth cancer with a global cancer death rate,with approximately 1,360,000 cases per year and approximately 650,000 colorectal cancer deaths.In 2018,the GLOBOCAN online analysis tool predicted that approximately 606,840 new cases were generated in Asia.Therefore,the prevention and treatment of CRC is of great significance.It is a splice component responsible for processing precursor mRNA into mRNA.SNRPA1 is a biomarker for the prognosis of many types of cancer.In addition to its role in mediating RNA processing,studies of other functions of SNRPA1 have not been performed in colorectal cancer.Based on the current study of SNRPA1,we hypothesize that SNPRA1 should also play an important role in the progression of CRC.To validate this hypothesis,we designed and produced a shRNA lentivirus that knocks down the expression of SNRPA1 and detects the subsequent effects of SNRPA1 silencing on CRC cell function and explores the molecular mechanisms underlying it.Methods: 1.Design the target of siRNA,synthesize single-stranded DNA oligo containing interference sequence,and anneal pair to produce double-stranded DNA;then directly ligase into the digested lentiviral vector through its restriction enzyme sites at both ends;The prepared E.coli competent cells were identified by PCR,and the positive recombinants were identified by PCR,and the sequencing was carried out,and the sequencing results were compared with the correct clones for plasmid extraction.293 T cells were co-transfected with highly efficient recombinant vectors and viral packaging plasmids for viral packaging and production.The virus solution was collected,concentrated,purified,and 293 T cells were infected with a high quality virus solution.The virus titer was measured,and the experimental results were analyzed by real-time quantitative PCR and Western blot experiments.Finally,colorectal cancer cells were infected with high-quality viral fluid,the silencing efficiency of siRNA was detected,and stable transfected cell lines were screened using specific drugs.2.Infected colorectal cancer cells knocked down the expression of SNRPA1 gene,the cell growth status was detected by Celigo cell counting,and the ability of clone formation was detected.The effect of inhibiting SNRPA1 gene expression on the proliferation of human colorectal cancer cells was studied.The effect of inhibiting SNRPA1 gene expression on the viability of human colorectal cancer cells was examined by MTT assay.The effect of inhibition of SNRPA1 gene expression on apoptosis of human colorectal cancer cells was investigated by Caspase 3/7 assay.3.Inhibition of SNRPA1 gene expression on the biological characteristics of human colorectal cancer cells in animals by nude mice tumor formation experiments.Differential genes were screened by gene chip.Then,through IPA analysis results,genes related to tumor cell proliferation and apoptosis were found in the disease function analysis results,and genes that inhibit tumor cell proliferation or promote tumor cell apoptosis were selected,and the target genes were selected by reference data and the gene relationship was drawn.Network Diagram.The SNRPA1 gene was studied to influence the performance of which genes downstream,thereby affecting cell proliferation.Finally,the results of genomics were verified by real-time quantitative PCR and western blot experiments.Results: 1.The effective SNRPA1 siRNA sequence was successfully screened and the recombinant lentiviral SNRPA1 shRNA expression vector was successfully constructed.Transfection of human colorectal cancer cells RKO and HCT116 significantly down-regulated the expression levels of SNRPA1 mRNA and its protein.2.Compared with the control group,cell proliferation and cell viability of RKO or HCT116 cells in the SNRPA1 knockdown group were significantly inhibited.To confirm the effect of SNRPA1 knockdown on CRC cell apoptosis,activated caspase-3/7 in the knockdown group and the control group after shRNA lentiviral transduction was examined.The caspase 3/7 activity in sh KOPA1 lentiviral transduced RKO or HCT116 cells was significantly increased compared to the control group,indicating that RKO or HCT116 cells experienced an increase in apoptosis following sh SNRPA1 lentiviral transduction.Thus,SNRPA1 knockdown can promote apoptosis in RKO or HCT116 cells.3.The results of gene chip showed that after SNRPA1 gene knockdown,602 genes were up-regulated and 765 genes were down-regulated in RKO cells.Genes that inhibit tumor cell proliferation or promote tumor cell apoptosis were selected and the SNRPA1 gene was added to map the gene relationship.A total of 38 genes interacting with SNRPA1,including 17 up-regulated genes and 21 down-regulated genes,were found.It is found in the network that SNRPA1 may affect the proliferation of cells by affecting the expression of a series of downstream genes by acting on EGFR and HGF.After qRT-PCR verification of these genes,the results were basically consistent with the chip.In addition,the genes of interest were verified by immunoblotting.After SNRPA1 knockdown,the expression of NRP1 and PIK3R1 genes increased,and the expression of E2 FZ,VEGFC,MKI67 and CDK1 genes decreased.It indicated that SNPRA1 affects the development of CRC through interaction with EZH2,VEGFC,MKI67,NRP1,PIK3R1,CDK1 and other genes.Conclusion: Knockdown of SNRPA1 plays an important role in the development of CRC by upregulating NRP1,PIK3R1 and down-regulating E2 FZ,VEGFC,MKI67,CDK1,etc.,which affect the proliferation and apoptosis of CRC cells.This study found that SNRPA1 plays an important role in the development of CRC in addition to its involvement in RNA processes.These new findings will provide the basis for future development of new diagnostic or therapeutic targets in the diagnosis and treatment of CRC.