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  • Title: [Effect of antisense oligodeoxynucleotide of small subunit component of human ribonucleotide reductase on human choriocarcinoma cell line in vitro].
    Author: Cui JQ, Shi YF, Zhou HJ.
    Journal: Zhonghua Fu Chan Ke Za Zhi; 2004 Jul; 39(7):465-8. PubMed ID: 15347470.
    Abstract:
    OBJECTIVE: To study effect of the antisense oligodeoxynucleotide (ASODN) of small subunit of human ribonucleotide reductase (RRM2) mRNA on cell line of human choriocarcinoma in vitro. METHODS: Two 20-mer gapmer ASODNs with a full phosphorothioate backbone were artificially synthesized, which were complementary to nucleotides 626-645 (a coding region) and 1572-1591 (a 3'untranslated region) of RRM2, respectively. ASODNs were transfected into JAR cells through oligofectamine. The survival rate was assessed by methyl thiazolyl tetrazolium (MMT) assay, and RRM2 expression was detected by immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) methods. RESULTS: Antisense oligodeoxynucleotide one (ASODN1) targeting the coding region significantly inhibited growth of JAR cells in a dose- and time-dependent manner and downregulated RRM2 expression in a time-dependent manner. ASODN1 at 100 nmol/L could inhibit significantly cell growth (P = 0.000), and the effects of ASODN1 on JAR cell proliferation were enhanced with increase of ASODN1 concentration and reached the peak point at 400 nmol/L concentration (P = 0.000). Cell growth was significantly inhibited by 200 nmol/L of ASODN1 after 24 h of treatment (P = 0.000). The effect of ASODN1 was at the maximum at 48 h (P = 0.000), and began to decrease at 72 h of treatment. RRM2 expression started to reduce after ASOND1 treatment for 12 hours, and was obviously downregulated at 24 h of treatment, and decreased to the lowest level at 48 h (P < 0.05). RRM2 expression began to recover at 72 h of treatment. Antisense oligodeoxynucleotide two (ASODN2) corresponding to 3'untranslated region and control oligodeoxynucleotides had no significant effect on the proliferation and RRM2 expression, but the combination of ASODN2 and ASODN1 was more effective on reducing RRM2 expression and inhibiting cell proliferation than ASODN1 alone. CONCLUSIONS: ASODN1 of RRM2 can inhibit the proliferation of JAR cells and downregulate RRM2 transcription and translation in a selective and specific manner. The combination of multiple ASODNs targeting different regions of RRM2 mRNA would be an important approach to improve antisense effectiveness.
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