| Hepatocellular carcinoma(HCC)is one of the leading malignant tumors with the highest morbidity and mortality in China,which seriously threatens the health of Chinese.Regorafenib,a multi-kinase oral inhibitor,is a second-line drug approved by the FDA for the treatment of HCC.A number of clinical trials have confirmed that Regorafenib is beneficial to those patients with advanced liver cancer,especially bringing hope for HCC patients who are ineffective to sorafenib.However,Regorafenib is only beneficial to a few HCC patients,and early sensitive patients usually develop acquired resistance in a short period of time.At present,the regulatory mechanism of regorafenib resistance in liver cancer has not been fully elucidated.Therefore,in-depth study of the mechanism of regorafenib resistance in liver cancer may pave the way for improving its efficacy.Cancer stem cells(CSC)have extremely powerful self-renewal and multi-directional differentiation potential,and they are the leading cause of tumor malignant progression,recurrence and drug resistance.The mechanism by which liver cancer stem cells(LCSC)regulate the sensitivity of regorafenib remains to be elucidated,and it is not clear how liver cancer stem cells make differentiated liver cancer cells(non-LCSC)resistant through information exchange.CRISPR Screen is an emerging high-throughput screening technology for studying tumor targeting genes in recent years.It is fast,efficient,and unbiased,and can be used as a powerful weapon for screening tumor drug resistance genes.Therefore,we plan to screen the genes that regulate regorafenib resistance in liver cancer stem cells based on CRISPR high-throughput screening technology and further explore its resistance mechanisms.Part Ⅰ Screening the new resistant gene to regorafenib for liver cancer stem cell based on CRISPR Screen Objective:In order to determine whether liver cancer stem cells are resistant to regorafenib,CRISPR Screen high-throughput screening technology was used to screen and verify new genes that regulate regorafenib resistance in liver cancer stem cells.Methods:1.The liver cancer stem cells were enriched by spheroid culture,and the expression of stem markers was detected by RT-qPCR to verify that tumor cell spheroids can be used as a representative of liver cancer stem cells.CCK-8(Cell counting kit-8)was used to detect the difference of sensitivity to regorafenib between liver cancer cells and liver cancer stem cells.2.Infect liver cancer cell lines with human genome-wide activation lentiviral libraries based on CRISPR/Cas9 technology.After treatment with regorafenib and placebo,the next generation sequencing,analyzing the negative enrichment of regorafenib treatment group sg RNA(small guide RNA)target genes.3.CRISPR/Cas9 technology was used to knockout the expression of target drug resistance genes in liver cancer stem cells,and CCK-8 was used to detect cell viability to verify the effect of target genes on the sensitivity of liver cancer stem cells to regorafenib.Results:1.The mRNA expression of stemness markers in HCC cells is higher than that in adherent cells,and the sensitivity of LCSC to regorafenib is lower than that in adherent HCC cells.2.Throught CRISPR Screen and high-throughput sequencing analysis,RAB27 A is identified as a gene which sg RNA is negatively enriched in the regorafenib treatment group cells.3.After knocking out RAB27 A,the sensitivity of liver cancer stem cells to regorafenib increases significantly.Conclusion:Liver cancer stem cells are resistant to regorafenib,and RAB27 A is a positive gene that regulates the resistance of liver cancer stem cells to regorafenib.Part Ⅱ The effect of RAB27 A on regorafenib resistance of liver cancer stem cells Objective:To study the effect of RAB27 A on the sensitivity of liver cancer cells to regorafenib.1.To study the effect of RAB27 A on the amount of exosomes secreted by liver cancer stem cells.2.To study the effect of exosomes secreted by liver cancer stem cells on the sensitivity of differentiated liver cancer cells to regorafenib.Methods:1.Western blot was used to detect the effect of RAB27 A knockout on the amount of exosomes secreted by liver cancer stem cells and the changes in stemness markers.Tumor sphere culture and soft agar clone formation assays were used to detect the effect of RAB27 A knockout on the self-renewal ability of liver cancer cells.2.The same amount of exosomes extracted from the culture supernatant of LCSCs and non-CSCs were used to stimulate adherent HCC cells,and CCK-8 was used to detect regorafenib sensitivity in adherent hepatocellular carcinoma cells.3.CCK-8 was performed to detect changes in the regorafenib sensitivity of liver cancer cells after RAB27 A knockout or treatment with exosome inhibitor.4.Mouse xenograft tumor model was used to study the change of regorafenib sensitivity in vivo after RAB27 A knockout.Results:1.After RAB27 A knockout,the amount of exosomes secreted by LCSC is significantly reduced,the expression of stemness marker NANOG is down-regulated,and the number of tumor spheres and soft agar clones is significantly reduced.2.Compared with the stimulation of exosomes derived from adherent cultured liver cancer cells,the stimulation of exosomes derived from LCSC significantly inhibited the sensitivity of adherent cells to regorafenib.3.After RAB27 A knockout or treatment with exosome inhibitor,the sensitivity of liver cancer cells to regorafenib increases in vitro.4.After knocking out RAB27 A,the growth of subcutaneous xenograft tumors was significantly inhibited,and knocking out RAB27 A and regorafenib synergistically inhibited the growth of subcutaneous xenografting tumors.Conclusion:1.Liver cancer stem cells rely on RAB27 A to secrete exosomes.2.The exosomes derived from liver cancer stem cells can induce the expression of NANOG in differentiated liver cancer cells,leading to resistance to regorafenib.3.Knockout of RAB27 A can enhance the sensitivity of liver cancer cells to regorafenib in vivo and in vitro. |
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