This feature makes miRNA an ideal strategy for anti-tumor gene therapy; tumors can be treated by cloning the full pre-miRNA sequence into a vector and delivering it in lentiviral particles (26). patients receiving sorafenib. Using the online tool miRDB, we predicted that has-microRNA-4277 (miR-4277), an online miRNA targets the 3UTR of the transcript of and a systemic analysis, Feng et?al. (18) and Shao et?al. (19) found that sorafenib can act as a ligand/agonist to activate PXR/NR1I2 (pregnane X receptor/nuclear receptor subfamily 1 group I member 2) and induce expression of downstream genes involved in chemoresistance, including and (ATP-binding cassette, sub-family B, member 1). Ultimately, this accelerates elimination of the therapeutic and results in drug resistance through negative feedback regulation (18, 19). Although related studies have expanded our understanding of PXR in HCC, much remains unclear; PXR is not the only metabolism-related nuclear receptor in HCC cells, and the CAR/NR1I3 (constitutive androstane receptor/nuclear receptor subfamily 1 group I member 3) may have similar functions to PXR (20, 21). By inhibiting the activity of PXR alone, CAR may have a compensatory effect on the function of PXR. Moreover, targeting oxidative metabolism, which is mediated by CYP3A4 in the initial step of sorafenib elimination in HCC cells, represents a promising approach to enhance the sensitivity of these cells to targeted agents (22, 23). This makes CYP3A4 more advantageous to target compared with PXR or CAR. Both PXR and CAR mediate the expression of CYP3A4 to eliminate sorafenib. By Keratin 16 antibody inhibiting CYP3A4, compensatory effects between PXR and CAR can be avoided. MicroRNA is a type of small non-coding RNA transcribed by RNA polymerase II (24C27). In mammalian cells, miRNA can directly affect the 3UTR of the target mRNA to degrade it in a sequence-specific manner and silence gene expression (28C30). Because of this feature, miRNAs are widely used as anti-cancer therapeutics. By predicting miRNAs that target certain sets of oncogenes, one can identify novel anti-cancer miRNAs that can be added to lentiviral particles to reduce expression of the target oncogene and sensitize cells to targeted agents. Our study used the online tool miRDB to identify miR-4277, a potential repressor of CYP3A4 NKY 80 expression. We infected HCC cells with lentiviral particles containing pre-miR-4277 and confirmed the effect of miR-4277 on CYP3A4 and the elimination of sorafenib. Materials and Methods Cell Lines and Reagents The HCC cell lines, MHCC97-H, HepG2, BEL7402 or SMMC7721, were grown in our lab and described previously (18, 19). The clinical specimens of advanced HCC were also descripted in our previous work (18, 19). The use of human subjects was approved by the ethics committee of the Fifth Medical Center, General Hospital of Chinese PLA (Peoples Liberation Army). All assays were carried out in accordance with the Helsinki Declaration. Sorafenib, lenvatinib, cabozantinib, regorafenib, anlotinib, and apatinib were chemically synthesized by Dr. Shuang Cao at the Wuhan Institute of Technology, Wuhan City, Hubei Province of China. The potential cyp3a4s inhibitor, ketoconazole, amprenavir or diltiazem, NKY 80 was also gifts from Dr. Shuang Cao at the Wuhan Institute of Technology, Wuhan City, Hubei Province of China. All agents were initially prepared as powders purified to >99% by using the HPLC (high performance liquid chromatography) ( Supplemental Table?1 ). The miR-4277 was a microRNA potentially targeting to an online tool, miRDB, and the full-length sequences of has-pre-miR-4277, wild-type and with the mutation of the 1st miR-4277 targeting site], CYP3A4Mut2 [the vector of with the mutation of the 2nd miR-4277 targeting site], or CYPMut [the vector of with the mutation of the 1st and 2nd miR-4277 targeting site]). Quantitative PCR The endogenous mRNA levels of in HCC clinical specimens were identified using quantitative polymerase chain reaction (qPCR) in accordance with methods described by Wang et?al. (20) and Ma et?al. (20, 25). The primers used were: (1) experimentation, HCC cells were cultured and subcutaneously injected into mice to generate tumors. When tumor volume reached 2000 mm3, a solution of sorafenib was directly injected into the tumors. After injection, the tumors were excised at a series of time-points. Next, sorafenib was extracted from MHCC97-H cells or tumors using the acetonitrile (ACN). The sustaining amount of sorafenib at each time-point was measured using liquid chromatographyCmass spectrometry/mass spectrometry (LC-MS/MS) and the half-life of sorafenib was determined (18, 19, 34, 35). Assessment of Cell Survival After transfection or treated with potential inhibitor of mRNA Expression Are Associated With Poor Prognosis in HCC Patients Receiving Sorafenib Though it has been suggested that participates in the resistance of HCC cells to sorafenib, the clinical significance of requires further analysis. As shown in Figure?1 , the endogenous mRNA levels of NKY 80 were examined in 52 clinical specimens from patients with advanced HCC ( Figure?1 and Table?1 ). According to the median expression level of in HCC tissue samples, patients were divided into two.
