Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

Effects of GOLPH3 gene silencing mediated by lentivirus on proliferation,invasion and metastasis of human gastric cancer cell line SGC-7901

AIM：To investigate the effect of Golgi phosphoprotein 3 (GOLPH3) gene silencing on the proliferation, invasion and metastasis of human gastric cancer SGC-7901 cell in vitro. METHODS：SGC-7901 cells were transfected with lentiviral vector carrying GOLPH3 shRNA to construct a stable GOLPH3-silencing cell line LV-GOLPH3-RNAi. The expression of GOLPH3 at mRNA and protein levelss were detected by real-time PCR and Western blotting, respectively. The cell proliferation was analyzed by MTT assay. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities, respectively. RESULTS：The stable GOLPH3-silencing cell line was successfully established. The expression of GOLPH3 at mRNA and protein levels was reduced significantly (P<0.05), leading to the inhibition of cell proliferation in LV-GOLPH3-RNAi group compared with scrambled group and blank control group, as well as the capacities of migration (56.7±1.5 vs 186.0±3.4 and 183.3±4.2, P<0.05) and invasion (33.5±3.0 vs 85.0±3.9 and 83.1±4.4, P<0.05). CONCLUSION：GOLPH3 silencing suppresses the capacities of proliferation, migration and invasion of human gastric cancer SGC-7901 cells, suggesting that GOLPH3 may be a potential tumor marker and independent prognostic factor.     

Effects of heterogously-expressed RON receptor tyrosine kinase on motile/invasive ability of human colorectal cancer cell line RKO

AIM: To investigate the roles of overexpression of RON receptor tyrosine kinase in motile/invasive ability of human colorectal cancer cell line RKO. METHODS: A eucaryotic expression vector pDR2 containing full-length wt-RON cDNA was transfected into the colorectal cancer cell line RKO and a stable expression clone was obtained. The motile/invasive ability was tested by wound healing test and the transwell migration assay. The expression of E-cadherin was measured by Western blotting. RESULTS: Motile ability of transfected RKO was greatly promoted by transwell chemotaxis assay (P<0.01). The wound healing time showed statistical difference as of (42.50±4.12) h, (69.50±2.52) h and (70.50±3.42) h, respectively in transfected group, untransfected group and vector control group. After knocking down RON by siRNA, the motile became less than that in control group (P<0.01). E-cadherin expression in transfected RKO was decreased significantly due to pDR2-wt-RON transfection. CONCLUSIONS: Overexpression of wt-RON led to the decrease in expression of E-cadherin and decreased cancer cell-cell adhension. At the same time, migration/invasion ability was promoted. Taken together, abnormal accumulation of RON might play potential roles in invasion/metastasis of colorectal cancer. RNAi can block motile/invasion ability mediated by RON.     

Construction of lentiviral vector-mediated siRNA knockdown of ER-α36and its action on gastric cancer cell growth

AIM: To construct a lentiviral vector for stable delivery of the ER-α36gene and to detect its effect on SGC7901 cell growth. METHODS: The efficient RNAi targeting sequences identified for the ER-α36gene were screened. The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA. Then it was digested by XhoI and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector. PCR was used to screen the positive clones and sequence. The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line. Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect. 17β-estrodial at concentration of 1×10-10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined. RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors. Immunofluorescence assay demonstrated that transfection efficiency was above 80%. Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mRNA and protein levels with tetracycline (TeT) simulating as revealed by real-time PCR and Western blotting. Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silenceER-α36expression are constructed successfully and can be used to study the role of ER-α36 in gastric cancer. The ER-α36is related with many kinds of cancer cell growth, including gastric cancer cells.     

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.     

Effects of notch1 gene on proliferation and cell cycle of human glioma U251 cells

AIM: To investigate the effect of notch1 gene on the change of proliferation and cell cycle in human glioma U251 cell line. METHODS: The lentiviral vectors, which express notch1 shRNA or notch1 intracellular domain (NICD), were constructed and transfected into U251 cells, respectively. RT-PCR and Western blotting were applied to monitor the validity of down-regulation of notch1 expression and over-expression of NICD. MTT assay was performed to examine the cell proliferation. Flow cytometric analysis was used to detect the cell cycle. RESULTS: The lentiviral vectors, which expressed notch1 shRNA and NICD, were efficient in silencing notch1 expression and over-expression of NICD. Down-regulation of notch1 gene by RNAi inhibited the cell proliferation remarkably (P<0.01), arrested cell cycle at G1 phase (P<0.01) and decreased the cell number of S phase (P<0.01). Over-expression of NICD enhanced the cell proliferation significantly (P<0.01), promoted the cell cycle at G1 phase (P<0.05) and increased the cell number of S phase (P<0.01). CONCLUSION: notch1 gene, which leads to change the proliferation and cell cycle in human glioma U251 cell line, is likely to be potential molecular target for glioma in gene therapy.     

CD147 affects autophagy of prostate cancer PC-3 cells

AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.     

Effects of GATA6 silencing mediated by lentivirus on apoptosis of human liver cancer Huh-7 cells

AIM: To explore the effect of GATA6 gene silencing on apoptosis of hepatocellular carcinoma Huh-7 cells. METHODS: RNA interference vectors of the target gene GATA6 mediated by lentivirus were constructed in vitro to transfect the hepatocellular carcinoma cell line Huh-7. The apoptotic rate of transfected cells was measured by flow cytometry. The protein expression of GATA6, NF-κB and Bcl-2 in transfected cells was determined by Western blotting. RESULTS: The transfection efficiency was 57.4%. The mRNA and protein expression of GATA6 reduced significantly after the carcinoma cell line Huh-7 being transfected by RNA interference vectors mediated by lentivirus. The apoptotic rate of the carcinoma cells with silent GATA6 gene was significantly increased (P<0.05). The protein expression levels of NF-κB and Bcl-2 were also significantly decreased. CONCLUSION: Lentiviral vector-mediated RNA interference of GATA6 has an inhibitory effect on the expression of the gene itself, and promotes the apoptosis of hepatocellular carcinoma cells. Regulation of the apoptosis-related protein expression by the NF-κB signaling to influence the proliferation and apoptosis of hepatocellular carcinoma cells might be one of the possible mechanisms.     

Silencing IDH-2 gene by siRNA-IDH-2 inhibits human small cell lung carcinoma growth

AIM：To investigate the effect of silencing isocitrate dehydrogenase 2 (IDH-2) gene by small interfering RNA (siRNA) on the biological characteristics of human small cell lung cancer cell line NCI-H446. METHODS：IDH-2 expression was knocked down in human small cell lung cancer cell line NCI-H446 by siRNA-IDH-2. The expression level of IDH-2 was determined by real-time PCR and Western blotting. The cell proliferation was measured by CCK-8 assay, the protein expression of MAPK p42 was detected by Western blotting, and the cell cycle was analyzed by flow cytometry. The migration was observed using Transwell cell migration system. BALB/c nude mice were subcutaneously injected on the back with NCI-H446 cells transfected with siRNA-IDH-2/negative control siRNA or non-transfected cells to study the tumor growth. RESULTS：siRNA-IDH-2 remarkably down-regulated the expression of IDH-2 and MAPK p42 in the NCI-H446 cells. siRNA-IDH-2 inhibited both the proliferation and migration abilities of NCI-H446 cells, and the cell cycle was arrested in S phase as compared with negative control group. Additionally, the volume of xenograft tumors in siRNA-IDH-2 group was significantly decreased as compared with control group. CONCLUSION：siRNA-IDH-2 down-regulates the expression of IDH-2 in NCI-H446 cells, reduces the cell migration efficiency and inhibits the tumor growth in vitro and in vivo.     

miR-335 regulates proliferation of human osteosarcoma MG-63 cells by targeting Sp1

AIM：To investigate the regulatory role of miR-335 on the proliferation of human osteosarcoma cell line MG-63. METHODS：Luciferase reporter gene assay was used to detect the specific binding ability of miR-335 to Sp1 3’-untranslated region (UTR). Real-time PCR and Western blotting were used to evaluate the expression of Sp1 mRNA and protein, respectively. MTT assay was used to analyze the proliferation of MG-63 cells. RESULTS：The luciferase reporter gene assay showed that miR-335 targeted Sp1 3’-UTR. Western blotting and real-time PCR showed that miR-335 inhibited the protein expression of Sp1, but had no effect on the mRNA expression of Sp1. Transfection of Sp1 expression plasmid increased the protein and mRNA expression of Sp1. MTT assay showed the viability of MG-63 cells transfected with miR-335 was significantly decreased compared with negative control. Transfection of Sp1 expression plasmid partly reversed the inhibitory effect of miR-335 on the proliferation of MG-63 cells. CONCLUSION: miR-335 inhibits the proliferation of human osteosarcoma MG-63 cells, which may be related to its targeting on Sp1 3’-UTR and the subsequent down-regulation of Sp1 expression.     

Effect of SMYD3 over-expression on miR-124 expression and proliferation ability in human intrahepatic cholangiocarcinoma cells

AIM：To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on miR-124 expression and proliferation ability of human intrahepatic cholangiocarcinoma cells. METHODS：Transient transfection of SMYD3 eukaryotic expression plasmid into human intrahepatic cholangiocarcinoma cell line HCCC-9810 were performed. The expression of SMYD3 at mRNA and protein levels was measured by qRT-PCR and Western blotting, respectively. The expression of miR-124 was detected by qRT-PCR, and the methylation status of miR-124 gene was determined by methylation-specific PCR. Cell proliferation was examined by CCK-8 assay and colony formation experiment. RESULTS：After transfected with SMYD3 eukaryotic expression plasmid, the over-expression of SMYD3 in HCCC-9810 cells was observed. Compared with the blank cells, the expression level of miR-124 was significantly decreased and miR-124 gene promoter methylation was significantly increased. In addition, SMYD3 over-expression significantly promoted the proliferation of HCCC-9810 cells. CONCLUSION：The transient transfection of SMYD3 plasmid increases the methylation of miR-124 gene promoter and induces under-expression of miR-124. Over-expression of SMYD3 promotes the proliferation of cholangiocarcinoma cells.     

Effects of p21-activated protein kinase 2 down-regulation on proliferation and apoptosis of human breast cancer cells

AIM：To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interference on the proliferation and apoptosis of human breast cancer cells. METHODS：The short hairpin RNA (shRNA) targeting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection. The knockdown efficiency was assessed by Western blotting. The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays. The cell apoptosis induced by staurosporine was detected by flow cytometry. RESULTS：The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells (P<0.01). Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01). Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION：Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemotherapeutic drug-induced cell apoptosis, suggesting that PAK2 might be a new therapeutic target in breast cancer treatment.     

Up/down-regulation of miR-21 changes biological function of colon can-cer cells and sensitivity to cetuximab

AIM: To explore the effects of miR-21 on biological behavior of colon cancer cells and their sensitivity to epidermal growth factor receptor monoclonal antibody cetuximab. METHODS: Lentiviral vectors were constructed to generate up- and down-regulations of miR-21 lentiviruses (LV-miR-21 and LV-anti-miR-21, respectively), and the corresponding negative control viruses (LV-miR-21 NC and LV-anti-miR-21 NC, respectively) were also constructed. The viruses were used to infect human colon cancer RKO cells. The changes of the miR-21 expression level, the cell proliferation, the colony-forming ability, the cell apoptosis and the sensitivity of the cells to cetuximab were detected by real-time PCR, MTT assay, soft agar colony assay, flow cytometry and CCK-8 assay. RESULTS: The lentivirus titers of LV-miR-21, LV-miR-2 NC, LV-anti-miR-21 and LV-anti-miR-21 NC were 3.0×1012 TU/L, 6.0×1011 TU/L, 2.0×1012 TU/L and 8.0×1011 TU/L, respectively. The infection efficiency was over 80% by the observation of green fluorescence. The miR-21 expression level, the cell proliferation, and the colony-forming ability in LV-miR-21 group were significantly higher than those in LV-anti-miR-21 group. The early apoptotic rate and the inhibitory rate of cetuximab for the cells in LV-anti-miR-21 group were higher than those in LV-miR-21 group. CONCLUSION: miR-21 promotes the proliferation of colon cancer cells. Down-regulation of miR-21 enhances the sensitivity of the colon cancer cells to the targeted therapy drug cetuximab.     

Effects of shRNA targeting SIAH2 gene on proliferation and apoptosis of human hepatoma cell line HepG2

AIM：To investigate the effects of shRNA-mediated seven in absentia homolog 2 (SIAH2; one of ubiquitin ligases) gene silencing on cell cycle and apoptosis of human hepatoma cell line HepG2. METHODS：The specific recombinant vector pGenesil-SIAH2 was transiently transfected into HepG2 cells with Lipofectamine 2000. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of SIAH2. MTS assay was employed to evaluate the cell proliferation. Flow cytometry was used to determine the cell cycle and apoptosis of the transfected cells. RESULTS：Compared with control groups, the mRNA and protein levels of SIAH2 were reduced by pGenesil-SIAH2 transfection in HepG2-SIAH2 group. The proliferation of HepG2-SIAH2 cells was significantly inhibited. The percentage of G1-phase cells and the early apoptotic rate were significantly higher in HepG2-SIAH2 cells. CONCLUSION: Tansfection of pGenesil-SIAH2 effectively inhibits the proliferation of HepG2 cells, arrests the cells in G1 phase and induces apoptosis, indicating an experimental basis of SIAH2-targeting gene therapy for hepatocellular carcinoma.     

Effect of tanshinone IIA on expression of cell cycle regulators and proliferation of pancreatic cancer cells

AIM：To observe the effect of tanshinone IIA on the expression of cell cycle regulators and the proliferation of pancreatic cancer cell line BX-PC-3. METHODS： The pancreatic cancer cell line BX-PC-3 was treated with tanshinone ⅡA at various concentrations for 48 h. The inhibition of proliferation was measured by MTT method. The change of the cell cycle was detected by flow cytometry. The protein levels of cyclin A and cyclin D2 were determined by Western blotting. RESULTS：Tanshinoone IIA significantly inhibited the proliferation of BX-PC-3 cells in a dose-dependent manner. The cancer cells were arrested in stage G0/G1 after treated with tanshinone IIA at low dose. The protein levels of cyclin A and cyclin D2 were decreased after drug intervention. CONCLUSION：Tanshinone IIA inhibits the proliferation of human pancreatic cancer cell line BX-PC-3 and the expression of cell cycle-promoting factors (cyclin A and cyclin D2), which may be the mechanism of attenuating the proliferation of pancreatic cancer cells.     

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.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.