Studies On The Biomarker Of MDR In CRC And The Marker Involved In ES2 Reversing MDR

Cancer is a serious threat to human health,and the research and development of antineoplastic drugs is an important component in the field of medical research.Although great progress has been made in the research of antineoplastic drugs,almost all patients will inevitably develop resistance to chemotherapeutic drugs in clinic,which will affect the therapeutic efficacy.Drug resistance,especially multidrug resistance(MDR),is one of the main reasons for the failure of treatment,which seriously affects the quality of life and survival time of cancer patients.The mechanism involved in the occurrence and development of MDR is complex and changeable,but the abnormal expression of ATP binding cassette(ABC)transporter represented by ABCB1,ABCC1 and ABCG2,which increases the pumping of anticancer drugs,is the most widely reported.Colorectal cancer(CRC)is one of the most common malignant tumors all over the world.The high expression of ABCB1 is one of the main factors of MDR in CRC patients with chemotherapy failure,but the regulatory factors related to drug resistance or other potential resistance mechanisms in CRC are unknown.In order to explore these problems,we first used proteomics and transcriptomics methods to screen and identify potential protein targets related to drug resistance phenotype in parent cells HCT8 and MDR cells HCT8/T.The results showed that the expression of insulin-like growth factor-2 m RNA-binding protein 3(IGF2BP3)was upregulated in HCT8/T cells,and si IGF2BP3 significantly increased the sensitivity of HCT8/T cells to DOX.In order to explore the relationship between IGF2BP3 and the sensitivity of chemotherapeutic agents,we constructed stable cell lines and detected the IC₅₀ of the compounds.The results showed that the overexpression of IGF2BP3 promoted the expression of ABCB1and decreased the sensitivity of cells to ABCB1 substrates.On the contrary,The knockout of IGF2BP3 reduced the expression of ABCB1 in vitro and increased the sensitivity to ABCB1 substrates.In addition,the anti-tumor effect of NVB and PTX on IGF2BP3-depleted HCT8/T xenograft models was significantly stronger than that of control HCT8/T xenograft models.In many CRC cell lines,the expression of IGF2BP3was positively correlated with the expression of ABCB1,and negatively correlated with the sensitivity of DOX,which further supported the above findings.In terms of mechanism,IGF2BP3,as a N6-methyladenosine(m6A)reader,recognized and bound the m6A modified region of ABCB1 m RNA,resulting in enhanced stability,up-regulation of m RNA and protein expression of ABCB1,and finally drug resistance to chemotherapeutic drugs.In summary,this part of the study demonstrated that IGF2BP3bound ABCB1 m RNA,and up-regulated ABCB1 expression by enhancing its stability to make CRC cells produce MDR in an m6A-dependent manner.This suggests that IGF2BP3 may be a candidate biomarker for MDR in CRC.Interfering with the expression of IGF2BP3 may become a therapeutic strategy to overcome CRC drug resistance and provide a new idea for the development of antineoplastic drugs.It is urgent to develop new antineoplastic drugs with the characteristics of reversing tumor MDR.Natural products with high biosafety are important sources of MDR reversal agents,and these compounds and their structural derivatives are important components in this field.Our previous studies have found that ES2,a macrocyclic diterpene from Euphorbia officinalis,can significantly increase the sensitivity of drug-resistant cell KB/VCR to chemotherapeutic drugs in vivo and in vitro,and then reverse MDR.ES2 is a potential MDR reversal agent without toxicity,but the specific mechanism involved in its efficacy is still unclear.In order to explore the relevant markers that can indicate the reversal of MDR by ES2,we screened five potential differentially expressed genes through proteomic identification and bioinformatics analysis in KB and KB/VCR cells.After RT-q PCR and Western blotting verification,it was found that only integrin subunit beta 4(ITGB4)and eukaryotic elongation factor 2 kinase(e EF2K)genes were consistent with proteomics data at transcriptional and translational levels.However,changing the expression of the two genes did not affect the sensitivity of cells to chemotherapeutic drugs.In this part of the study,it was found that ITGB4 and e EF2K are not the causes of MDR,so they are not the upstream factors of ES2 reversing MDR process,and can not be used as related markers indicating MDR reversal.Therefore,the changes in the expression of ITGB4and e EF2K could only indicate the characteristics of MDR or the reversal result of ES2in KB/VCR cells.