Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Construction of FAK-shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

AIM: To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase (FAK) gene and to establish a cell line with stable knockdown of FAK.METHODS: The oligonucleotides that transcribed to short hairpin RNA (shRNA) targeting FAK gene were synthesized in vitro, cloned into retroviral vector pSuper.retro and transfected into Phoenix cell line. The stable clones were screened and high-titer virus was produced. The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant. The stable LM3 cell line, which showed significantly to silence FAK and associated proteins, was selected by puromycin.RESULTS: The recombinant retroviral vector was successfully constructed. Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting. Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells. The cell cycle was blocked at G₀/G₁ phase, and apoptosis was increased. The proliferation rate also decreased significantly.CONCLUSION: FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells. Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.     

Construction of shRNA eukaryotic expression vectors targeting PERK gene and effect of PERK gene knockdown on apoptosis in endoplasmic reticulum stress-induced L02 hepatocytes

AIM: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting the gene of RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase ( PERK ), and to observe the effect of PERK gene knockdown on apoptosis of human normal hepatic L02 cells treated with thapsigargin. METHODS: Three shRNA expression vectors targeting PERK gene, named PERK1-shRNA, PERK2-shRNA and PERK3-shRNA, and one non-homologous negative control expression vector (HK-shRNA) were constructed based on the nucleotide sequence of PERK and the criteria of designing small interfering RNA (siRNA), and were identified by enzyme digestion and DNA sequencing analysis. After L02 hepatocytes were transfected with the plasmids, the PERK expression was determined by RT-PCR and Western blotting, and the plasmid with the best inhibitory effect on PERK expression was screened. The cell viability and apoptotic rate of L02 hepatocytes transfected with PERK-shRNA under endoplasmic reticulum stress (ERS) were measured by the methods of MTT and flow cytometry,respectively. RESULTS: Four shRNA expression vectors were constructed. The mRNA and protein expression levels of PERK gene decreased significantly in L02 hepatocytes transfected with PERK1-shRNA, PERK2-shRNA and PERK3-shRNA as compared with those in control cells (P<0.05). The interfering effect of PERK1-shRNA on PERK gene expression was the best. PERK knockdown by PERK1-shRNA increased the viability and inhibited the apoptosis of L02 cells under ERS.CONCLUSION: The shRNA expression vectors targeting PERK gene are constructed, and PERK gene knockdown may inhibit apoptosis in L02 hepatocytes under ERS.     

Effect of CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons

AIM: To construct recombinant lentiviral vector with short hairpin RNA (shRNA) of CREB gene, and to investigate the effect of CREB gene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neurons. METHODS: Three lentiviral vectors pLentiLox3.7 (PLL) inserted shRNA fragments targeting CREB gene were co-transfected with the packaging plasmids psPAX2 and pMD2.G to the 293T cells, and the virus particles, which was infected with the primary cortical neurons, was encapsulated. The protein expression of CREB was detected by Western blot. The mitochondrial morphology, cell apoptosis and the expression of Bcl-2 and Bax were evaluated by the methods of MitoTracker red, TUNEL and Western blot in OGD/R induced cortical neurons after CREB gene silencing. RESULTS: The pLL-CREB-shRNA1 was the most effective shRNA, which inhibited 80% CREB gene expression in the cortical neurons. The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1 plasmid. In addition, the expression of Bcl-2 was decreased, the expression of Bax, and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1. CONCLUSION: CREB shRNA recombinant lentiviral vector specifically inhibits the expression of CREB gene. CREB gene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

Inhibition of survivin expression in BGC-823 cells by RNA interference

AIM: To investigate the feasibility and specificity of gastric carcinoma gene therapy by utilizing RNA interference (RNAi) to inhibit survivin expression in vitro and in vivo. METHODS: Small interference RNA (siRNA) homologous to survivin was designed. pTZU6+1-siRNA-survivin vector was constructed and transfected into BGC-823 cells. The transplanted BGC-823 tumor in nude mice was established to induce RNAi. The changes of survivin gene expression, tumor cell cycle and cell apoptosis were detected by flow cytometry, RT-PCR, Western blotting, immunochemistry and TUNEL. RESULTS: The expression of survivin was obviously inhibited by RNAi in vitro. The phase of cell cycle indicated the reduction of S phase, while G1/G0 phase increased. Cell apoptosis was obvious. Both the mRNA level and the protein expression of survivin decreased obviously. The tumor size reduced after treated with pTZU6+1-siRNA-survivin vector in vivo. The expression of survivin decreased in siRNA treatment group. In contrast, little change in control group in vitro and in vivo was observed. CONCLUSION: RNA interference down-regulates survivin gene expression, inhibits BGC-823 cell proliferation and induces cell apoptosis with good specificity, which may be a possible new approach for neoplasm gene therapy.     

Effects of fibulin-3 expression on malignant pleural mesothelioma cells

AIM: To observe the effects of over-expression and silencing of fibulin-3 on malignant pleural mesothelioma (MPM) cell line SMC-1, and to search for a new method for the treatment of MPM. METHODS: The vectors for fibulin-3 over-expression and short hairpin RNA (shRNA) were constructed, and control vector, fibulin-3 over-expression vector (Exp), over-expression control vector (Exp-NC), interference vector (shRNA1, shRNA2, shRNA3 and shRNA4), and interference control vector (shRNA-NC) were transfected into the SMC-1 cells. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of fibulin-3 before and after interference was determined by real-time PCR and Western blot. RESULTS: Compared with control group, the number of the SMC-1 cells in G₂ phase in Exp group was increased (P<0.05), while that in shRNA2 group was decreased (P<0.05). The results of apoptosis analyzed by flow cytometry showed that compared with control group, the apoptotic rate in Exp group was decreased (P<0.05), while that in shRNA2 group was increased (P<0.05). The mRNA and protein expression levels of fibulin-3 and mesothelin in Exp group were up-regulated, while those in shRNA group were down-regulated (P<0.05).CONCLUSION: The vectors for over-expression and silencing of fibulin-3 are successfully constructed, proving that transfection of Exp and shRNA effectively changes fibulin-3 expression and the SMC-1 cell growth. Fibulin-3 may be a target molecule for MPM treatment.     

Role of ANX A2 in APL-induced expression of tissue factor in THP-1 cells

AIM: To construct a lentiviral vector harboring RNAi sequence targeting human annexin A2 (ANX A2) and to investigate the functions of ANX A2 in antiphospholipid antibody (APL)-induced tissue factor (TF) expression in monocytes. METHODS: Four different short hairpin RNAs (shRNA) targeting ANX A2 gene were constructed and cloned into the pGCSIL-GFP vector. After identification with PCR and sequencing, the effective interference sequence was further determined by Western blotting analysis. The recombinant lentivirus LV-RNAi-ANX A2 harvested from 293T cells was transferred into THP-1 cells and the ANX A2 mRNA and protein expression on THP-1 cells were examined. The transferred-THP-1 cells were stimulated by APL/β2GPI compound, and the TF mRNA and TF activity was assayed. RESULTS: The RNAi sequences targeting the human ANX A2 gene were successfully inserted into the lentiviral vector and the high-performance RNA interference sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×1012 TU/L. THP-1 cells infected with LV-RNAi-ANX A2 showed almost lockout of ANX A2 both at mRNA and protein levels. The TF expression was also significantly decreased in the transferred-THP-1 cells induced by APL/β2GPI compound. CONCLUSION: The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP-1 cells and further inhibits the TF expression induced by APL/β2GPI compound, which indicates that ANX A2 do play an important role in APL-induced TF expression on monocytes.     

Construction of short hairpin RNA lentiviral vector targeting MAG gene and its silencing effect on MAG gene

AIM：To construct lentiviral vector-based short hairpin RNA (shRNA) targeting myelin-associated glycoprotein (MAG) gene and to evaluate its inhibitory effect on the expression of MAG gene. METHODS：Three shRNA fragments targeting MAG gene (shRNA1, shRNA2 and shRNA3) were designed and cloned into lentiviral vector pWPI. The three recombinant plasmids were identified by enzyme digestion and sequencing. Positive plasmids were co-transfected with pCDNA3-MAG-FLAG into 293T cells, and the most effective shRNA for knockdown of MAG gene was screened by Western blotting. Cells transfected with empty pWPI served as a control. Oligodendrocytes were infected with recombinant lentivirus that was produced by 293T packaging cells co-transfected with the most effective shRNA, pAX2 and pMD2G. After 48 h, the expression of MAG protein was measured by Western blotting. RESULTS：The MAG shRNA lentiviral vectors were confirmed by double enzyme digestion and sequencing, and shRNA2 showed the highest inhibitory efficacy among the three shRNA fragments. Recombinant lentivirus carrying shRNA-2 markedly decreased the expression of MAG protein in oligodendrocytes. CONCLUSION：Lentiviral vector-based shRNA targeting MAG gene specifically knocks down the gene expression, which provides a useful tool for investigating the role of MAG-specific shRNA in regulating myelination of central nerve system.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.     

Effect of RNA interference on Polo-like kinase-1 in A549 cells

AIM: To investigate whether RNA interference(RNAi) induced by small interference RNA(siRNA) could suppress Polo-like kinase-1 (Plk 1) expression and its effects in A549 cells.METHODS: A recombinant plasmid containing siRNA targeting Plk1 (psiRNA-hH1-Plk1) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1,cyclin B1 and p53 protein were detected by Western blotting.Cell proliferation was evaluated by direct cell counting,while cell cycle and apoptosis were examined by flow cytometry,and expression of α-tubulin was detected by immunofluorescence.RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression,and reflecting in lower kinase activity in A549 cells.The level of Plk1 protein was reduced by at least 70% after 48 h of psiRNA-hH1-Plk1 treatment relative to controls.Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion,and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin.Growth inhibition,G₂/M arrest and apoptosis were observed in psiRNA-hH1-Plk1 transfected group.CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.     

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.     

Effect of RNAi-mediated IGF1R gene silencing on growth,migration, and invasion of hepatocellular carcinoma cells

AIM: To investigate the effect of RNA interference (RNAi)-mediated insulin-like growth factor 1 receptor (IGF1R) gene silencing on the growth, migration, and invasion of hepatocellular carcinoma cells. METHODS: The most effective siRNA targeting IGF1R gene was designed and screened. After lentiviral expression vector pLVX-shRNA2-IGF1R carrying the most effective siRNA sequence was constructed, it was transfected into 293T cells and packed into pLVX-shRNA2-IGF1R lentivirus. Huh7 and Hep3B cells were infected with the pLVX-shRNA2-IGF1R lentivirus to screen the positive clone Huh7 cells and Hep3B cells with the lentivirus. These Huh7 cells and Hep3B cells were cultured to analyze the mRNA level of IGF1R, cell proliferation, cell cycle, cell apoptosis, cell migration/invasion, and the protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL. RESULTS: The mRNA expression of IGF1R in Huh7 cells and Hep3B cells with pLVX-shRNA2-IGF1R lentivirus was significantly reduced. The proliferation of these cells was remarkably inhibited, and the number in G₁ phase was increased significantly. The percentages of apoptotic cells were increased markedly, and the number of cell migration/invasion was decreased markedly. The protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL were decreased significantly compared with the blank control group and negative control group. CONCLUSION: The RNAi-mediated IGF1R gene silencing significantly suppresses the growth and the malignant biological characteristics of Huh7 cells and Hep3B cells, which may be involved in the reduced protein levels of the above genes induced by down-regulation of IGF1R expression.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

Dendritic cells transfected with tumor total RNA induce specific immune responses against hepatocellular carcinoma in vitro

AIM: To observe the ability of dendritic cells (DC) vaccine transfected with human hepatocellular carcinoma (HCC) total RNA induce specific cytotoxic T lymphocyte(CTL) response in vitro. METHODS: DCs generated from HCC patient’s peripheral blood mononuclear cells (PBMC) were incubated with recombinant human granulocyte macrophage colony-stimulationg factor (GM-CSF) and human interleukin (IL-4). Tumor total RNA was isolated from Hep G-2 cells and HCC cells. DCs transfected with tumor total RNA were used to induce specific CTL proliferation. Specific cytotoxicity was measured using MTT method. RESULTS: DC transfected with HepG-2 cell RNA and HCC RNA exhibited increased expression of CD83, CD86 and HLA-DR. The CTL from DCs transfected with HepG-2 cell RNA killed 5.84％， 14.26％， 25.19％, or 35.78％ of HepG-2 cells, and 5.26％， 11.67％， 14.68％, or 23.24％ of HCC cells, respectively, at an E/T ratio of 2.5, 5, 10, or 20. The CTL from DCs transfected with HCC cells RNA killed 4.65％， 12.23％， 15.61％, or 19.15％ of HepG-2 cells, and 7.20％， 12.83％， 27.21％, or 31.15％ of HCC cells,respectively, at an E/T ratio of 2.5, 5, 10, and 20. These CTL did not kill allogeneic malignant cells as human gastric carcinoma cells SGC-7901. CONCLUSION: DC transfected with tumor-derived total RNA could induce specific antitumor immune CTL response. These results suggest that CTL generation is applicable to adoptive immunotherapy of HCC.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.