Screening Drug-resistant Melanoma Cell Line Using Genome-wide SiRNa Library And Mechanical Exploring

Objectives: FDA approved BRAF inhibitor vemurafenib for the treatment of melanoma patients with BRAF V600 E mutation or advanced unresectable cancer in 2011. After a period of treatment in clinical practice, drug-resistant of vemurafenib appears. To solve this problem, we constructed a siRNA library covering the human genome, screened drug-resistant melanoma cell lines using siRNA library, sequenced by Illumina Hi Seq, verificate of single sRNA fragments after sequencing, and explore the infection conditions. The aim of this thesis is to further expect the mechanisms of clinical drug-resistance from gene silencing.Methods: We got our library fragment which contains 19 base randomly arranged firstly by chemical synthesis, PCR to amplify the fragment complementary sequences to form double-stranded DNA which is used as Gibson assembly insert cassette. Retroviral plasmid backbone pSOK is used as vector which will be inserted this cassette by Gibson assembly and some verification will be done.Three melanoma cell lines, A375, MDA-MB-435 and UACC62 with BRAF V600 E mutation were selected. Vemurafenib and trametinib which were FDA-approved for the treatment of BRAF V600 E mutation as first-line treatment of melanoma were used to screen drug-resistant cells. After explicit the lethal dose of the cells, we screened drug-resistant cells out by siRNA library. Crystal violet staining, IC50 detection, Q-PCR detection of resistance-associated genes, EMT-related genes were used to verify its resistant. Meanwhile, genomic DNA was extracted from these cells and PCR amplified fragment. All these PCR fragments were sequenced by Illumina HiSeq. According to the sequencing result, we picked up 11 single siRNA sequences, and constructed plasmids which can overexpress these fragments. Cells were transfected these plasmids and some drug-related testing were done.To make sure that one cell just has only one purpose retroviral infection, two types of retroviral plasmids(pSEN-GFP containing G418 resistance gene and pSEB-RFP containing BSD resistance gene) have been designed. Meanwhile, detection of viral titers, the best infection time and viral stability storage time were tested.Results:1. The number of siRNA library plasmid we constructed can reach to 1.08x106 just in once assembly, the positive rate of inserted cassette was 91.7%, and no sequence of these 15 plasmids which we selected randomly was repeated. These data showed that the siRNA library plasmid has been greatly covering 30,000 genes of the human genome.2. As to vemurafenib, the lethal doses of A375 and MDA-MB-435 cells are 25 uM, except UACC62(50uM). Meanwhile, as to trametinib, the lethal dose of these tree cells is 20 uM.3. We successfully screened out A375 V, A375 T, A375 VT and A375 TV cells with the lethal dose of anticancer drugs vemurafenib and trametinib.4. Compared with the control group A375, A375 V shows stronger drug-resistance to dabrafenib(a BRAF inhibitor like vemurafenib), trametinib(target MEK protein), and cisplatin and gemcitabine(used as clinical first-line cytotoxic drugs).5. The IC50 of A375 V was greatly enhanced to 5.09 uM, far higher than the control cells A375 and A375n19(with IC50 of 0.18 uM).6. 13 drug-resistant relative genes selected randomly and RAS/RAF/MEK pathway were up-regulated in drug-resistant cells(A375V, A375 T, A375 VT and A375TV). As to the EMT-related genes, compared with control cells A375n19, CDH1, CDH2, VIM, FOX2, and OCLN genes are up-regulated too.7. We successfully extracted genomic DNA of drug-resistant cell lines and designed the sequencing primers for PCR amplification to sequence by Illumina HiSeq.8. 11 single siRNA fragments selected randomly were transfected into A375. 9 of 11 single siRNA fragments can enhance the drug-resistance of A375. The result is verified by crystal violet staining.9. The expression of drug-resistant relative genes selected randomly, RAS/RAF/MEK pathway and EMT related genes is up-regulated in A375TBRs(single siRNA fragments overexpressed).10. Some candidate target genes at the downstream of five fragments repeat highest by bioinformatic analysis. Compared with control cells A375n19, these genes are up-regulated both in the resistant cell lines(A375V, A375 T, A375 VT and A375TV) and A375 TBRs.11. We blasted the lncRNAs targeted by TBR1, TBR3 and TBR 8, and designed corresponding primers(ncRNA-1 to ncRNA-25). Compared with the control group A375n19, the trend of these ncRNAs in the drug-resistant cell lines and A375TBR3/5/7/8/9/10 are the same.12. The best collection times of retroviral are 36 h, 48 h, 60 h and 72 h after packaging. As to A375 cell, the optimal infected time is about 4 hours.13. The titer of the retrovirus is declined gradually with the extension of storage time, and it decreased the most obviously after 4 days.14. The infection rate of different cells is different from each other even in the same retrovirus titer, cell density and infection time.