Chinese Journal of Biochemistry and Molecular Biology RNA.

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (8) : RNA 1), 2), 1) 3, 1) 1),2), ( 1), ; 2), ) RNA, (CTGF) RNA. EcoR Hind, ptm12 CTGF PCR CTGF, psos2hus psos2hus2ctgf. psos2hus2ctgf Sfi 5 CTGF 1 psos2 CTGFi1,pSOS2CTGFi2,pSOS2CTGFi3,pSOS2CTGFi4,pSOS2CTGFi5,pSOS2CTGFicontrol. HEK293, d, IPP. 5 CTGF RNA 0124 % % 2193 % % %. site 1 site 3. psos2hus,. CTGF RNA. RNA ; ; ; Q52212 A Fast Simplified Fluorescence2Based Method to Screen Specific RNAi Sequence of Connective Tissue Gro wth Factor LIANG Rui 1), KANG Quan 2), LUO Qing 1) 3, ZHAO Li2Hua 1) 1),2), J IN Xian2Qing ( 1) Research Institute of Children s Hospital of Chongqing Medical University, Chongqing , China ; 2) General Surgery of Children s Hospital of Chongqing Medical University, Chongqing , China) Abstract To set up a fast simplified fluorescence2based method to screen gene 2specific RNAi sequences of connective tissue growth factor (CTGF), a pair of primers based on CTGF gene with EcoR or Hind restriction site were designed and synthesized. PCR fragment was amplified with ptm12ctgf template and cloned into the plasmid psos2hus. Five specific interference sequences and a random controlled sequence were inserted into the psos2hus2ctgf, respectively. The recombinant plasmid ( psos2ctgfi ) was transfected into the cultured HEK293 cells. After 1, 3, 5 and 7 days,the expression of egfp was detected by the fluorescence microscopy at the same conditions. The intensity of egfp was measured by the IPP image software. The relative intensity of egfp in five sequence2specific RNAi were 0124 %, %, 2193 %, % and %, respectively at 5th day. Site 1 and site 3 sequence2specific silencing were distinct. This method was fast simplified and reliable. Effective CTGF2specific RNAi sequences were successfully screened. Key words RNA interference ; connective tissue growth factor (CTGF) ; eukaryotic expression vector ; egfp : ; : (No ) 3 Tel : ; E2mail : com ; E2mail com Received : February 23, 2009 ; Accepted : April 27,2009 Supported by National Natural Science Foundation of China (No ) 3 Corresponding author Tel : ; E2mail : com ; E2mail : com

2 ( connective tissue growth factor, CTGF) CCN,..,CTGF.,CTGF wnt. wnt., CTGF RNA CTGF. RNA ( RNA interference,rnai) RNA (double strand RNA,dsRNA), mrna,, [1 ]. (posttranscription gene silencing,ptgs). RNAi, [2 ]., RNA,. RNA, CTGF RNA Hind, EcoR, sfi NEB ; PCR,,DNA Table 1 The specific RNAi sequences to silence CTGF, PCR, T4 DNA, DNA2Hind marker TAKARA ; DNA CTGF ( GI :14219) TAKARA ; DMEM Gibco ;,, Hyclone ;Lipofectamine INVITROGEN ;ptm12ctgf psos2hus TC HE ; DH5 HEK CTGF Ambion (http :ΠΠ www. ambion. cornπ), Reynolds sirna [3 ]. http :ΠΠwww. ncbi. nlm. nih. gov CTGF cdna ( GI :14219), sirna. sirna, sirnas. sirnas 5 Blast, sirna. CTGF 5 sirna, 1. 5 CTGF sirna DNA Table CTGF dsrna psos2 CTGFi CTGF : ptm12ctgf, 5 2gaattcGTGAGCCTGGTGCTGGAC23 EcoR, 5 2aagcttACTTCCTGGCTTTACG23 Gene No. Sequences Target site NM Site 1 NM Site 2 NM Site 3 NM Site 4 NM Site 5 Sense strand 5 2aCCGATGGCGAGATCATGAAtttt 23 Antisense strand 5 2aTTCATGATCTCGCCATCGGtttt 23 Sense strand 5 2aAAGTGCATCCGGACACCTAtttt 23 Antisense strand 5 2aTAGGTGTCCGGATGCACTTtttt23 Sense strand 5 2aTCGCCAAGCCTGTCAAGTTtttt 23 Antisense strand 5 2aAACTTGACAGGCTTGGCGAtttt 23 Sense strand 5 2aTGCCTGCCATTACAACTGTtttt 23 Antisense strand 5 2aACAGTTGTAATGGCAGGCAtttt 23 Sense strand 5 2aAGAACTGTGTACGGAGCGTtttt 23 Antisense strand 5 2aACGCTCCGTACACAGTTCTtttt Hind, 935 bp. PCR : 94,4 min, 94,1 min 55, 1 min 72,5 min ;30, 72,5 min.,. psos2hus2ctgf : CTGF psos2hus, EcoR Hind,,T4 DNA. DH5, DH5,,

3 8 : RNA 761,. psos2hus2 CTGF,. psos2ctgfi : CTGF DNA, 1 l,, 48 l (100 mmolπl + 2 mmolπl + 30 mmolπl HEPES2KOH ph 714) 95 2 min,75 10 min, 4, DNA. psos2hus2ctgf Sfi,, psos2hus2ctgf, DNA 1 3,T4 DNA., psos2ctgfi1 5 psos2ctgfi1 control. HEK293 psos2ctgfi :HEK % DMEM 37,5 %CO h, psos2ctgfi HEK293 ( 018 g psos2ctgfi 2 l ), d, IPP,,. = Π 100 % psos2ctgfi 6 ( ),,,, Fig. 1 (762 ). 2.2 HEK293 ( e GFP) HEK293 6 psos2ctgfi 12 h Table 2 The intensity level of GFP in different vectors ( n = 3) psos2ctgfi1 psos2ctgfi2 psos2ctgfi3 psos2ctgfi4 psos2ctgfi5, egfp, 1 d egfp, 7 d. egfp 1 d, 7 d. 5 d egfp, Fig. 2 (762 ) Fig. 3. Fig. 3 RNAi effect in each group transduced into HEK293 cells with plasmids psos2ctgfi The intensity of egfp in each group was measured and analyzed by the IPP image software. The intensity of egfp in the control group was set as the standard while the relative egfp intensity in other groups were calculated. The egfp intensity in different groups was 0124 %, %, 2193 %, % and %, respectively,at 5 th day ( n = 3). Site 1 and site 3 sequence2 specific silencing were distinct CTGF egfp, egfp, Table 2.,5 CTGF. 5 d, 0124 % % 2193 % % %. site 1 site 3. Day 1 Day 3 Day 5 Day % % % 1168 % 0124 % 0141 % % 2112 % % % % % % 3154 % % % % % % 6170 % 2193 % 0129 % % 5119 % % 6198 % % 8132 % % 6115 % % % % 3168 % % % % 5174 % % 6112 % psos2tgficontrol 100 % 100 % 100 % 100 %

4 Fig. 1 Results of site 1 and site 3 specific sequences inserted into the plasmid psos2hus2ctgf The U6 promoter was chose as the sequencing prime. (A) Site1 : The insertion was shown in the site of bp ; (B) Site3 : The insertion was shown in the site of bp Fig. 2 Expression of e GFP in each group at 5th day after the plasmids psos2ctgfi transfected into HEK293 cells ( 100) 018 g plasmids of psos2ctgfi in each group were mixed gently with 2 l lipo2fectamine according to the instructions. The mixture was transduced into the HEK293 cells which were passaged hours ago with the confluence of around 80 %. The expression of egfp was observed under the aid of fluorescence microscopy at the fixed timepoints. Here presents the expression of egfp at 5 d after transduction. (A) psos2ctgfi1 ; (B) psos2ctgfi2 ; ( C) psos2ctgfi3 ; (D) psos2ctgfi4 ; ( E) psos2ctgfi5 ; ( F) psos2ctgficontrol. The expression of egfp was dramatically deceased in (A) and (C), which suggests psos2ctgfi1 and psos2ctgfi3 were of high efficacy in CTGF silencing Fig. 4 The schematic representation of plasmid psos2hus Expression of sirna duplex was driven by the convergent opposing pol promoters, H1 and U6. Expression of GFP was driven by human EF1 promoter. A polylinker site immediately following the GFP coding region ( with a stop codon) was designed to clone the gene of interest [4 ]

5 8 : RNA psos2hus, Fig. 4. H1 U6., H1 U6 2 RNA, dsrna. Sfi,,sfiI TTTT,, RNA,. H1 U6 dsrna, shrna mrna [4 ]. egfp CTGF, hef1 egfp CTGF mrna. egfp,, mrna egfp egfp CTGF. mrna egfp, RNA mrna CTGF, RNAi, mrna, egfp [4 ]., egfp RNA.,,, egfp. CTGF CCN, 1991, CTGF(hCTGF). hctgf 6q23. 1, 5 4, 349, 38 kd,, ( IGF) (TSP) 1 vonwil1ebrand C C 4.,CTGF ;,CTGF.,CTGF. CTGF,.,CTGF,, [527 ], CCN [8 ]. CTGF,, CTGF CTGF [9212 ]. RNAi [13 ]. egfp, CTGF RNA, CTGF. ( References) [ 1 ] Kim D, Rossi J. RNAi mechanisms and applications [ J ]. Biotechniques, 2008,44(5) : [ 2 ] Whitehead KA, Langer R, Anderson D G. Knocking down barriers : advances in sirna delivery [J ]. Nat Rev Drug Discov,2009,8(2) : [ 3 ],,,. sirna pro 2. 0 : sirna [J ]. ( Fang X, Du Z P, Cao Y C, et al. sirna pro 210 :An online tool for rational design of sirna[j ]. Chin J Biochem Mol Biol),2007,23(9) : [ 4 ] Luo Q, Kang Q, Song W X, et al. Selection and validation of optimal sirna target sites for RNAi2mediated gene silencing [J ]. Gene, 2007, 395(122) : [ 5 ] Bennewith KL, Huang X, Ham C M, et al. The role of tumor cell2 derived connective tissue growth factor ( CTGFΠCCN2) in pancreatic tumor growth[j ]. Cancer Res, 2009,69(3) : [ 6 ] Pandey D P, Lappano R, Albanito L, et al. Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF[J ]. EMBO J, 2009,28 (5) : [ 7 ] Eguchi T, Kubota S, Kawata K, et al. Different transcriptional strategies for ccn2πctgf gene induction between human chondrocytic and breast cancer cell lines[j ]. Biochimie, 2007, 89 (3) : [ 8 ],. CCN1 143B [J ]. (Si W K, He T C. Over expressing exogenous CCN1 and its effect on growth and migration of human osteosarcoma cell lines 143B [J ]. Chin J Biochem Mol Biol),2006,22(7) : [ 9 ] Pan L H, Beppu T, Kurose A, et al. Neoplastic cells and proliferating endothelial cells express connective tissue growth factor (CTGF) in glioblastoma[j ]. Neurol Res, 2002,24(7) : [10 ] Mullis T C, Tang X, Chong K T. Expression of connective tissue growth factor ( CTGFΠCCN2 ) in head and neck squamous cell carcinoma[j ]. J Clin Pathol, 2008,61 (5) : [11 ] Shimo T, Kubota S, Goda T, et al. Clinical significance and pathogenic function of connective tissue growth factor (CTGFΠCCN2) in osteolytic mandibular squamous cell carcinoma [ J ]. Anticancer Res, 2008, 28 (4C) : [12 ] Lin B R, Chang C C, Che T F, et al. Connective tissue growth factor inhibits metastasis and acts as an independent prognostic marker in colorectal cancer[j ]. Gastroenterology, 2005, 128(1) :9223 [13 ] Kawasaki H, Taira K. Short hairpin type of dsrnas that are controlled by trna ( Val ) promoter significantly induce RNAi2 mediated gene silencing in the cytoplasm of human cells[j ]. Nucleic Acids Res, 2003, 31 (2) :