Inhibition Of CYP1B1 By MiR-200b-3p Increases Sensitivity Of Paclitaxel In The Treatment Of Hepatocellular Carcinoma

Liver cancer can be divided into primary liver cancer and secondary liver cancer.China is a high incidence area of liver cancer,and primary liver cancer is currently the second cause of cancer death in China.Hepatocellular Carcinoma(HCC)accounts for the highest proportion of primary liver cancer.The high intratumoral heterogeneity of liver cancer seriously reduces the efficacy of chemotherapy drugs.Therefore,it is urgent to focus on improving the sensitivity of HCC to chemotherapy drugs.Drug metabolizing enzyme CYP1B1 is a member of cytochrome P450 superfamily,which can metabolize exogenous substances(polycyclic aromatic hydrocarbons and aromatic amines)and endogenous substances(estrogen and arachidonic acid).The expression of CYP1B1 in breast cancer,ovarian cancer,prostate cancer and colon cancer is higher than that in normal tissues,and it has also been reported that CYP1B1 is related to HCC resistance of taxanes,cisplatin and gemcitabine.Mi RNA is a kind of noncoding single-stranded RNA with a length of about 22 nucleotides,which is encoded by endogenous genes.It binds to AGO protein to form RNA-induced silencing complex(RISC),and then specifically binds to the m RNA of the target gene to degrade it,or inhibits the translation process to inhibit gene expression.In our previous research,it was found that the downregulation of CYP1B1 can alleviate the insensitivity of HCC to paclitaxel(PTX).In this paper,we explore the miRNA targeting CYP1B1,and verify whether miR-200b-3p that we chose has the effect of sensitizing PTX at HCC cellular level and in patient-derived xenografts(PDX)model.First of all,explore the miRNA targeting CYP1B1.7 miRNAs that may target CYP1B1 were obtained through database miRDB and Target Scan prediction.QRT-PCR and Western Blot were carried out to study their effects on expression of CYP1B1,and miR-200b-3p,with the strongest inhibitory effect,was preliminarily screened out.Then,the dose-response relationship of miR-200b-3p on CYP1B1 was investigated in SNU-387 and Li-7 cells,and m RNA expression levels of miR-200b-3p and CYP1B1 in tumor tissues and paired adjacent tissues from 9 HCC patients were detected.Results show that the two have a negative correlation.Database analysis supports this conclusion.Subsequently,whether miR-200b-3p has the effect of sensitizing PTX was verified on human HCC cells SNU-387 and Li-7.Overexpression of miR-200b-3p and administration of PTX were simultaneously carried out to observe tubulin polymerization,cell cycle arrest and apoptosis induction of PTX.Experiments were divided into 4 groups: control,miR-200b-3p alone,PTX alone and combination therapy.The immunofluorescence of α-tubulin showed that the polymerization of tubulin increased in PTX group,and was further enhanced after combined medication.Cell cycle was detected by flow cytometry,and it was found that the proportion of cells blocked in G2/M phase in PTX group increased,which was also further enhanced by combined medication.These results suggest that miR-200b-3p can sensitize PTX in HCC cells.Finally,whether miR-200b-3p can sensitize PTX was verified on PDX model.Tumor tissue of HCC patient was inoculated subcutaneously into BALB/c Nude mice to establish the PDX model.Set Saline group,miR-200b-3p group,PTX group and combined drug group to investigate the sensitivity of PDX model to PTX.Through real-time monitoring of tumor growth and the weighing of tumor mass,it was found that there was no tumor inhibition in single drug group,while the tumor growth rate decreased significantly in combined drug group.The immunofluorescence of α-tubulin on tumor showed that the fluorescence intensity of the combined drug group was stronger than that of PTX group,indicating that miR-200b-3p enhanced the tubulin polymerization of PTX in HCC.At the same time,the preliminary safety of this combined drug regimen was evaluated,and the weight of mice,the content of oxidative stress factor and the morphology of HE staining in liver and kidney were also compared.Results showed that there were no obvious side effects after combination therapy.