DNA-PKcs短发夹RNA载体的构建及其表达

目的构建DNA-PKcsshRNA表达载体,观察该基因的抑制对A2780细胞增殖活性的影响。方法将针对人DNA-PKcs基因的不同部位设计的shRNA插入到真核表达质粒并转染人卵巢A2780细胞,RT-PCR和WesterBlot检测其对该基因mRNA和蛋白水平的抑制效率,MTT法测定DNA-PKcs基因的抑制对肿瘤细胞增殖活性的影响。结果 DNA测序分析证实DNA-PKcs shRNA表达载体pSIRENDNA-PKcs shRNA构建成功并在细胞中表达,转染重组载体的A2780细胞DNA-PKcs的表达在mRNA和蛋白水平均明显下降;重组载体转染细胞增殖活性明显降低。结论成功构建DNA-PKcs shRNA表达载体为进一步研究DNA损伤修复基因DNA-PKcs奠定了基础;DNA-PKcs表达的抑制可能会导致肿瘤细胞增殖活性降低。     

RNA干扰技术抑制smad4基因表达对宫颈癌细胞增殖的影响

目的：探讨用RNA干扰技术下调smad4基因表达对宫颈癌细胞增殖的影响。方法：用DNA重组技术将针对人smad4基因不同部位所设计的三对短发夹状RNA（shorthairpinRNA，shRNA）序列克隆到真核表达质粒pGenesil-1中，构建smad4shRNA表达载体pGenesil—smad4一shRNA1、2、3。脂质体介导转染人宫颈癌细胞株HeLa，经G418筛选抑制smad4表达的稳定细胞克隆。MTT法、克隆形成实验、流式细胞术检测抑制smad4基因对宫颈癌细胞增殖的影响。结果：成功构建分别携带三段shRNA及空载体对照的重组质粒pGenesil—smad4-shRNA1、2、3和pGenesil-con，三种shRNA重组质粒中pGenesil—smad4-shRNA2可明显降低细胞内smad4mRNA及smad4蛋白表达，筛选出的HeLa／shRNA2细胞增殖能力明显增强。结论：应用RNA干扰技术能筛选出特异而高效阻断smad4基因表达及功能的shRNA，smad4基因表达下调能明显促进宫颈癌细胞生长。     

肺癌A549细胞中RNAi抑制MeCP2表达的实验研究

目的：探讨RNA干扰下调MeCP2基因后对人肺癌A549细胞RASSF1A和C/EBPα基因表达及细胞增殖能力的影响。方法：根据MeCP2的碱基序列设计并合成短发夹RNA（shRNA）,构建质粒表达载体,用阳离子脂质体法体外转染A549细胞,筛选出稳定转染的细胞株,RT-PCR法检测MeCP2抑制效果及RASSF1A和C/EBPα表达,MTT法检测转染后细胞增殖能力。结果：成功构建了靶向MeCP2的真核表达载体pSilencer-MeCP2/Ⅰ和pSilencer-MeCP2/Ⅱ,与空白组和阴性组相比,pSilencer-MeCP2/Ⅰ和pSilencer-MeCP2/Ⅱ组均可有效抑制MeCP2基因mRNA的表达,抑制率分别为47.6%和70.3%。且在上述两组中C/EBPα表达分别上调约115%和135%、细胞增殖能力明显减弱,与空白组和阴性组比较差异具有统计学意义（P〈0.05）,而RASSF1A的表达在各组细胞中未见明显变化（P〉0.05）。结论：肺癌A549细胞中MeCP2基因的表达可被RNA干扰载体有效抑制,进而导致细胞中部分与增殖和凋亡相关基因的表达改变,细胞增殖能力减弱。     

Bcl-2短发夹状RNA对NB4细胞株生长的抑制作用

目的：构建Bcl-2短发夹状RNA（short hairpin RNA，shRNA）序列的表达载体并研究其对早幼粒白血病细胞株NB4生长的抑制作用。方法：针对Bcl-2基因构建编码shRNA序列的重组表达载体．经酶切电泳和DNA测序鉴定。采用脂质体介导的转染方法将重组的RNAi质粒转入NB4细胞后．通过荧光显微镜和流式细胞仪观察转染效率，采用Western Blot检测Bcl-2蛋白表达水平；采用MTT法测定细胞增殖情况。结果：编码Bcl-2 shRNA的RNA序列被正确地插入到预期位点。将两个Bcl-2 shRNA载体分别转入NB4细胞后，Bcl-2蛋白表达水平均降低．与转染阴性shRNA及未转染组比较，均有显著性差异（P〈0．05）。转染Bcl-2 shRNA1、shRNA2载体入NB4细胞在72、96h细胞生长明显受到抑制，分别与转染阴性shRNA、单纯载体组及未转染组比较，差异有显著性（P〈0．05）。结论：构建的两个Bcl-2 shRNA均可特异性地抑制NB4细胞生长。     

RNA干扰hTERT基因治疗喉鳞状细胞癌的实验研究

目的:探讨RNA干扰hTERT(人端粒酶逆转录酶)基因对人喉鳞状细胞癌的治疗作用.方法:根据hTERT cDNA序列构建表达hTERT mRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA1、pshRNA2.将shRNA质粒分别转染人喉癌Hep-2细胞株及荷瘤裸鼠瘤体内.以激光共聚焦显微镜观察质粒在Hep-2细胞及瘤体内的表达;以MTT法观察质粒对Hep-2细胞增殖的抑制作用;以蛋白印迹法(Western blot法)检测Hep-2细胞中hTERT蛋白的表达;以免疫组化SP法检测裸鼠瘤体内hTERT蛋白的表达.结果:pshRNA1、pshRNA2转染Hep-2细胞及pshRNA1转染瘤体后,共聚焦显微镜下见大量的癌细胞表达绿色荧光,hTERT蛋白表达明显下降.细胞受pshRNA1、pshRNA2转染后,其生长活性受到明显抑制.体内抑瘤实验表明,与空质粒载体组(pshRNA4)和生理盐水组相比,pshRNA1组移植瘤生长明显受到抑制.结论:表达shRNA的、hTERT基因特异的真核表达质粒能有效转染体内、外喉鳞癌细胞,并可有效抑制人喉鳞癌细胞的生长,为今后应用RNA干扰基因治疗喉癌提供重要的实验参考.     

重组人血管内皮生长因子165对K562白血病细胞生物学活性的影响

目的：探讨人重组血管内皮生长的因子165对人K562白血病细胞生物学活性的影响。方法：利用pcDNA3.1（＋）质粒构建人重组含VEGF165的真核表达载体,脂质体转染至K562白血病细胞,并用G418筛选阳性克隆;以RT-PCR测定重组质粒载体VEGF165基因的表达;MTT法测定K562白血病细胞的生长;建立裸鼠皮下移植人K562肿瘤模型,测定肿瘤体积的大小;免疫组化检测肿瘤的微血管密度（MVD）。结果：成功构建pcDNA3.1（＋）-VEGF165表达质粒。K562白血病细胞转染重组质粒后,明显增加K562细胞中VEGF165 mRNA的表达,转染重组质粒的K562细胞增殖明显快于对照组;移植重组pcDNA3.1（＋）-VEGF165质粒转染的K562细胞之后,荷瘤鼠移植瘤生长明显快于对照组;并且移植瘤组织微血管密度明显高于对照组（P〈0.05）。结论：利用pc DNA3.1（＋）真核表达质粒可成功构建含人VEGF165的重组质粒,重组质粒转染K562细胞后,体内外均明显促进K562细胞增殖。     

APE1shRNA重组质粒的构建及对人乳腺癌细胞生长的影响

目的设计构建靶向脱嘌呤脱嘧啶核酸内切酶1（APE1/ref-1）特异性短发卡RNA的真核表达载体,并转染人乳腺癌T47D细胞。方法设计、合成针对APE1的特异性的短链寡核苷酸,构建APE1特异性shRNA的重组质粒,稳定转染人乳腺癌T47D细胞;采用聚合酶链反应和免疫印迹法检测转染前后T47D细胞中APE1的表达。采用四甲基偶氮唑盐法观察T47D细胞的生长情况。结果经酶切和测序鉴定,成功构建APE1特异性shRNA的重组质粒,并能够转染T47D细胞。转染后,APE1在mRNA和蛋白水平表达分别下降约53.1%和60.5%,T47D细胞增殖活性受到抑制。结论运用pGenesil-1.1质粒载体构建的靶向APE1特异性shRNA的重组质粒可以成功转染T47D细胞,有效抑制APE1的表达,并抑制肿瘤细胞的生长。     

CUL5基因的RNA干扰真核表达载体的构建

目的：构建CUL5基因的RNA干扰真核表达载体,探索CUL5在鼻咽癌放射治疗中加速再增殖的作用。方法：根据GenBank数据库提供的CUL5基因mRNA序列,按照Tuschl设计原则,设计选择双链小干扰RNA（small interfering RNA,siRNA）,再设计合成为能表达其小发卡结构RNA（small hairpin RNAs,shRNA）的DNA序列,并与pGPU6／GFP／Neo质粒定向连接,构建真核表达载体,并进行限制性内切酶酶切和DNA测序鉴定。结果：经限制性内切酶酶切和DNA测序证实质粒中插入的为所需序列,通过RT—PCR证实其能够干扰CNE2细胞的CUL5基因表达。结论：CUL5基因RNA干扰真核细胞表达载体的构建成功。     

RNA干扰沉默α1，3岩藻糖转移酶-Ⅶ基因对人肝癌细胞增殖的影响

 目的：研究RNA干扰沉默α1,3 岩藻糖转移酶-Ⅶ （α1,3FucT-Ⅶ） 基因对人肝癌细胞增殖的影响。 方法：设计合成α1,3FucT-Ⅶ特异性RNA干扰质粒,并用脂质体转染导入人肝癌H7721细胞。RT-PCR检测细胞中α1,3FucT-Ⅶ mRNA的表达。流式细胞术测定细胞表面唾液酸化的Lewis X抗原（SLex）的表达及细胞周期。细胞生长曲线通过MTT测定。p27Kip1的表达检测采用Western blot。 结果：成功构建了α1,3FucT-Ⅶ特异的shRNA真核表达质粒载体。RNA干扰下调α1,3FucT-Ⅶ表达能抑制H7721细胞生长,表现为细胞生长速度明显较对照组细胞慢,S期细胞百分数明显降低, p27Kip1的表达增加。 结论：α1,3FucT-Ⅶ可能通过调节p27Kip1的降解影响H7721细胞增殖。     

shRNA沉默RAGE基因表达对前列腺癌细胞增殖的抑制作用

目的:构建针对人晚期糖基化终产物受体(receptor for advanced glycation end products , RAGE)基因的特异性短发夹RNA(small hairpin RNA, shRNA)表达载体,探讨其对前列腺癌细胞DU145增殖的抑制作用.方法:设计并合成4种针对RAGE基因的特异性短链寡核苷酸,构建含shRNA RAGE的表达载体,转染高表达RAGE的亚克隆细胞株sub DU145-2C1.荧光显微镜下观察细胞转染后的情况,实时荧光定量PCR(real-time fluorescence quantitative -PCR, RFQ-PCR)检测转染shRNA后对RAGE mRNA表达的影响,Western印迹法检测对RAGE蛋白表达的影响,CCK-8法检测对细胞增殖的影响,划痕实验观察对细胞迁移能力的影响.结果:构建获得的shRNA RAGE表达载体能够有效抑制RAGE基因的表达(P<0.05),其中以shRNA RAGE -1(R1)的抑制作用最强,其对RAGE mRNA表达的抑制率为84%,对蛋白表达的抑制率为27%.细胞增殖结果显示,转染shRNA RAGE后细胞增殖能力明显降低;而细胞迁移能力则无明显变化.结论:shRNA RAGE能有效下调RAGE基因的表达水平,抑制细胞增殖.     

PCNA的shRNA的真核表达载体的构建及对子宫颈癌细胞的干预性研究

目的构建PCNA的小发夹结构RNA(shRNA)的真核表达载体并在Hela细胞表达,同时观察PCNA的shRNA对人Hela细胞株体外增殖的影响及生物学特性的改变。方法将PCNA的cD-NA的shRNA引物插入真核表达载体pGenesil-1,构建真核表达质粒pGenesil-1-PCNA1-4,并通过酶切和测序等方法进行鉴定,将真核表达质粒pGenesil-1-PCNA1-4转染子宫颈癌细胞,运用Westernblot检测PCNA的蛋白表达,流式细胞仪分析细胞周期变化。结果PCNA的shRNA能显著抑制人Hela细胞的生长,阻滞细胞G0/G1—S期,PCNA的蛋白表达同时受到抑制。结论PCNA的shRNA能显著抑制人Hela细胞的生长,其抑制作用可能通过阻断PCNA蛋白表达实现,实验结果为进一步研究PC-NA的shRNA对子宫颈癌的基因治疗奠定了基础。     

食管癌中uPA、uPAR蛋白表达及其临床意义

 目的探讨食管癌中uPA、uPAR蛋白表达与临床病理因素间的关系及其对单纯放疗预后的影响。方法应用免疫组化SP法检测59例食管癌和41例食管癌旁组织中uPA、uPAR蛋白表达情况,分析蛋白表达与临床病理因素间的关系及其对食管癌放疗预后的影响。结果59例食管癌组织中uPA、uPAR蛋白阳性表达率分别为76.27%、77.97%,41例食管癌旁组织中阳性表达率分别为48.78%、43.90%,癌组织和癌旁组织中两种蛋白的阳性表达率比较差异均有统计学意义(P<0.01)。胸部CT扫描显示食管病变层面椎前三角形间隙消失者uPA蛋白强阳性表达率为70.37%,明显高于三角形间隙存在者的43.75%,两者比较差异有统计学意义(P=0.040)。发生远处转移者uPA蛋白阳性表达率比无远处转移者也显著升高,分别为100%和68.89%,两者比较差异有统计学意义(P=0.042)。进一步分析发现uPA、uPAR蛋白表达与食管癌预后未见明显相关性,而uPA、uPAR两种蛋白的表达呈显著性正相关。结论食管癌中uPA蛋白高表达可能与肿瘤局部浸润及远处转移的发生有关。     

COX-2 Gene increases tongue cancer cell proliferation and invasion through VEGF-C pathway

COX-2 induces the proliferation and invasion of oral squamous cell carcinoma. In the present study, the role of the COX-2 gene in the tongue cancer cell proliferation and invasion was investigated. A short hairpin RNA (shRNA)method was used to knock down COX-2 gene expression and investigate the relationship between COX-2 and VEGF-C, and the role of the COX-2 gene for proliferation and invasion was also investigated in the tongue cancer cell Tca8113. COX-2 gene overexpressed in tongue cancer cell line. Suppressing the expression of COX-2 by shRNA could decrease cell proliferation comparing with control shRNA. Nevertheless, depressing COX-2 gene expression by shRNA reduced VEGF-C expression on both mRNA and protein levels. VEGF-C gene expression could be regulated by COX-2 gene. Our results suggested that COX-2 played essential roles in the proliferation and metastasis of tongue cancer, and COX-2 could serve as a potential chemotherapy target for tongue cancer.     

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.     

CtBP1基因shRNA真核表达载体的构建及其干扰效果的初步鉴定

目的：构建C-末端结合蛋白-1（CtBP1）基因RNA干扰（RNAi）的真核表达载体,转染人子宫内膜癌耐药细胞株B-MD-C1（ADR＋/＋）,初步鉴定其干扰效果。方法：以人CtBP1基因为靶基因,以pGenesil-1质粒为载体,根据GenBank数据库提供的CtBP1基因核苷酸序列,选择设计两条siRNA干扰序列,将带9个碱基茎环结构的干扰序列插入带有绿色荧光蛋白（EGFP）基因、卡那霉素（Kanr）抗性基因及U6启动子的真核表达载体pGenesil-1中,构建针对CtBP1基因的shRNA真核表达载体,转化大肠杆菌DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。确认载体构建成功后,用脂质体LipofectamineTM2000将重组质粒瞬时转染人子宫内膜癌耐药细胞株B-MD-C1（ADR＋/＋）,在荧光显微镜下观察绿色荧光蛋白表达,计数转染细胞数,计算转染效率,RT-PCR法检测载体对CtBP1基因的表达抑制效果。结果：构建针对CtBP1基因的pGenesil-1-CtBP1小发夹RNA（pGenesil-1-CtBP1-shRNA）,经限制性内切酶酶切、PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到B-MD-C1（ADR＋/＋）细胞表达绿色荧光蛋白（EGFP）,证实重组质粒己转染入细胞,转染效率达到60%-70%;RT-PCR结果显示B-MD-C1（ADR＋/＋）细胞中CtBP1基因被特异抑制,基因表达抑制率达40%以上,与转染试剂对照组、空白对照组和阴性序列对照组间的差异均具有统计学意义（P均〈0.05）。结论：成功构建CtBP1基因的shRNA表达载体,可以有效抑制B-MD-C1（ADR＋/＋）细胞上CtBP1基因表达。为进一步研究CtBP1基因功能进而逆转肿瘤的多药耐药奠定基础。     

An inducible short-hairpin RNA vector against osteopontin reduces metastatic potential of human esophageal squamous cell carcinoma in vitro and in vivo.

PURPOSE: To elucidate the clinical significance of osteopontin and the effect of conditional down-regulation of osteopontin in esophageal squamous cell carcinoma (ESCC), we investigated osteopontin expression in tumors and tested an inducible osteopontin-short-hairpin RNA (shRNA) expression vector in an ESCC cell line. EXPERIMENTAL DESIGN: Osteopontin mRNA expression was extracted from gene expression profiles of 23 tumors determined by cDNA microarray and was analyzed. Paraffin sections of 144 tumors were immunohistochemically investigated. Osteopontin protein expression in 34 cell lines was examined by Western blot. A doxycycline-inducible osteopontin-shRNA vector was stably transfected into HSA/c cells to assess the role of osteopontin in cell motility, invasion in vitro, tumor formation, and lymph node metastasis in nude mice. RESULTS: cDNA microarray revealed that high osteopontin mRNA expression was associated with poor survival of ESCC patients (P = 0.029). In immunohistochemistry, osteopontin protein expression was associated with poor prognosis (P < 0.001), distant lymph node metastasis (P = 0.0004), tumor staging (P = 0.027), and histologic grade (P = 0.024). Multivariate analysis showed that osteopontin overexpression was the strongest independent prognostic factor among nine clinicopathologic variables (P < 0.001). Among cell lines tested, 30 had overexpressed osteopontin protein compared with a normal esophageal epithelial cell line. An inducible shRNA vector against osteopontin successfully down-regulated osteopontin expression by 71% to 88% and repressed cell motility by 69% to 97%, cell invasion by 59% to 71%, tumor formation by 56% to 92%, and lymph node metastasis by 50% to 67% in HSA/c cells. CONCLUSIONS: Our findings suggest that osteopontin overexpression may play an important role in progression of ESCC and osteopontin could be a potential target of ESCC therapy.     

TFPI-2基因克隆及其在胰腺癌细胞中的表达

目的克隆人TFPI-2基因全长cDNA,构建其真核表达载体,并将其转染到人胰腺癌细胞Panc-1中,检测其表达。方法用RT-PCR法从人胎盘组织中扩增人TFPI-2基因,并将其与真核表达载体pEGFP-C1连接,构建真核表达载体pEGFP-C1-TFPI-2。将构建的重组载体转染到人胰腺癌细胞系Panc-1细胞,Westernblot检测TFPI-2在Pane-1细胞中的表达。结果RT-PCR成功的扩增出一条约708bp的片断,扩增片断与载体连接后,经限制性内切酶酶切电泳分析和DNA序列测定证实该基因已经成功构建到载体上,转染Panc-1细胞后,荧光显微镜观察到稳定转染细胞发出较强绿色荧光,Westernblot技术证明TFPI-2基因能在Panc-1细胞中稳定表达。结论成功构建了人TFPI-2基因的真核表达载体pEGFP-C1-TFPI-2,获得了稳定表达TFPI-2的Panc-1细胞,证实TFPI-2基因能够在人胰腺癌细胞系Panc-1中高效、稳定的表达,为进一步研究其胰腺癌迁移、浸润及转移中的作用打下基础。     

Knockdown of HBx by RNAi inhibits proliferation and enhances chemotherapy-induced apoptosis in hepatocellular carcinoma cells

The present study was designed to research on RNA interference hepatitis B virus x gene approach to hepatocellular carcinoma (HCC) therapy. Previously, we constructed and identified shRNA eukaryotic expression vectors (pshRNA-X220) specific to HBx gene, pshRNA-MOCK (control); and established HCC cell lines with stable expression shRNA eukaryotic vector targeting HBx gene—21543 cell lines (MHCC97-H of expressing shRNA against HBx), HK3 cell lines (MHCC97-H by transfected with pshRNA-MOCK). We examined the expression of HBx gene after RNA interference by semi-quantitative RT-PCR and assessed the effect of HBx knocked down on cell growth by proliferation assay using kit-8 (CCK8). As well as, we analyzed cell cycle distribution by flowcytometry and examined cell apoptosis using TUNEL assay. The HBx mRNA expression level is reduced, and cells growth was significantly stopped in 21543 cell lines. Cells with HBx knockdown were more sensitive to 5-fluorouracil/cisplatin. RNA interfering HBx induced an obvious time and dose-dependent inhibitory in comparison with the control cells. Meanwhile, RNA interferenced targeting HBx, in combination with chemotherapy can effectively induce apoptosis in hepatocellular carcinoma cells and restricts cell proliferation.     

CD147 shRNA真核表达质粒的构建及鉴定

目的针对CD147基因的不同部位,构建不同CD147 shRNA表达质粒载体,在转录后水平抑制CD147的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆.方法用DNA重组技术将针对人CD147基因的不同部位所设计的3对shRNA序列克隆到真核表达质粒pGE-1中,构建CD147 shRNA表达质粒载体pGE-1 CD147shRNA1、2、3.用阳离子脂质体介导转染人卵巢癌高转移细胞系HO-8910pm,经G418筛选后获得克隆进行鉴定.结果3个CD147 shRNA表达载体pGE-1CD147shRNA1、2、3经PCR、限制性酶切及部分序列分析证明基因插入正确.半定量RT-PCR、Western blotting均证实:转染细胞8910 pm/pGE-1 CD147shRNA,与亲本细胞相比,CD147的表达在mRNA和蛋白水平均明显下降.结论成功构建了CD147 shRNA表达载体pGE-1 CD147shRNA,并筛选出特异而高效地阻断CD147表达的克隆.此实验结果为进一步研究CD147蛋白分子的生物学功能及应用奠定了基础,并对shRNA设计靶序列的选择有一定指导意义.     

Induction of S100A4 gene expression inhibits in vitro invasiveness of human squamous cell carcinoma, KOSC-3 cells

S100A4 is considered functionally involved in metastasis and invasiveness of rodent and human mammary tumors. We screened the expression of S100A4 in human squamous cell carcinoma cell lines, and found 2 cell lines which were highly invasive, but did not express any noticeable extent of S100A4. To examine whether the expression of S100A4 regulated invasiveness of squamous cell carcinoma, we transfected S100A4 cDNA into KOCS-3 and HSC-4 squamous cell carcinoma cells. The transfectants from KOSC-3 cells expressing sense S100A4 decreased invasiveness by 80% compared with cells of the wild type or those with the vector only.