Construction Of A B-Raf Mutant Colon Cancer Cell Line Based On Gene Editing Technology And Preliminary Investigation Of The Possible Mechanism Of Antitumor Activity Of Andrographolide

Objective:Colon cancer is one of the common malignancies,and timely diagnosis and treatment regimens have substantially improved patient survival,but several challenges remain,such as recurrence,metastasis and drug resistance,and its treatment remains a major challenge Andrographolide（Andro）,a diterpene lactone obtained from the medicinal plant Andrographis paniculata by isolated extraction and purification,is the main active ingredient of Andrographis paniculata with a variety of biological activities,including antibacterial,anti-inflammatory,anti-infective,and anticancer.The results of previous studies showed that Andro could inhibit the proliferation,migration and invasion of SW480 cells,but its specific mechanism awaits further studies.The B-Raf gene is an important proto-oncogene located on the long arm of human chromosome 7,and among the mutations in this gene Mutations in V600E are the most common and occur in a variety of tumors.It has been found that B-Raf V600E mutations can cause sustained activation of the Ras/Raf/MEK/ERK signaling pathway,which is one of the important causes of colon cancer development and progression.In this study,we constructed a B-Raf V600E mutant SW480 cell line by gene editing technology and preliminarily investigated the possible mechanism of the anti-tumor activity of Andro.Methods:Part 1,AAVS1-all-in-one-CRISPR plasmids were constructed using molecular cloning techniques,and liposomes were cotransfected into HEK-293T cells with ZFN and TALEN plasmids that identified and cleaved the AAVS1 genomic sequence and the AAVS1-all-in-one-CRISPR plasmid.After obtaining monoclonal cells using puromycin screening,stable cell lines with complete integration of the CRISPR-Cas9 gene editing reporter system into the AAVS1 safe harbor were obtained using inoculin S screening,red fluorescence detection,Cas9 Western Blot,and genomic PCR identification.The EGFP repair template single-stranded DNA and EGFP sgRNA were transfected into the above cell lines using liposome transfection technique,and the loss of fluorescence of EGFP was genetically edited,and the expression of EGFP was detected by fluorescence microscopy fluorescence.Part 2,this study was designed to target the single guide RNA（sgRNA）of B-Raf V600E gene and the B-Raf V600E,donor repair template was transfected into SW480 cells with Cas9 targeted integration of AAVS1 locus,while using AAV virus as a delivery vector for B-Raf mutant donor repair template with chemically modified sgRNA.The purified Cas9 protein was co-transfected into SW480 cells.The B-Raf V600E gene was detected for mutation by PCR and PCR sequencing.Part 3,Firstly the dose range of Andro was initially detected by MTT assay.Then a wound healing assay was performed using the IC₅₀ value of Andro concentration to examine the effect of Andro on the metastatic and invasive ability of wild-type and mutant SW480 cell line at this does.Finally,the expression of B-Raf protein SW480 cells was examined by Western blot to investigate the possible mechanism of IC₅₀value Andro against cancer.Results:In the first part,monoclonal cells with both puromycin and insecticide S resistance and expressing red fluorescent protein were obtained,the successful expression of Cas9 protein was detected by Western Blot,and the target gene was successfully integrated into the genomic AAVS1 locus as identified by genomic PCR.after Cas9-mediated EGFP gene editing,the cells recovered green fluorescence as detected by fluorescence microscopy,Cas9 has editing function and this system was successfully established.In the second part,the sgRNA targeting the B-Raf V600E gene was designed,Cas9 was guided to perform targeted cleavage,and the B-Raf V600E donor repair template was given,and the B-Raf V600E mutant SW480 cell line was finally obtained by cellular genomic DNA extraction,PCR amplification of the DNA fragment of the target gene and PCR product sequencing.In the third part,the MTT results showed that the semi-inhibitory concentration(IC₅₀)values of Andro to inhibit the cell viability of swild-type and B-Raf V600E mutant SW480 cells were44.6μM and 36.5μM,respectively.Wound healing assay results showed that Andro had a stronger inhibitory effect on B-Raf V600E mutant SW480 cells.Western blot results showed that the expression of B-Raf protein was elevated in B-Raf V600E mutant cells and significantly decreased in B-Raf V600E mutant cells after following the treatment of Andro.Conclusion:The B-Raf V600E mutant SW480 cell line constructed using gene editing can be used for anti-tumor drug screening and the study of anti-tumor mechanism.Andro inhibited the metastatic proliferation invasive ability of both wild-type and mutant SW480 colon cancer cells,and the inhibitory effect was stronger in the mutant cell line,suggesting that the mechanism of Andro on the inhibition of colorectal cancer might be related to the reduction of B-Raf protein expression level.The results of the study lay the foundation for exploring new mechanisms of Andro for the treatment of colon cancer and can contribute to the development of new anticancer drugs targeting the B-Raf V600E mutation.