Effects of down-regulation of AEG-1 expression on cell cycle and invasion of human cervical carcinoma cells

AIM: To investigate the effects of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell cycle and invasion of human cervical carcinoma SiHa cells.METHODS: The protein expression of AEG-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, HeLa cells, SiHa cells and CaSki cells. Control siRNA or AEG-1 siRNA was transfected into SiHa cells, and the protein expression of AEG-1 in SiHa cells was detected by Western blotting. The changes of cell cycle distribution and cell invasion were determined by flow cytometry and Boyden chamber, respectively. The protein levels of cyclin D1, cyclin-dependent kinase 2(CDK2) and matrix metalloproteinase-9 (MMP-9) were analyzed by Western blotting.RESULTS: The protein expression of AEG-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues (P<0.05). Meanwhile, the protein expression of AEG-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which SiHa cells displayed the highest AEG-1 protein level (P<0.05). In addition, AEG-1 siRNA effectively down-regulated the protein expression of AEG-1 in SiHa cells, which led to increase the percentage at G0/G1 phase and reduced the invasion of SiHa cells. Furthermore, the protein levels of cyclin D1, CDK2 and MMP-9 in AEG-1 siRNA group were markedly lower than those in non-treatment group and control siRNA group (P<0.05).CONCLUSION: Over-expression of AEG-1 may be closely associated with the occurrence and development of cervical carcinoma, and the AEG-1 down-regulation-mediated cell cycle arrest and attenuation of invasion may be tightly related to the down-regulations of cyclin D1, CDK2 and MMP-9 at protein levels.     

Effect of ARMCX1 on proliferation and apoptosis of cervical cancer SiHa cells

AIM: To observe the effect of Armadillo repeat-containing X-linked protein 1 (ARMCX1) on the proliferation and apoptosis of human cervical cancer SiHa cells by knock-down of ARMCX1 expression with small interfering RNA. METHODS: After knock-down of ARMCX1 expression, the cell cycle distribution and apoptosis of SiHa cells were detected by flow cytometry. The proliferation of SiHa cells was observed by plate colony formation assay after knock-down of ARMCX1 for 10 d. The protein levels of cell proliferation-and apoptosis-related molecules were determined by Western blot. RESULTS: After knock-down of ARMCX1 expression in the SiHa cells, the cell colony formation ability was significantly inhibited (P < 0.05), the cell cycle was arrested in S phase, and the protein levels of cyclin E and cell division cycle 25A (Cdc25A) in the SiHa cells were decreased. Meanwhile, knock-down of ARMCX1 expression promoted the apoptosis of SiHa cells, significantly reduced the protein expression of Bcl-2, and significantly increased the protein levels of Bax and active caspase-3 (P < 0.05). CONCLUSION: Knock-down of ARMCX1 expression inhibits the proliferation of SiHa cells and induces apoptosis.     

siRNA inhibits HPV16-DNA replication,tumor volume and expression of P16 and P53 in SCID mice with SiHa cell cervical carcinoma in vivo

AIM: To investigate the effect of targeting gene therapy on mouse SiHa cell cervical carcinoma by transfecting siRNA into the tumor with solid-phase method in vivo. METHODS: A sense strand siRNA (21 nt) for human papilloma virus type 16 (HPV16) was designed. siRNA-Lipo2000-carbomer gum was prepared. Forty SCID mice with SiHa cell cervical carcinoma were divided into experimental group (n=32) and control group (n=8). The diameter and volume of the tumors were measured before treatment. The mice in experimental group were treated with siRNA-Lipo 2000-carbomer gum for 7 d. The control mice were treated with Lipo 2000-carbomer gum for 7 d. The mice in experimental group and control group were sacrificed at the time points of 4 d, 8 d, 12 d and 16 d after treatment. The diameter and volume of the tumors were measured again. The HPV16-DNA in the tumor tissues was measured by PCR. The protein levels of P16 and P53 in the tumors were determined by the method of immunohistochemistry. RESULTS: Compared with control group, the titer of HPV16-DNA decreased significantly at the time points of 8 d and 12 d after transfection in experimental group (P<0.05). The protein expression of P16 in experimental group showed decreased tendency 8 d and 12 d after transfection, but without statistical difference. The protein expression of P53 significantly decreased 8 d and 12 d after transfection. The tumor volume in experimental group was significantly decreased as compared to that in control group at the time point of 12 d (P<0.05). Transfection of siRNA for 12 d resulted in attenuating dyskaryosis and karyoplasmic ratio of the tumor cells.CONCLUSION: Solid-phase transfaction of siRNA in vivo inhibits HPV-DNA replication and growth of SiHa cell cervical carcinoma.     

Downregulation of DEK induces cell apoptosis via inhibition of NF-κB signaling pathway in gastric carcinoma SGC-7901 cells

AIM: To investigate the effect of DEK downregulation on the apoptosis of gastric carcinoma SGC-7901 cells, and to explore its associations with NF-κB signaling pathway and apoptosis related proteins. METHODS: SGC-7901 cells with different treatments were divided into 3 groups including untreated group, control siRNA group and DEK siRNA group. The expression of DEK at mRNA and protein levels in the SGC-7901 cells was detected by real-time PCR and Western blot. The cell apoptosis was examined by flow cytometry. Furthermore, the activities of caspase-3 and caspase-9 in the SGC-7901 cells were investigated by Caspase-Glo^®-3/9 kit. Finally, the expression of key regulatory protein p65 of NF-κB signaling pathway and apoptosis-related proteins Bcl-2 and Bax in the SGC-7901 cells was investigated by Western blot. RESULTS: Compared with untreated group and control siRNA group, the expression of DEK at mRNA and protein levels was significantly downregulated in DEK siRNA group (P<0.05). In addition, the ratios of early phase apoptosis and total apoptosis in DEK siRNA group were markedly higher than those in untreated group and control siRNA group (P<0.05). Most notably, the decrease in p65 and Bcl-2 proteins, increase in Bax protein and the increases of caspase-3 and caspase-9 activities were observed in DEK siRNA group. CONCLUSION: Downregulation of DEK mediates cell apoptosis of gastric carcinoma may be tightly associated with NF-κB signaling pathway.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

Clinical significance of STMN1 expression in cervical cancer and effect of inhibition of its expression on viability and apoptosis of cervical cancer SiHa cells

AIM: To investigate the clinical significance of stathmin 1 (STMN1) expression in cervical cancer and the influence of its expression on the viability and apoptosis of cervical cancer cells. METHODS: Western blot was used to detect the protein expression of STMN1 in cervical cancer tissues, and the relationship between the expression and clinical characteristics of cervical cancer was analyzed. STMN1-siRNA was transfected into cervical squamous-cell carcinoma SiHa cells. The protein levels of STMN1, STAT3, p-STAT3 and survivin were determined by Western blot after transfection for 48 h. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. DCFH-DA probe was used to detect the level of reactive oxygen species (ROS). RESULTS: The protein expression of STMN1 in cervical cancer tissues was significantly higher than that in paracancerous tissues (P<0.01). The STMN1 protein expression level was not correlated with age and histological types of cervical cancer patients, but was related to clinical stage, histological differentiation and lymph node metastasis (P<0.01). Transfection with STMN1-siRNA significantly reduced the expression of STMN1 in SiHa cells. Compared with control group, the cell viability in STMN1-siRNA group was significantly decreased, the apoptotic rate and ROS content were increased, and the protein levels of p-STAT3 and survivin were down-regulated (P<0.01). However, no significant difference of the STAT3 protein level was observed between STMN1-siRNA group and control group. CONCLUSION: STMN1 is highly expressed in cervical cancer, and its expression is related to clinical stage, histological differentiation and lymph node metastasis. Inhibition of STMN1 expression reduces the viability and promotes apoptosis of cancer cells by down-regulating STAT3 signaling pathway.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Mortalin expression in cervical squamous-cell carcinoma and its effect on tumor metastasis

AIM: To investigate the significance of mortalin expression in clinical pathology of cervical squamous-cell carcinoma. METHODS: Immunofluorescence staining was used to detect the location of mortalin in human cervical squamous-cell carcinoma SiHa cells. The protein expression of mortalin was detected in 59 cases of normal cervical epithelial tissues and 93 cases of cervical squamous-cell carcinoma tissues by immunohistochemical staining, and its correlation with clinicopathological features of cervical squamous-cell carcinoma was also analyzed. MTT assay was used to evaluate the optimal concentration and dosing time of mortalin inhibitor MKT-077. After the protein expression of mortalin in SiHa cells was inhibited, wound-healing and migration assays were performed. The protein expression of epithelial-mesenchymal transition (EMT)-related molecules was determined by Western blot. RESULTS: Immunofluorescence staining showed that mortalin was located in the cytoplasm of SiHa cells. The positive rate and strongly positive rate of mortalin in the cervical squamous-cell carcinoma patients were 88.7% (55/62) and 61.3% (38/62), respectively, and they were significantly higher than those in normal cervical epithelial tissues (23.7% and 5.1%, P < 0.01). Additionally, mortalin expression was statistically correlated with the histological grade, clinical stage and lymph node metastasis. After inhibiting the expression of mortalin in the SiHa cells by MKT-077, the results of wound-healing and migration assays showed that the migration ability of SiHa cells was down-regulated. The protein expression of E-cadherin was up-regulated, and vimentin and Snail were significantly down-regulated. CONCLUSION: Mortalin over-expression is an effective biomarker for prediction of malignant potential and poor prognosis of cervical squamous-cell carcinoma.     

Effect of HIPK2 gene on viability,apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation

AIM: To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reoxygenation (H/R). METHODS: HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipofectamine^TM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca²⁺ fluorescence intensity were analyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS: Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca²⁺ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION: Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epithelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.     

siRNA targeting TRIM29 gene induces apoptosis of nasopharyngeal carcinoma cells by inhibiting PI3K/AKT signaling pathway

AIM:To investigate the effect of TRIM29 gene expression silencing on the apoptosis and PI3K/AKT signaling pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were divided into blank group, negative control (NC) group (transfected negative control siRNA) and si-TRIM29 group (transfected TRIM29 specific siRNA). The viability of the 5-8F cells transfected with si-TRIM29 for 0~96 h was measured by CCK-8 assay. The apoptotic rate and the protein levels of TRIM29, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, t-AKT and p-AKT in the 5-8F cells transfected with si-TRIM29 for 48 h were determined by flow cytometry and Western blot, respectively. PI3K/AKT signal specific inhibitor LY294002 at 10 μmol/L and si-TRIM29 alone or in combination were treated with the 5-8F cells, and the cells were divided into blank group, LY294002 group and LY294002+si-TRIM29 group. The apoptotic rates in the 3 groups were detected by flow cytometry. RESULTS:The protein expression of TRIM29 in the 5-8F cells transfected with TRIM29 siRNA was significantly lower than that in blank group (P<0.05). Compared with blank group, the cell viability was significantly decreased, the apoptotic rate was significantly increased, the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax were significantly increased, and the protein levels of Bcl-2 and p-AKT were significantly decreased in si-TRIM29 group (P<0.05). The apoptotic rate in LY294002 group was higher than that in blank group, while that in LY294002+si-TRIM29 group was even higher than that in LY294002 group (P<0.05). CONCLUSION:Silencing of TRIM29 gene expression induces apoptosis of nasopharyngeal carcinoma 5-8F cells by inhibiting PI3K/AKT signaling pathway.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effects of shRNA-mediated hWAPL silencing on proliferation and apoptosis of human cervical cancer CaSki cells

AIM：To investigate the role of human wings apart-like (hWAPL) protein in proliferation and apoptosis of human cervical cancer CaSki cells through hWAPL gene silencing by specific short hairpin RNA (shRNA) duplexes. METHODS：The relative hWAPL mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell proliferation was detected by MTT assay, and the apoptosis was determined by Annexin V-PE and Hoechst 33258 staining. Western blotting was used to analyze the expression of cleaved caspase-3, p21 and p27. The effect of hWAPL gene silencing on the in vivo tumorigenic capacity of CaSki cells was investigated in a tumor-bearing nude mouse model. RESULTS：Real-time fluorescence quantitative PCR and Western blotting showed that hWAPL mRNA and protein expression in CaSki cells was efficiently inhibited by hWAPL shRNA. The shRNA-mediated hWAPL silencing inhibited the proliferation and induced the apoptosis of CaSki cells. Additionally, the expression levels of cleaved caspase-3, p21 and p27 were up-regulated in hWAPL knockdown cells. Knockdown of hWAPL also inhibited the in vivo tumorigenic capacity of CaSki cells. CONCLUSION：hWAPL is involved in the regulation of the proliferation and apoptosis of CaSki cells in vitro and in vivo, and might serve as a therapeutic target in cancer treatment.     

Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM: To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells. METHODS: The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues, and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot. Small interfering RNA (siRNA) targeting Rab1A was designed, synthetized and transfected into the breast carcinoma MDA-MB-231 cells. After validation of efficiency of Rab1A gene expression knock-down, the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay, wound healing assay, Transwell assay and flow cytometry. The protein levels were determined by Western blot. RESULTS: Rab1A was expressed in normal breast tissue and cells at low level, and at high level in the cancer tissues and cancer cells (P<0.05). Compare with control group, after knock-down of Rab1A expression, the viability of MDA-MB-231 cells was significantly inhibited (P<005), the abilities of migration and invasion were reduced (P<0.05), the apoptosis was decreased (P<0.05), the percentage of G²/M phase was increased, the protein levels of p53, Bax, cleaved caspase-3 and PTEN were significantly increased (P<0.05), and the protein levels of Bcl-2, cyclin D1, cyclin B1, matrix metalloproteinase 2 (MMP2), p-AKT and mTOR were significantly decreased (P<0.05). CONCLUSION: Rab1A modulates the breast carcinoma cell viability, inhibits the migration and invasion abilities, induces G² arrest and effectively regulates the cell growth-, cell cycle-and apoptosis-related proteins. Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway, and Rab1A may function as a potential target in breast carcinoma treatment.     

Transfection of siRNA into rabbit cervical cells in transformation zone by adopting solid phase in vivo inhibits HPV-DNA replication in SCID mouse with cervical carcinoma

AIM: To investigate the possibility of transfecting siRNA into rabbit cervical cells in transformation zone by the method of solid phase in vivo and to verify the effectivity of siRNA transfection by modifying the permeability of the cervical epithelium. METHODS: A sense strand small-interference RNA (siRNA) for human papillomavirus type 16 (21 bp) was designed and labeled with Cy3. siRNA-Lipo2000-carbomer gum was prepared. Twelve rabbits were included in the study and divided into experimental group and control group. In order to modify the permeability of cervical epithelium, hypertonic saline solution at concentration of 200 mmol/L was used to infuse the cervix in the experimental rabbits for 10 min, and normal saline was used for the control animals. The siRNA-Lipo2000-carbomer gum was applied to the surface of the rabbit cervix. Twenty-four hours later, the rabbits were sacrificed, and the cervix was isolated, cut into 2 parts, one part was for rapid frozen sectioning and the efficiency of transfection was observed under fluorescence microscope, another part was prepared by paraffin embedding and sectioning, and the form of cervical histiocytes was observed. Twelve SCID mice with SiHa cell cervical tumor, divided into experimental group and control group, were also used in the study. The mice in experimental group were treated with siRNA-Lipo2000-carbomer gum for 7 d. The control mice were treated with Lipo2000-carbomer gum only. Five days later, the mice were sacrificed and the tumor was collected, and the HPV16-DNA was measured by PCR. RESULTS: (1) Red fluorescence (Cy3) in cervical epithelium was observed in all rabbits. However, no different effect of siRNA transfection was found between the ways of modifying the cervical epithelium permeability. (2) No abnormal change such as flare, swelling and ulcer at all cervical tissue was observed, the cervical cell form was normal. (3) The titer of HPV16-DNA was decreased significantly after siRNA transfection (P<0.05). CONCLUSION: Transfection of siRNA into rabbit cervical epithelium in vivo is successful by using the method of solid phase and inhibits the processes of HPV-DNA, indicating that using RNAi is a practical way to treat HPV infection in human cercix and to decrease the incidence of cercical carcinoma.     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

SPK1 interferes with LLC cell apoptosis by regulating Bcl-2/Bax pathway

AIM: To investigate whether sphingosine kinase 1 (SPK1) interferes with apoptosis of Lewis lung cancer (LLC) cells by regulating the Bcl-2/Bax pathway. METHODS: The SPK1 gene siRNA eukaryotic expression vector was constructed, and transfected into the LLC cells. The transfected LLC cells was observed under a fluorescence microscope. The apoptotic rate of LLC cells after transfection was analyzed by flow cytometry. The expression levels of SPK1, Bcl-2 and Bax in LLC cells after transfection were detected by Western blot. The protein levels of Bax and Bcl-2 were measured by ELISA. RESULTS: Transfected LLC cells emitted green fluorescence under a fluorescence microscope. Apoptosis in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01). Western blot analysis showed that the expression of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group, and the expression of Bcl-2 was lower than that in siRNA-SPK1-Neg group. The ELISA results showed that the protein level of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01), and the protein level of Bcl-2 in siRNA-SPK1 group was significantly lower than that in siRNA-SPK1-Neg group (P<0.01). CONCLUSION: The expression of SPK1 in LLC cells is related to the apoptotic rate. SPK1 may interfere with the apoptosis of LLC cells via Bcl-2/Bax pathway.     

Molecular mechanism of miR-1246 enhancing radiosensitivity of cervical cancer cells

AIM: To investigate the molecular mechanism of microRNA-1246(miR-1246) enhancing radiosensitivity of cervical cancer cells. METHODS: Cervical cancer lines HeLa, CaSki, C33A and SiHa were transfected with miR-1246 mimic and negative control mimic (NC-mimic) using Lipofectamine 2000 kit, and the expression level of miR-1246 in cervical cancer tissue, normal tissue, cervical cancer cell lines and endometrial epithelium cell line ESC was detected by real-time PCR. The transfected cells were exposed to X-ray radiation. The cell viability and migration rate were measured respectively by MTT assay and Transwell method. The protein levels of γH2AX, ATM, p-ATM and p-p53 were monitored by immunofluorescence and Western blot. RESUITS: Higher miR-1246 level was found in normal tissue and ESC cells, while lower miR-1246 level was found in HeLa, SiHa, C33A and Caski cells and cervical cancer tissues. The expression level of miR-1246 in the cells transfected with miR-1246 mimic was significantly higher than that in the cells transfected with NC-mimic (P<0.05). The cell viability and migration rate of the cervical cancer cells with miR-1246 over-expression were notably lower than those of the cells transfected with NC-mimic (P<0.05) under the same conditions. The results of immunofluorescence indicated that the protein expression level of γH2AX significantly increased in the cervical cancer cells with miR-1246 over-expression exposed to radiation compared with the negative control (P<0.05). The protein expression level of γH2AX was significantly increased in the cervical cancer cells with miR-1246 over-expression, while the protein levels of p-ATM and p-p53 were significantly decreased as compared with the negative control group (P<0.05). CONCLUSION: miR-1246 is highly expressed in normal tissue and normal endometrial epithelial cells, while is low expressed in the cervical cancer tissues or cells. miR-1246 over-expression inhibits growth and migration, and significantly enhances radiosensitivity of cervical cancer cells. The molecular mechanism is possibly related to inhibiting ATM pathway and DNA damage repair.