[Construction of deltaNp63 specific small hairpin RNA expressing plasmid and its role in bladder cancer--a preliminary study].
OBJECTIVE
To construct deltaNp63 specific small hairpin RNA (shRNA) expressing plasmid,to examine its inhibitory effect to the expression of deltaNp63 protein and mRNA in transitional cell carcinoma of the bladder (TCCB) , its effect on TCCB cells cycle and proliferation.


METHODS
DeltaNp63 specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form double strand DNA fragments and this fragment was cloned into Pgenesil-1 plasmid. The recombinant deltaNp63-shRNA expression construct was confirmed by using Pst I + Sal I double digestion and by sequencing. Fluorescence staining was used to confirm the success of transfection in TCCB cells under the fluorescence microscope. The inhibitory effect of deltaNp63-shRNA construct was examined with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry (FCM). The cellular proliferation of TCCB cells was assayed by tetrazolium bromide (MTT) colorimetry.


RESULTS
The deltaNp63-shRNA expression plasmid was successfully constructed and transfected into TCCB cells. It can effectively reduce the expression of deltaNp63 protein and mRNA. The reduction rate of deltaNp63 mRNA was 63.0%, and the G0/G1 ratio was increased and S phase was decreased in transfected TCCB cells. The cellular proliferation was also lower in transfected 5637 cells in comparrison with that of non-transfected TCCB cells.


CONCLUSIONS
A deltaNp63-shRNA expression plasmid, constructed from Pgenesil-1 plasmid, can successfully be transfected into TCCB cells and can effectively inhibit the expression of deltaNp63 protein and mRNA. It also can take part in regulation of the cell cycling and inhibit the cellular proliferation of TCCB cells.
He
Yun-feng
YF
Department of Urological Surgery, The First Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400016, China.
Wu
Xiao-hou
XH
Luo
Chun-li
CL
Tian
Dai-yin
DY
Zhang
Liang-suo
LS
Gao
Fei
F
D002295:Carcinoma, Transitional Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins