Mechanism Underlying Drug Resistance Of HepG2/ADM Cells Silently Reversed By Lentivirus-mediated BCO47440Gene During Chemotherapy

Purpose and significance:Primary liver cancer， which consists predominantly of hepatocellular carcinoma（HCC),is the most common gastrointestinal tumor in the world, and its incidence rate andmortality are high in cancer. Primary liver cancer is a very great health hazard to our country’spopulation. At present, the efficacy of combined therapy which mainly consists surgicalresection is not good, the5-year survival is only about5%. Poor efficacy of adjuvantchemotherapy is one of the main treatment difficulty, the reason is mainly due to the primaryliver cancer multidrug resistance （multidrug resistance, MDR). Therefore chemotherapyplays an important role in the treatment of primary liver cancer. With the continuousimprovement of adjuvant chemotherapy in recent years, the efficacy of chemotherapy hadimproved. But there is still a large proportion of patients who are not sensitive tochemotherapy because the tumor cells of intrinsic and （or) acquired multidrug resistant.Many studies have shown that the carcinogenesis of liver cancer is a multi-step process whichinvolves multiple genes. Abnormal activation or inactivation genes such as oncogene、tumorsuppressor genes、cell cycle regulating genes、apoptosis genes are the molecular basis of livercancer. And its clinical biological behaviors including resistance to chemotherapy are theresults of the antagonistic/promote balance disorders. Genomics operations to interfere withthe balance of the disorder at the molecular level may be the key to solve hepatocellularcarcinoma multidrug resistance.A functional gene discovered in recent years called BC047440was highly expressed inliver tissue and positively correlated with the degree of malignancy of liver cancer in theprevious study in our laboratory. In our experiment, we silenced BC047440gene inHepG2/ADM cell lines with lentivirus vector by the RNAi technology, and had constructedBC047440shRNA interference model successfully.We observe the biological characteristicsafter the silencing of BC047440gene in HepG2/ADM cells line, then we detected the genes those are related to BC047440by Western Blotting and Real time PCR methods in proteinand mRNA levels, to explore the mechanism that BC047440gene reverse HepG2/ADMchemotherapy resistance. And based on the important role of the gene on the development oftumor cell,we can investigate the way that reverse liver cancer multidrug resistance bypromoting tumor cell apoptosis or through the block in the tumor cell cycles, or inhibitingtumor doubling time after the silencing of BC047440gene in HepG2/ADM cell line.Methods:1．We selected a appropriate titer of lentiviral particles to infect HepG2cells, then viewedthe expression of green fluorescent protein by the ordinary light microscope48、72hours later.We chosed the Multiplicity of Infection（MOI) whose expression of green fluorescenceintensity is higher and the condition of the cells are better as the optimal MOI value.2． We separated HepG2/ADM cells into BC047440-shRNA group,control-shRNAgroupand HepG2group, secondly, added the corresponding recombinant lentivirus and controllentivirus, and extracted the total protein of each group96hours later as well as detectedBC047440protein by Western Blotting.3．We cultured each group cells with doxorubicin whose final concentration is2.5ug/ml.Then we measured the absorbance values [D （460)] in the wavelength of460nm24h、48h、72h later by cell counting kit-8（CCK-8) assays, and calculated the growth inhibition ratio.4．We detected cell cycles by flow cytometry assay, and analyzed the changes in thedistribution of cell cycle. We cultured each group cells with0.04ug/ml doxorubicin for48h,and then we detected apoptosis ratio by flow cytometry assays.5．After silencing BC047440by by lentiviral vector, the changes of NF-κB protein levelwere detected by Western Blotting analysis, the expressions of Surviving mRNA and CCNL1mRNA were detected by Real time PCR.Results：1． The BC047440RNA interference model in HepG2/ADM cell line had beenconstructed successfully. The BC047440protein of BC047440-shRNA group was about71.5%of HepG2/ADM group，about70.0%of control-shRNA group，respectively.2．The cell growth inhibiting ratio of adriamycin in BC047440-shRNA group was significantly enhanced.3． The data of flow cytometry assay showed that the apoptotic cells increasedsignificantly in BC047440-shRNA group，and cell cycle was distributed mainly in the Sphase，and the G0/G1phase was reduced.4．NF-κB protein expression levels of BC047440-shRNA group was about67.69%ofHepG2/ADM group and67.39%of control-shRNA group，respectively.5．Detection of Survivin mRNA expression found in BC047440-shRNA group was about37%of control-shRNA group and42%of HepG2/ADM group, detection of CCNL1mRNAexpression in BC047440-shRNA group was about3.5times than control-shRNA group and2.4times than HepG2/ADM group，respectively.Conclusion:1． The BC047440RNA interference model in HepG2/ADM cell line had beenconstructed successfully.2． Silencing BC047440gene can reverse multidrug resistance of adriamycin inHepG2/ADM cell line.3．The results reveal that silencing BC047440gene can reverse multidrug resistance ofadriamycin in HepG2/ADM cell line by NF kappa B signaling pathway and related genesSurvivin and CCNL1，it suggest that BC047440gene may be one of the important moleculesthat reverse multidrug resistance of hepatocellular carcinoma.