Effect of RhoC silencing on migration and invasion of oral squamous cell carcinoma / 中国病理生理杂志

AIM:To explore the expression of RhoC in oral squamous cell carcinoma(OSCC)and its effects on the malignant biological behavior of OSCC cells.METHODS:The UALCAN and K-M plotter databases,alongside tis-sue sample analyses,facilitated understanding RhoC expression in cancer and its links to clinicopathological traits.Two small interfering RNAs(RhoC-siRNA)were constructed according to the RhoC gene sequence.The mRNA and protein ex-pression levels of RhoC in OSCC cells were determined.The protein levels of FAK,p-FAK,MAPK,p-MAPK,matrix me-talloproteinase-2(MMP-2)and MMP-9 were also examined by Western blot.Furthermore,the invasion and migration of OSCC cells were analyzed by Transwell assay and scratch test.Finally,the pulmonary metastasis model of nude mice was established.RESULTS:The results of the databases showed that RhoC was highly expressed in OSCC tissues,which was closely related to pathological stage,pathological grade and lymph node metastasis,but not significantly related to the sur-vival rate of patients.Furthermore,compared with paracancer tissues,the mRNA and protein expression levels of RhoC were increased in OSCC tissues(P<0.01).Silencing of RhoC prominently reduced the migration and invasion of OSCC cells as well as the protein levels of p-FAK,p-MAPK,MMP2 and MMP9(P<0.05).The protein levels of MAPK and FAK were unchanged(P>0.05).The fluorescence intensity of the experimental group was significantly lower than that of the control group,and the results of HE staining showed that the number of lung nodules in the experimental group was sig-nificantly reduced(P<0.05).CONCLUSION:RhoC can effectively influence the migration and invasion of OSCC cells,and its potential mechanism may be related to FAK/MAPK/MMPs signaling pathway.

[Effects of geranylgeranyltransferaseâ silencing on the proliferation of tongue squamous cancer cells].

Objective This study aims to investigate the effect of geranylgeranyltransferaseⅠ (GGTase-Ⅰ) on the proliferation and growth of tongue squamous cancer cells. Methods Three small interfering RNAs (siRNAs) were designed on the basis of the GGTase-Ⅰ sequence in GeneBank. These siRNAs were then transfected into tongue squamous cancer cells Cal-27. The mRNA and protein expression of GGTase-Ⅰ and RhoA were examined by real-time quantitative polymerase chain reaction and Western blotting, respectively. The expression of Cyclin D1 and p21 were examined by Western blotting. The proliferation and growth ability were analyzed by cell counting kit-8 assay and flow cytometry. Results The mRNA and protein expression of GGTase-Ⅰ in Cal-27 was reduced significantly after the GGTase-Ⅰ siRNAs were transfected (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05). Cyclin D1 expression decreased, whereas p21 expression increased significantly. The cell cycle was altered, and the growth-proliferative activity was inhibited (P<0.05). Conclusion GGTase-Ⅰ siRNA can inhibit the expression of GGTase-Ⅰ and the proliferative activity of tongue squamous cancer cells. GGTase-Ⅰ may be a potential target for gene therapy in tongue squamous cell cancer.

[Effects of geranylgeranyltransferase â gene silencing by RNA interference on the migration and invasion of tongue carcinoma].

OBJECTIVE: RNA interference was used to silence geranylgeranyltransferase Ⅰ(GGTase-Ⅰ) in vitro and to study the effect of GGTase-Ⅰ on the migration and invasion of tongue squamous cancer cells. METHODS: Three small interfering RNAs (siRNA) were designed according to the GGTase-Ⅰ sequence by Genebank and were transfected into tongue squamous cancer cells Cal-27 to knock down GGTase-Ⅰ expression. The tested cells were divided into three groups, as follows: the RNA-interfered groups (GGTase-Ⅰ siRNA1, GGTase-Ⅰ siRNA 2, GGTase-Ⅰ siRNA 3), a negative control group (disrupted by random sequence NC-siRNA), and a blank control group. GGTase-Ⅰ and RhoA gene expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The optimum interference group was screened by qRT-PCR and Western blot and was assigned as the experimental group. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were examined by Western blot. GTP-RhoA expression of protein was examined by GST-pull down. The migration and invasion abilities were analyzed by wound healing assay and Transwell motility assay. RESULTS: GGTase-Ⅰ mRNA and protein expression in Cal-27 decreased significantly after transfection of GGTase-I siRNA (P<0.05). No significant difference of RhoA gene expression was detected. MMP-2, MMP-9, and GTP-RhoA protein expressions decreased significantly (P<0.05). The migration and invasion abilities were inhibited (P<0.05). CONCLUSIONS: To inhibit GGTase-Ⅰ expression, the migration and invasion abilities of tongue squamous cancer cells should also be inhibited. Further studies on GGTase-Ⅰ may provide novel effective molecular targets for tongue squamous cancer cells.

Effects of geranylgeranyltransferase Ⅰ gene silencing by RNA interference on the migration and invasion of tongue carcinoma / 华西口腔医学杂志

<p><b>OBJECTIVE</b>RNA interference was used to silence geranylgeranyltransferase Ⅰ(GGTase-Ⅰ) in vitro and to study the effect of GGTase-Ⅰ on the migration and invasion of tongue squamous cancer cells.</p><p><b>METHODS</b>Three small interfering RNAs (siRNA) were designed according to the GGTase-Ⅰ sequence by Genebank and were transfected into tongue squamous cancer cells Cal-27 to knock down GGTase-Ⅰ expression. The tested cells were divided into three groups, as follows: the RNA-interfered groups (GGTase-Ⅰ siRNA1, GGTase-Ⅰ siRNA 2, GGTase-Ⅰ siRNA 3), a negative control group (disrupted by random sequence NC-siRNA), and a blank control group. GGTase-Ⅰ and RhoA gene expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The optimum interference group was screened by qRT-PCR and Western blot and was assigned as the experimental group. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were examined by Western blot. GTP-RhoA expression of protein was examined by GST-pull down. The migration and invasion abilities were analyzed by wound healing assay and Transwell motility assay.</p><p><b>RESULTS</b>GGTase-Ⅰ mRNA and protein expression in Cal-27 decreased significantly after transfection of GGTase-I siRNA (P<0.05). No significant difference of RhoA gene expression was detected. MMP-2, MMP-9, and GTP-RhoA protein expressions decreased significantly (P<0.05). The migration and invasion abilities were inhibited (P<0.05).</p><p><b>CONCLUSIONS</b>To inhibit GGTase-Ⅰ expression, the migration and invasion abilities of tongue squamous cancer cells should also be inhibited. Further studies on GGTase-Ⅰ may provide novel effective molecular targets for tongue squamous cancer cells.</p>

Protective effect of mitochondria-targeted antioxidant SS31 on early brain injury following subarachnoid hemorrhage in rats / 中南大学学报(医学版)

Objective:To evaluate protective effects of SS31 on early brain injury (EBI) induced by subarachnoid hemorrhage (SAH) in rats.Methods:A total of 96 Sprague-Dawley rats were randomly divided into 4 groups:A sham group,an SAH group,an SAH+vehicle group (SAH+V),and an SAH+SS31 group.The SAHinduced prechiasmatic cistern rat model was established in this study.Neurological deficit scores were evaluated at 24 h after SAH.The SS31 (5 mg/kg) as well as equal volume of vehicle were administrated intraperitoneally at 2 h after SAH.The neurological scores,brain edema,blood-brain barrier (BBB) permeability,apoptosis,malondialdehyde (MDA),glutathione peroxidase (GPx) activity,superoride dismutase (SOD) activity,and the expression ofcytosolic cytochrome c (Cyt C) and Bax were analyzed at 24 h after SAH.Results:Treatment with SS31 could significantly reduce MDA levels,and restored the activities of GPx and SOD in the cortex following SAH when compared with the SAH+V group.In addition,Bax SS31 trearment increased or decreased the levels of mitochondrial Cyt C or Bax,respectively.Moreover,SS31 treatment ameliorated brain edema and Evans blue dye extravasation,improved neurological deficits,and decreased neuronal apoptosis at 24 h after SAH.Conclusion:SS31 could alleviate EBI after SAH through its antioxidant property and ability in inhibition of neuronal apoptosis.

[Effects of RhoA silencing on proliferation of tongue squamous cancer cells].

OBJECTIVE: This study investigated the effect of RhoA silencing through RNA interference on proliferation and growth of tongue cancer cells, as well as explored the possible mechanisms of this effect. METHODS: SSC-4 tongue cancer cells were cultured in vitro and then transfected with small interfering RNA to knock down RhoA expression. The tested cells were divided into three groups: experimental group (experimental group 1: transfected with RhoA-siRNA-1; experi-mental group 2: transfected with RhoA-siRNA-2), negative control group (transfected by random sequence NC-siRNA), and blank control group (transfected with Lipofectamine). The expression levels of RhoA mRNA were respectively measured by quantitative real-time polymerase chain reaction and western blot assay. Moreover, the expression levels of cyclin D1, p21, and p27 and RhoA protein were evaluated by Western blot assay. Proliferation and growth potentiality were analyzed through evaluation of doubling times and methyl thiazolyl tetrazolium assessment. RESULTS: The expression levels of RhoA gene and protein of experimental groups significantly decreased following siRNA transfection compared with those in the negative and blank control groups. The expression of cyclin D1 decreased significantly and that of p21 and p27 increased significantly. The doubling time was extended and the growth potentiality decreased. CONCLUSIONS: The results indicated that RhoA silencing can inhibit proliferation of tongue cancer cells, whereas RhoA affects cell proliferation by regulating the cell cycle pathway. Thus, RhoA is a potential target in gene therapy for tongue cancer.

Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene / 中南大学学报(医学版)

OBJECTIVE@#To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
@*METHODS@#Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. 
@*RESULTS@#DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. 
@*CONCLUSION@#An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.

Effects of RhoA gene silencing by RNA interference on invasion of tongue carcinoma / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of RhoA down-regulation by RNA interference on the invasion of tongue carcinoma Tca8113 and SCC-4.</p><p><b>METHODS</b>Determination of the human RhoA sequence as well as the design and constructionof a short specific small interfering RNAs (siRNA) were performed. The siRNA of RhoA gene was transfected into humantongue squamous cell carcinoma Tca8113 and SCC-4 cells line by Lipofectamine 2000. Quantitative real-time polymerasechain reaction was used to examine the mRNA expressionlevels of RhoA. Protein expressions of mRNA, galectin-3,and matrix metalloproteinase (MMP)-9 were evaluated byWestern blot. Transwell invasion assay was performed toassess the invasion ability of tongue carcinoma.</p><p><b>RESULTS</b>RhoA expressions in Tca8113 and SCC-4 cells were reducedsignificantly after transfection of RhoA-siRNA. Protein levels f galectin-3 and MVP-9 were also down-regulated significantly. Invasion ability was inhibited as well.</p><p><b>CONCLUSION</b>RhoA-siRNA can effectively inhibit RhoA expression in Tca8113 and SCC-4 cells. The invasion ability of tongue carcinoma cells decreased with down-regulation of the protein expressions of galectin-3 and MMP-9, indicating that RhoA-siRNA can inhibit invasion of tongue carcinoma. Results show that RhoA may play an important role in the processes of invasion and metastasis of tongue carcinoma.</p>

Expression of Cbfα1/p56 subtype in bone marrow mesenchymal stem cells from rat mandible / 中国组织工程研究

BACKGROUND:Unlike the ilium derived from the paraxial mesoderm, the mandible from cranial neural crest has a unique mechanism. Core binding factorα1 (Cbfα1) is a key transcription factor for skeletogenic process. However, the role of Cbfα1/p56 subtype in mandible tissue is yet not clear. OBJECTIVE:To research the expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from rat mandible in vitro. METHODS:Bone marrow mesenchymal stem cells from rat mandible and ilium were in vitro isolated and purified by primary culture. The characteristics of bone marrow mesenchymal cells were compared through the methods of enzyme linked immunosorbent assay and real-time PCR, including growth curve, alkaline phosphatase activity and relative mRNA expression of Cbfα1 subtypes. RESULTS AND CONCLUSION:Bone marrow mesenchymal cells from rat mandible and ilium were successful y obtained. Bone marrow mesenchymal cells from the mandible proliferated more rapidly, alkaline phosphatase activity of which was higher than iliac cells. The relative mRNA expression of Cbfα1/p56 subtype in bone marrow mesenchymal cells from the mandible was more than that in iliac cells at 6 days of culture (P0.05). The results showed that Cbfα1/p56 is very significant in the early osteogenic differentiation of bone marrow mesenchymal cells from the mandible.