Background & objective: MicroRNA (miRNA), a group of non-coding small RNA (20-25 nt) involved in post-transcriptional regulation, regulate gene expression and closely relate to cancer pathogenesis. This study was to clone miRNA from osteosarcoma cell line SOSP-9607, and identify the expression of some functional genes.
Methods: Low molecular weight RNA fraction (< or =200 nt) was extracted from SOSP-9607 cells, and polyadenylated by poly(A) polymerase. Then a 5' RNA adapter was ligated to poly(A)-tailed RNA using T4 RNA ligase. RNAs were reversely transcribed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product of 109 bp was recovered and cloned into pCR 4-TOPO vector. After sequencing, database searching, and expression profiling, the expression of miRNA in SOSP-9607 cells was sieved. The expression of novel and some known miRNAs was examined by Northern blot with small RNAs (< or =200 nt) isolated from SOSP-9607 cells, osteosarcoma tissue, and HeLa cells.
Results: A total of 182 clones were subsequently characterized through DNA sequencing and database searching; 47 clones (correspond to 25 species) out of the 182 clones from SOSP-9607 cells were identified as miRNAs. Two novel miRNAs (miR-165 and miR-166) were discovered among other 23 known miRNAs, which were predicted in Nature. Northern blot confirmed that the 2 novel miRNAs and 3 solid cancer miRNAs (miR-21, miR-20a,miR-17-5p) were stably expressed in SOSP-9607 cells and osteosarcoma tissue, but miR-166 was not expressed in HeLa cells.
Conclusion: We have cloned miRNAs for SOSP-9607 cells and identified parts of the functional ones, which imply that miRNAs may closely relate to the cancer pathogenesis.