[Cloning and identification of microRNA from human osteosarcoma cell line SOSP-9607].
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.


CONCLUSIONS
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.
Gao
Jie
J
Department of Orthopedic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, 710038, P. R. China.
Yang
Tong-Tao
TT
Qiu
Xiu-Chun
XC
Yu
Bing
B
Han
Jian-Wei
JW
Fan
Qing-Yu
QY
Ma
Bao-An
BA
D001483:Base Sequence; D015152:Blotting, Northern; D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics
D035683:MicroRNAs; D012334:RNA, Neoplasm