Identification Of Differential Expression Of MiR-130a And MiR-212in The HepG2and HepG2/Adr

Backgroud and Objective:Liver cancer is one of the most common malignant tumors in the world.Its incidence showed an increasing trend with high death rate. As animportant treatment for patients with advanced liver cancer, the efficacyof Systemic chemotherapy is not satisfactory, mainly due to liver cancermultidrug resistance. Hepatocellular carcinoma complicated mechanismsof multidrug resistance, the current study suggests that the principalmechanisms include membrane transporter mediated drug efflux,apoptosis mediated apoptosis genes, various enzyme-mediated resistanceand so on. The liver cancer multidrug resistance mechanisms mediatedmembrane transport proteins is the most important mechanism. P-gpprotein is one of the ABC transporter proteins with high expression in inhepatocellular carcinoma,and the high expression in resistant cells ismore obvious.The expression of ABCG2protein is related to drugs.Indrug environment its increased expression can speed up the drug efflux ofhepatoma cells.So P-gp and ABCG2protein in liver cancer membranetransport protein mediated drugs efflux pump mechanisms play animportant role. miRNA is a single-stranded small RNA molecules consisting of21-25nucleotides, widely found in eukaryotes, are a class ofshort RNA sequences of evolutionarily highly conserved non-codingproteins involved in post-transcriptional level regulation and regulationin a variety of biological processes such as cell proliferation,differentiation, apoptosis and so on, associated with many humandiseases. Currently a large number of studies have shown that miRNA asa tumor suppressor gene or oncogene and tumor occurrence anddevelopment have a close relationship. Has been reported hsa-mir-130aand hsa-mir-212through a variety of different mechanisms involved inmulti-drug resistant tumors, but rare reports of liver cancer-related, andmany of the mechanism is not clear. Our study was to analysis miRNAexpression profiles in HepG2and HepG2/Adr(doxorubicin-resistantHepG2cells) by miRNA array,searching for the special miRNAsassociated with liver caner drugresistant membrane protein andverificating the related expression of special candidate’s miRNAs byqPCR technology to understand which miRNA may participate in theregulation of liver cancer multidrug resistance protein and its codinggene.The study will reveal the mechanism of drug resistance ofhepatocellular carcinoma,help us find a new way to improve the curativeeffect of combined treatment of hepatocellular carcinoma.Metheds:1. MTT assay was used to determine the sensitivity of HepG2/Adr andHepG2cells to ADM. 2. Using the Exiqon miRNA expression profil chip contains3100maturehuman, mouse, rat, and all three species-related virus miRNAs and25human-related miRPlus miRNAs,the expression of miRNAs in HepG2cells and HepG2/Adr was detected. Hierarchical clustering analysis wasapplied for determing HepG2/Adr differentially expressed miRNAsspectrum. According to the relevant literature, We selected miR-130a andmiR-212as candidate miRNAs.3. Verifications of altered RNAs expression including miR-130a,miR-212, MDR1and ABCG2were performed in HepG2and HepG2/Adrby real-time qRT-PCR. Data analysis was performed using the2-Ctmethod.4. Data are presented as the means±standard deviation. One-wayANOVA and t-test were employed to analyze difference of meansbetween groups using SPSS17.0software(SPSS Inc.,Chicago,IL,USA).Statistical significance was set as P<0.05.Results:1. The resistance index to ADM in HepG2/Adr cells was2.968-foldhigher than that in HepG2cells, so HepG2/Adr cells had a strongerresisitance to ADM.2. The miRNA profiling microarray results shown that a set ofHepG2/Adr-specific miRNAs expression spectrum which contain236differentially expressed miRNAs, of which118miRNAS upregulated and118miRNAs downregulated. Inclusion criteria were raised and lowered more than2times. The miR-130a and miR-212were significantlydifferent in HepG2and HepG2/Adr cells.3. Using qPCR method, We found that MDR1was detecteddownregulated in HepG2cells and the relative expression to HepG2/Adris0.094(P<0.05); ABCG2was detected downregulated in HepG2cellsand the relative expression to HepG2/Adr is0.47(P<0.05); miR-130a wasdetected upregulated in HepG2cells and the relative expression toHepG2/Adr is4.057(P<0.05); miR-212was detected upregulated inHepG2cells and the relative expression to HepG2/Adr is5.434(P<0.05).The results were statistically significant.Conclusion:1. The HepG2/Adr have a specific miRNA expression profile.Thesespecific miRNAs may be involved in the regulation of liver cancermultidrug resistance.2. MDR1mRNA and ABCG2mRNA were upregulation in HepG2/Adr.MDR1and ABCG2may be involved in the mechanism of membraneprotein-mediated liver cancer multidrug resistance.3. miR-130a and miR-212were downregulation in HepG2/Adr. Whetherthe two miRNAs involved in the regulation of liver cancer membraneproteins still needs to confirm by funcional tests.