Negative Transfection Group Research Articles

COL11A1 regulates PI3K/Akt/GSK-3β pathway and promotes human lung adenocarcinoma primary cell migration and invasion

Objective: To investigate the role and mechanism of COL11A1 in lung adenocarcinoma migration and invasion. Methods: Surgical pathological tissues of 4 patients with lung adenocarcinoma admitted to the Affiliated Hospital of Guizhou Medical University from September to November 2020 were used. Immunohistochemical methods were used to identify lung adenocarcinoma tissues, para-cancerous tissues and parallel transcriptome sequencing. Genetic prognostic analysis was conducted by TCGA and GTEx databases.The expression level of COL11A1 gene in lung adenocarcinoma and adjacent tissues was detected by Western blotting.The primary human lung adenocarcinoma cells cultured. The COL11A1 siRNA was transfected into primary human lung adenocarcinoma cells, then the transcriptome sequencing of differential genes was performed,and KEGG enrichment analysis of differential gene enrichment pathway was conducted. Protein expression and phosphorylation were detected by Western blot method. Cell migration was detected by scratch healing test. Cell proliferation was detected by CCK8 method and invasion ability was detected by Transwell method. Results: Ten differentially expressed genes were screened by transcription sequencing in lung adenocarcinoma. Prognostic analysis of single gene showed that COL11A1 gene expression level was correlated with survival rate (P<0.001). The expression of COL11A1 in lung adenocarcinoma was higher than that in adjacent tissues by Western blot (P<0.001). Transcriptome sequencing of COL11A1 siRNA transfection into primary human lung adenocarcinoma cells showed that differential genes were concentrated in PI3K-akt pathway. The expression of tumor suppressor gene PTEN in siRNA transfection group was significantly higher than that in control group and negative transfection group by Western blot. The expression of Aktp-Akt 473 p-Akt 308 p-PTENp-PDK1p-c-Rafp-GSK-3 β was down-regulated (all P<0.05).Compared with the negative control group, the ability of migration, proliferation and invasion of primary human lung adenocarcinoma cells in siRNA transfection group decreased (all P<0.05). COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells. Conclusion: COL11A1 regulates PI3K/Akt/GSK-3 β pathway to promote migration and invasion of primary human lung adenocarcinoma cells.

Effect of BECN1 Expression on Proliferation and Invasion of Human Myeloma Cell Line RPMI-8226

To investigate the effect of BECN1 expression on proliferation and invasion of human multiple myeloma (MM) cell line RPMI-8226. RPMI-8226 cells were cultured in vitro, the recombinant plasmid pcDNA3.1-BECN1 was constructed and transfected into RPMI-8226 cells, then the cells were divided into three groups: control group, negative transfection group and BECN1 transfection group. The expression of BECN1 mRNA in cells of each group was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the effect of BECN1 overexpression on cell proliferation inhibition rate was detected by cell counting kit 8 (CCK-8); the effect of BECN1 overexpression on the colony formation rate was detected by plate cloning assay; the effect of BECN1 overexpression on cell invasion was detected by Transwell assay; the effects of BECN1 overexpression on the expression of cell proliferation, invasion and autophagy-related proteins were detected by Western blot. The expression of BECN1 mRNA in BECN1 transfection group was significantly higher than that in control group and negative transfection group (P<0.05); the inhibition rate of cell proliferation and the expression of autophagy-related proteins Beclin1, Atg5 and invasion-related protein E-cadherin in BECN1 transfection group were significantly higher than those in control group and negative transfection group; the colony formation rate, invasion number and the expression of proliferation-related proteins CyclinD1, β-catenin and invasion-related protein N-cadherin were significantly lower than those in control group and negative transfection group (P<0.05). Overexpression of BECN1 can inhibit the proliferation and invasion of human MM cells RPMI-8226, which may be a potential research target of MM.

Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract.

Cigarette smoking contributes to the development of pulmonary artery hypertension (PAH). As the basic pathological change of PAH, pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the molecular mechanism underlying this process remains not exactly clear. The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking. Human PASMCs (HPASMCs) were divided into 6 groups: 0% (control group), cigarette smoking extract (CSE)-treated groups at concentrations of 0.5%, 1%, 2%, 5%, 10% CSE respectively. HPASMCs proliferation was observed after 24 h. HPASMCs were divided into two groups: 0 (control group), 0.5% CSE group. The mRNA and protein expression levels of transient receptor potential channel 1 (TRPC1) and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting. The intracellular calcium ion concentration was measured by the calcium probe in each group. In the negative control group and TRPC1-siRNA transfection group, the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected. Data were compared with one-way ANOVA (for multiple-group comparison) and independent t-test (for two-group comparison) followed by the least significant difference (LSD) test with the computer software SPSS 17.0. It was found that 0.5% and 1% CSE could promote the proliferation of HPASMCs (P<0.05), and the former was more effective than the latter (P<0.05), while 3% and above CSE had inhibitory effect on HPASMCs (P<0.05). The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5% and 1% CSE groups were significantly higher than those in the control group (P<0.05), while those in 3% CSE group were significantly decreased (P<0.05). Moreover, the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group (P<0.05). It was concluded that low concentration of CSE can promote the proliferation of HPASMCs, while high concentrations of CSE inhibit HPASMCs proliferation. These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.

Inhibiting MiR-34α reduces retinal cell apoptosis and downstream NF-κB pathway in diabetic retinopathy rats through regulating HMGB1 expression.

This is a research aimed to study the effect of micro ribonucleic acid (miR)-34α on the retinal cell apoptosis in diabetic retinopathy (DR) rats and its key molecular mechanism. Sprague-Dawley rats were randomly divided into healthy group (H group, N.=5), diabetes group (D group, N.=5), diabetes + negative control transfection group (N group, N.=5) and diabetes + miR-34α inhibitor transfection group (M group, N.=5). The rat model of diabetes was established via intraperitoneal injection of 2% streptozotocin solution (60 mg/kg). After 72 h, the urine glucose and blood glucose were detected, and the urine glucose above 3+ and the blood glucose concentration >16.7 mmol/L indicated the successful modeling. After the rats were normally fed for 4 months, the changes in expression of miR-34α in retinal tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR), the pathological changes in retinal tissues were observed via hematoxylin-eosin (HE) staining, and the retinal cell apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the changes in the number of cells containing active caspase-3 in retinal tissues were determined through immunohistochemistry, and the changes in expressions of caspase-3, high mobility group box 1 (HMGB1) and nuclear factor-κB (NF-κB) in retinal tissues were determined through Western blotting. Compared with those in H group, the cell density declined, and the cells were arranged disorderly with swelling in each retinal layer, the expression of miR-34α in retinal tissues was increased, the retinal cell apoptosis was enhanced, the number of cells containing active caspase-3 in retinal tissues rose, and the expressions of caspase-3, HMGB1 and NF-κB in retinal tissues were increased in D group, N group and M group (P<0.05). Compared with those in D group and N group, the cell density rose, and the cells were arranged less disorderly with milder swelling in each retinal layer, the expression of miR-34α in retinal tissues declined, the retinal cell apoptosis was weakened, the number of cells containing active caspase-3 in retinal tissues was decreased, and the expressions of caspase-3, HMGB1 and NF-κB in retinal tissues were reduced in M group (P<0.05). Inhibiting miR-34α reduces the retinal cell apoptosis in DR rats through regulating the HMGB1 expression and downstream NF-κB pathway.

MiR-20a Promotes kidney injury in sepsis rats through autophagy.

The aim of this work was to study the effects of micro ribonucleic acid (miR)-20a on acute kidney injury (AKI) in sepsis rats and its key molecular mechanism. Sprague-Dawley rats were randomly divided into healthy rat group (H group, n=3), sham group (S group, n=3), sepsis rat group (D group, n=3), sepsis rat + negative control transfection group (N group, n=3) and sepsis rat + miR-20a inhibitor transfection group (M group, n=3). At 6 h, 12 h and 24 h, serum creatinine (Scr) and blood urea nitrogen (BUN) were detected, the changes in miR-20a expression in kidney tissues were determined via reverse transcription-polymerase chain reaction (RT-PCR), the expression of autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) was measured via Western blotting, and the inflammatory cell infiltration in kidney tissues was detected using hematoxylin-eosin (HE) staining. There was no obvious change in each index in S group compared with H group. D group, N group and M group had higher levels of Scr, BUN and LC3 in kidney tissues than S group. The levels of Scr, BUN and LC3 in kidney tissues were lower in M group than those in N group. MiR-20a may cause AKI in sepsis rats via activating autophagy.

Proliferation inhibition and apoptosis promotion induced by deletion of TMPO in A549

Objective: To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism. Methods: TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis. Results: The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11), (0.99±0.10), (0.36±0.04), respectively, the protein levels were (0.27±0.02), (0.29±0.03), (0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04), (0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06), (0.46±0.08), (0.37±0.04), the OD values at 72 h were (0.75±0.08), (0.73±0.07), (0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07), (1.06±0.08), (0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the proportion of G(0)/G(1) phase cells in blank control group, negative control group and transfection group were (62.55±2.03)%, (61.24±3.15)%, (47.35±2.44)%, respectively. The proportion of cells in S phase were (17.12±1.31)%, (17.70±2.01)%, and (20.81±2.06)%, respectively. The proportion of cells in G(2)/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G(0)/G(1) phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G(2)/M phase of transfection group was significantly higher than those of blank control and negative control group (P<0.05). The apoptosis ratio of the transfection group was (34.10±2.69)%, significantly higher than (2.96±0.03)% of the blank control and (3.01±0.04)% of the negative control group (P<0.05). Western blot analysis showed that PCNA, Notch1 and mTOR proteins were down-regulated while cleaved caspase-3 protein was up-regulated in A549 cells after deletion of TMPO. Conclusion: The inhibition of TMPO gene expression induced by small interfering RNA can significantly inhibit the proliferation and induce apoptosis of A549 cells, and the mechanism is associated with the inhibition of the activation of Notch1/mTOR signaling pathway.

Effect of geniposidic acid on SHP-LRH-1 signaling pathway in cholestasis rats

To explore the effect of geniposidic acid (GPA) on the signal pathway of small heterodimer dimer receptor (SHP) and liver receptor homologue 1 (LRH-1) in cholestasis rats induced by alpha-naphthalene isothiocyanate (ANIT). Methods: Fifty SD rats were randomly divided into five groups: a blank group, an ANIT group, an ANIT+GPA (100 mg/kg) group, an ANIT+GPA (50 mg/kg) group, and an ANIT+GPA (25 mg/kg) group (n=10 in each group). The GPA were intragastrically given to rats for 10 days, and the control group and the ANIT group were given normal saline. At the eighth day of administration, all rats except the blank group were given 65 mg/kg ANIT once until the tenth day. After the last administration, serum total cholesterol (TC), triglyceride (TG) and total bile acids (TBA) were measured. The primary hepatocytes (RPH) were isolated from normal rats and cultured. The cells were divided into a blank group, an ANIT (40 μmol/L) group, an ANIT (40 μmol/L)+GPA (4.00 mmol/L) group (A4.00G group), an ANIT (40 μmol/L)+GPA (1.00 mmol/L) group (A1.00G group), and an ANIT (40 μmol/L)+GPA (0.25 mmol/L) group (A0.25G group). The mRNA transcription levels of SHP and cholesterol 7 alpha hydroxylase (CYP7A1) in RPH were detected by real-time-PCR, and the protein levels of SHP and CYP7a1 were detected by Western blotting. In the LRH-1 silence experiment, the RPH were divided into a blank group, a negative transfection group, a siRNA-LRH group (ZR group), a siRNA-LRH+GPA (4.00 mmol/L) group (ZR4.00G group), a siRNA-LRH+GPA (1.00 mmol/L) group (ZR1.00G group) and a siRNA-LRH+GPA (0.25 mmol/L) group (ZR0.25G group). The protein and mRNA levels of SHP, CYP7a1, LRH-1 were detected. In the over-expression experiment, the RPH were also divided into a blank group, a negative transfection group, a LRH-1 over-expression plasmid group (OE group), a LRH-1 over-expression plasmid+GPA (4.00 mmol/L) group (OE4.00G group), a LRH-1 over-expression plasmid+GPA (1.00 mmol/L) group (OE1.00G group), and a LRH-1 over-expression plasmid+GPA (0.25 mmol/L) group (OE0.25G group). The protein and mRNA levels of SHP, CYP7a1 and LRH-1 were detected. Results: Compared with the blank control group, TC and TBA were significantly increased (both P<0.01) in the ANIT group, but there was no difference in TG; compared with the ANIT group, the contents of TC and TBA in the AG100 and AG50 groups were significantly reduced (all P<0.01). Compared with the blank control group, the proteins and mRNA levels of SHP were significantly decreased (P<0.01), while CYP7a1 were dramatically increased (P<0.01) in the ANIT group; compared with the ANIT group, the proteins and mRNA levels of SHP in the A4.00G group and the A1.00G group were significantly increased (both P<0.01), while the levels of CYP7a1 proteins and mRNA levels were evidently decreased in the A4.00G and A1.00G groups (both P<0.01). Compared with the negative transfection group, the proteins and mRNA levels of CYP7a1 and LRH-1 were dramatically restrained (all P<0.01), while there was no change in SHP in the ZR group; compared with the ZR group, the proteins and mRNA levels of SHP were significantly increased (all P<0.01), while LRH-1 and CYP7a1 were not changed in the ZR4.00G, ZR1.00G and ZR0.25G groups. Compared with the negative transfection group, the protein and mRNA levels of CYP7a1 and LRH-1 were significantly suppressed in the OE group (all P<0.01). Compared with the OE group, the protein and mRNA levels of SHP were evidently increased in the OE4G and OE1G groups (all P<0.01), while LRH-1 and CYP7a1 were not changed in the OE4G, OE1G and OE0.25G groups. Conclusion: The over-expression of LRH-1 in RPH can up-regulate the mRNA and protein levels of CYP7a1. GPA can improve the biochemical and liver pathology of ANIT-induced cholestasis rats, which may be related to the decrease of CYP7a1 by activating SHP through LRH-1 in RPH.

Expression of miR-490-5p, miR-148a-3p and miR-608 in bladder cancer and their effects on the biological characteristics of bladder cancer cells.

Changes in the expression of miR-490-5p, miR-148a-3p and miR-608 in bladder cancer tissues were studied. A total of 30 patients with bladder cancer who had surgical resection in the Hunan Provincial People's Hospital (Changsha, China) from April 2015 to August 2016 were selected. RT-qPCR was used to detect the expression levels of miR-490-5p, miR-148a-3p and miR-608. The expression vectors of miR-490-5p, miR-148a-3p and miR-608 were respectively transfected and divided into three groups: blank cell group, gene transfection group (groups A-C) and negative transfection group (NC group). CCK8 was used to detect cell proliferation and flow cytometry was used to detect the condition of apoptosis of each group, and the Transwell chamber was used to detect the invasion ability of the cells. After the transfection, the expression level of miR-490-5p in group A was significantly higher than that in the NC and blank groups, and the expression level of miR-148a-3p in group B was significantly higher than that in the NC and blank groups. The expression level of miR-608 in group C was significantly higher than that in the NC and blank groups (P<0.001). The survival rates of the cells in groups A-C were significantly lower than those in the NC and blank groups at 48 and 72 h (P<0.001). After the transfection, the number of invasive cells and the apoptosis rates in groups A-C were significantly higher than those in the NC and blank groups (P<0.05). miR-490-5p, miR-148a-3p and miR-608 promoted proliferation of bladder cancer T24 cells and inhibited apoptosis of the cells and showed potential to become a new target for the future treatment of bladder cancer.

Retinoic acid affects apoptosis of neurons injured by hypoxic-ischemic brain damage via retinoic acid receptor alpha

Objective To explore the effect of the retinoic acid (RA) on the apoptosis of neurons caused by hypoxic ischemic brain damage (HIBD). Methods Seventy-two newborn Sprague-Dawley rats were randomly divided into an RA deficiency (RAD) group, an RA normal (RAN) group and a control group, each of 24. The HIBD model was established in the RAD and RAN groups using Rice′s method. The left common carotid artery was exposed, ligated and cut, inducing hypoxia. In the control group the left common carotid artery was exposed without any other treatment. Three and 7 days after the operation, neuron apoptosis in the brain tissue was evaluated using TUNEL staining. The degree of HIBD was quantified using modified neurological severity scores (mNSS) 7, 14, 21 and 28 days after the operation. Primary neurons were cultured in vitro, and oxygen glucose deprivation (OGD) was induced, then control, OGD and RA+ OGD groups were formed. The gene transcription and the protein activity of retinoic acid receptor alpha (RARα), GDNF (glial cell line-derived neurotrophic factor) and Caspase-8 were examined with polymerase chain reactions (PCR) and Western blotting. The RA+ OGD group was exposed to RA and SiRNA adenovirus, and divided into a silenced group and a negative transfection group according to the infection. Results The average mNSS of the RAD group was significantly higher than that of the RAN group. TUNEL staining showed that the apoptotic cells in the cortex increased from day 3 to 7 after the operation, but significantly more in the RAD group than in the RAN group. The gene transcription and protein activity of RARα and GDNF in the RA+ OGD group were significantly higher than in the OGD group, and those of Caspase-8 were significantly lower. The gene transcription and protein activity of RARα and GDNF in the silenced group were significantly lower than in the negative transfection group, while those of Caspase-8 were just the opposite. Conclusion RA can inhibit the apoptosis of primary neurons after HIBD by up-regulating the expression of GDNF and down-regulating that of Caspase-8 via RARα. Key words: Retinoic acid receptor alpha; Neuron apoptosis; Adenovirus; Glial cell-derived neurotrophic factor; Caspase-8

Expression of SET8 in renal cell carcinoma and the effects of SET8 silencing on proliferation and migration of 786-O cells

Objective To investigate the expression of lysine methyltransferase SET8 protein in the clear cell carcinoma of kidney and the effects of silencing SET8 gene by shRNA on proliferation and migration of human renal cell carcinoma cell line 786-O in vitro. Methods Immunohistochemistry(IHC) was employed to detect the expression of SET8 in 156 tissue samples of clear cell renal cell carcinoma in the study between December, 2006 and December, 2011.Four pairs of SET8-shRNA were designed and synthesized. Transfection was performed with cationic lipid vectors(LipofectamineTM2000). The transfection efficiency was observed by fluorescence confocal microscopy. The effecive shRNA sequences were screened by Western blot. After transfection with effective shRNA sequences, 786-O cells were divided into 3 groups: (1)SET8-shRNA transfection; (2)negative control transfection; (3)blank control.The assays of methyl thiazolyl tetrazolium(MTT), colony formation and scratch wound were applied to examine variations in cell proliferation, colony formation and migration. Results The positive rate of SET8 expression was 98.7%(154/156), and its expression was closely associated with TNM staging, the size of tumor and lymph node metastasis(all P<0.05). The transfection efficiency was around 70% with a concentration of SET8-shRNA 2μg/L and lipofectamine 2000 4μl/well. SET8-shRNA2 was chosen as the effective sequences with a suppression ratio up to 64%. At 48h post-transfection, the expression of SET8 protein was significantly lower in SET8-shRNA2 group than that in negative control transfection group and blank control group(t=-38.174, P<0.01; t=-58.766, P<0.01). While SET8 gene was silenced by shRNA in SET8-shRNA transfection group, the proliferation, colony formation and migration ability were decreased markedly. Conclusion SET8 protein plays a significant role in the carcinogenesis and progression of renal cell carcinoma. SET8-shRNA can effectively inhibit the expression of SET8 protein in 786-O cells and thus suppress malignant phenotypes of RCC. Key words: Lysine methyltransferaseSET8; Clear cell renal cell carcinoma; 786-O cell; Cell proliferation; Colony formation; Migration

Lentivirus mediated RNAi silence esophageal MDC1 Eca109 cell gene expression of the influence of nude mouse transplantation tumor radiosensitivity

Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice, and to observe the histopathological and cellular changes in nude mice. Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector. Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1. After selection by copGFP reporter gene, cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M). The transfected cells were injected into nude mice. The mice were divided into ECA109 group, ECA109-N group, and ECA109-M group. Each group was divided into irradiation subgroup and non-irradiation subgroup. The changes in tumor size after irradiation were evaluated in each group. Western blot was used to measure the expression of CHK1, CHK2, and CHK2T68 in xenografted tumors. Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice. The variance analysis was used to compare the mean of multiple groups, and the SNK-q test was used in the two two groups. Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells. ECA109-M cells were obtained by stable transfection with the recombinant plasmid. All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week. There was no swelling and wound in inoculation sites. There was no significant difference in tumor size between different groups (P>0.05). The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P 0.05); however, the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P 0.05). Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity. Key words: RNA interference; Radiosensitivity; MDC1 gene; Implanted tumor in nude mice, ECA109 cell line; Tumor growth delay

Roles of glucose-regulated protein 78 in proliferation and migration of human colorectal carcinoma cell line RKO

To investigate the roles of glucose regulated protein 78(GRP78) in proliferation and migration of human colorectal carcinoma cell RKO. The colorectal carcinoma cell line RKO was transfected by lentiviral vector pLV-shRNA GRP78 and lentivirus vector pLV-control. MTT test and colony formation assay were used to evaluate the cell proliferation ability. Distribution of cell cycle was analyzed by flow cytometry. Cell migration ability was detected by scratches migration experiment. In vivo tumorigenicity ability was measured using subcutaneous tumor assay. Differentially expressed genes were detected by Affymetrix human genome-wide expression profile chip and confirmed by Western blot analysis. Compared with the negative vector transfection group, cell proliferation was inhibited in vitro and in vivo (P<0.05), while there was no significant difference in migration (P>0.05). Flow cytometry results showed that silencing GRP78 resulted in a significant increase in the proportion of cells in G1 phase, while the proportion of cells in S phase was significantly lower (P<0.05). The gene chip results showed that 397 genes were differentially expressed by at least 1.2 folds in GRP78 knocked-out RKO cells, including 258 up-regulated and 139 down-regulated ones. Bioinformatics analysis identified 3 genes (CDKN2B, MTOR and BIRC3) with specific expression in GRP78 down-regulated RKO cells, and the result was verified by Western blot. GRP78 knock-out inhibits the proliferation and growth of colorectal cancer cell, but has no obvious effect on migration invasion. Down regulation of GRP78 results in expression changes of lots of genes in RKO cells. GRP78 may exert its role in proliferation of RKO cell through regulating these genes.

The molecular mechanism of antiapoptosis of type II alveolar epithelial cell by microRNA-21-5p

To study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of type II alveolar epithelial cell (AECII) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis. Subculture AECII were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group (0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECII, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours). (1) AECII identification: fluorescence microscopy showed the presence of characteristic structure of AECII, i.e. microvilli and osmiophilic lamellar bodies. (2) Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECII. (3) The expression of miR-21-5p in AECII: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, all P<0.05). (4) The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours: (16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, all P<0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point. (5) Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, all P<0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P<0.05). (1) miR-21-5p has the function of anti-apoptosis of AECII. (2) Adenoviral vector is a successful gene transfer vector when transfected with AECII. (3) The anti-apoptosis of AECII by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.

Effects of microRNA-29b on invasion and proliferation of human high peritoneal metastatic gastric cancer cells

Objective To investigate the expression of microRNA-29b (miR-29b) in gastric cancer cell line GC9811 and high peritoneal metastatic gastric cancer cell line GC9811-P and its effect on invasion, proliferation and apoptosis. Methods The relative quantitative expression of miR-29b was detected by quantitative realtime polymerase chain reaction(qRT-PCR). GC9811 cells were divided into three groups, miRNA down-regulated and transfected with lentiviruses LV-miR-29b inhibitor group, negative control group with negative transfection, and untransfected blank control group. GC9811-P cells were divided into three groups, miR-29b up-regulated and transfected with lentiviruses LV-miR-29b group, negative control with negative transfection group, and untransfected blank control group. The cell invasion ability was detected with Transwell assay, the cell proliferation ability was measured by methyl-thiazolyl tetrazolium (MTT) test, the colony-forming ability was determined by plate colony formation assay, and the apoptosis was tested by flow cytometry. The expressions of miR-29b and secreted protein, acidic and rich in cystenie (SPARC) in gastric cell line GC9811-P, GC9811, MKN28M, MKN28NM and normal gastric cell line GES were determined by qRT-PCR, and the correlation was analyzed. Two independent samples t test or SNK-q test was performed for mean comparison between two groups, and one-way analysis of variance was used for mean comparison among three groups. Results The relative quantitative expression of miR-29b in GC9811-P (0.21±0.04) was significantly lower than that of GC9811 (1.00±0.03, t=28.140, P<0.01). After GC9811 cells transfected with lentiviruses LV-miR-29b inhibitor, the expression of miR-29b (0.21±0.04) was significantly lower than that of control group (0.89±0.07) and blank control group (1.00±0.04, q=12.76, 14.73, both P<0.01). Compared with negative group, the transmembrane cell number and the clonality of miRNA up-regulated group raised(274.33±9.03 vs 110.67±13.69, t=9.981, P<0.01; 131.33±4.91 vs 69.67±2.33, t=11.340, P<0.01), and the results of MTT test also shows the proliferation was increased. After GC9811-P cells transfected with LV-miR-29b, the expression of miR-29b (4.08±0.20) was significantly higher than that of negative control group (1.15±0.05) and blank control group (1.00±0.10, q=21.73, 22.81, both P<0.01). Compared with negative group, the transmembrane cell number and the clonality of miRNA up-regulated group reduced (51.33±5.55 vs 104.00±6.24, t=6.305, P<0.01; 48.00±5.51 vs 113.33±5.17, t=11.340, P<0.01), and the results of MTT test also shows the proliferation was weakened. Apoptosis assays demonstrated miR-29b promoted apoptosis; however, the difference was not statistically significant. The expression of miR-29b was negatively correlated with SPARC mRNA in gastric cancer cells (r=-0.97, P=0.03). Conclusions The low expression of miR-29b in high peritoneal metastatic gastric cancer cell inhibited the ability of invasion and proliferation. MiR-29b might be a new target of inhibiting peritoneal metastasis in gastric cancer. Key words: Stomach neoplasms; Peritoneal metastasis; Cell invasion; Cell proliferation; miRNA-29b

Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells

To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all P<0.05). The number of cell migration in the non-transfection group,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

Effect of silencing Bcl-2 expression by small interfering RNA on radiosensitivity of gastric cancer BGC823 cells

ObjectiveTo explore the influence of silencing Bcl-2 expression by small interfering RNA (siRNA) on Bcl-2 protein expression, cell apoptosis rate and radiosensitivity of gastric cancer BGC823 cells. MethodssiRNA segment for Bcl-2 gene was designed and synthesized, then was induced into gastric cancer BGC 823 cells by liposome transfection. Bcl-2 protein expression was detected by Western Blotting. After X radiation, flow cytometry and clone forming assay were used to determine the effects of RNA interference on BGC823 cell apoptosis rate and radiosensitivity. ResultAfter the transfection of Bcl-2 siRNA, the positive expression rate of Bcl-2 protein in BGC823 cells was (35.45±2.35)%. Compared with the control group and negative siRNA transfection group, the rate was significantly decreased (P<0.01). The apoptosis rate of BGC823-RNAi cell was (10.81±0.91)%, which was significantly higher than the control group and negative siRNA transfection group (P<0.01). After 48h X radiation, the apoptosis rate of BGC823-RNAi was (28.91±1.40)%, which was significantly higher than the control group and the group without radiation (P<0.01). During clone forming assay D0, Dq and SF2 values in Bcl-2 siRNA1 transfection group were all lower than those in the control group. The radiosensitivity ratio was 1.28 (the ratio of D0) and 1.60 (the ratio of Dq). ConclusionsSpecific siRNA of Bcl-2 gene can effectively inhibit the expression of Bcl-2 gene, enhance the radiosensitivity and apoptosis of gastric cancer BGC823 cells, having good clinical application perspective.

The Effect of Tumor Necrosis Factor-α-Induced Protein 8 Like-2 on Immune Response of CD4+T Lymphocytes in Mice after Thermal Injury

The status of cellular immunity has been shown to be associated with the occurrence and development of sepsis. Accumulating evidence has demonstrated that tumor necrosis factor-α-induced protein 8 like-2 (TIPE2) plays an important role in maintaining homeostasis of immune function. The present study, with the use of a controlled in vivo approach, demonstrated the effect of TIPE2 on cell-mediated immunity of CD4+ T lymphocytes in thermal injury murine model. One hundred and twenty-eight male mice were randomly allocated into four groups, which were sham burn group (n=48), burn group (n=48), burn with lentivirus-RNAi-TIPE2 transfection group (n=16), burn with negative control transfection group (n=16), and they were sacrificed at the designated time points. CD4+ T lymphocytes were isolated from the spleen using MACS microbeads. Phenotypes were analyzed by flow cytometry analysis, and cytokines were determined using ELISA kits. We found that the expression of TIPE2 was markedly increased in CD4+T lymphocytes in mice at 24,48 and 72 hours postburn. Down-regulation of TIPE2 by lentivirus-RNAi-TIPE2 attenuated the suppressive effect of CD4+T lymphocytes, which was associated with profound elevation of nuclear factor of activated T cell (NF-AT) activity. These results demonstrate that TIPE2 appear to be involved in the immune regulation of CD4+ T lymphocytes, and the decrease in TIPE2 expression on CD4+T lymphocytes in vivo can enhance peripheral T lymphocyte function after thermal injury. These data might provide a valid strategy to prevent the development of immunosuppressive state resulted from major burns.

Effects of hydrodynamics-mediated RNAi on Mfn2 expression, blood sugar and fat levels in mice

To investigate the effects of hydrodynamics-mediated RNAi for Mfn2 gene expression in liver and the levels of blood sugar and fat in mice. Fifty-six male BALB/c mice were randomly divided into normal control group (NC, n = 8), negative control group (HK, n = 24) and transfection group (Mfn2, n = 24) according to random digits table. 1.5 ml plasmid (negative control or Mfn2 shRNA, 75mug for each mouse) diluted into phosphate buffered solution (PBS) was injected into the HK and Mfn2 groups mice via hydrodynamic intravascular injection. Mfn2 mRNA and protein expression in hepatic tissue was detected by RT-PCR and Western-blot 24 hours, 72 hours and 120 hours respectively after injection. At the same time, the levels of fasted blood sugar (FBS) and triglyceride (TG) were measured. Compared with HK mice, the expressions of Mfn2 mRNA (1.00+/-0.03 vs 1.14+/-0.07, t = 4.027, P = 0.007; 1.01+/-0.053 vs 1.18+/-0.07, t = 4.234, P = 0.006) and protein (7.81+/-0.80 vs 8.01+/-0.08, t = 2.941, P = 0.042; 8.05+/-0.15 vs 8.56+/-0.014, t = 4.883, P = 0.039) decreased markedly in Mfn2 mice in 72 and 120 hours after injection. In the fasting state, in 24 hours after injection, FBS in Mfn2 group was significantly lower than that in HK group [(2.65+/-0.70 vs 5.28+/-0.82) mmol/L, t = 6.879, P value less than 0.01] and TG was also significantly higher than that in HK group [(1.96+/-0.32 vs 1.12+/-0.16) mmol/L, t = -6.711, P value less than 0.01]. No statistical differences found between the NC and HK groups for FBS and TG (F = 1.412, P = 0.26; F = 2.711, P = 0.14). The plasma glucose level in Mfn2 mice was significantly higher than that in HK mice [(7.23+/-0.82 vs 5.18+/-0.69) mmol/L, t = 2.050, P value less than 0.01; (7.00+/-0.67 vs 6.05+/-0.76) mmol/L, t = 3.57, P = 0.023] in 72 and 120 hours after injection. However, no differences found between the two groups for blood TG [(1.53+/-0.27 vs 1.37+/-0.18) mmol/L, t = 0.160, P = 0.23; (1.84+/-0.30 vs 1.52+/-0.37) mmol/L, t = 0.330, P = 0.503]. The data indicate that hydrodynamics- mediated RNAi for Mfn2 gene can effectively inhibit the expression of target gene in mice liver in 72 and 120 hours after shRNA administration, and the inhibition of hepatic Mfn2 can induce glycometabolic and fat metabolic disorder.

Effects of apoptosis protein XIAP inhibitor on the apoptosis and sensitivity of chemotherapy in A549 cell.

X-linked inhibitor of apoptosis protein (XIAP) is a newly discovered inhibitor of apoptosis protein which prevents apoptosis by inhibiting the activation of caspase. After down-regulating XIAP gene expression in A549 cells, a non-small cell lung cancer (NSCLC) cell lines, we investigated the role of XIAP specific siRNA in apoptosis and chemotherapy sensitivity. The mRNA levels of XIAP gene in A549 cells were assessed using a semi-quantitative reverse transcriptase-PCR (RT-PCR). The expression vector of XIAP small interfering RNA (XIAP siRNAwas constructed and transfected into A549 cells. The transfection was proved effective by the fluorescence microscope. Cell proliferation and cell killing rate after chemotherapeutics treatment were investigated by MTT assay. The rate of apoptosis was detected by flow cytometry assay. XIAP siRNA construction was proved successful by enzyme digestion and DNA sequencing. The transfection efficiency in A549 cells from positive transfection group and negative transfection group had no differences. Compared to those in cell from control group, the level of XIAP mRNA expression was significantly decreased, the inhibition activity of Cisplatin was significantly higher in cells from positive transfection group. Proliferation of cells from positive transfection group was significantly inhibited after 24, 48, 72, 96 hours . The rates cell killing and apoptosis in cells from positive transfection group caused by Cisplatin were significantly higher compared to those cells from control group. The increased expression of XIAP in NSCLC can inhibit the apoptosis of NSCLC cells and result in NSCLC chemotherapy drug resistance. XIAP siRNA could inhibit the NSCLC cell growth specifically, down-regulation of XIAP gene expression promote apoptosis and increase the chemotherapy sensitivity of NSCLC. XIAP siRNA sequence might become a therapeutic target of NSCLC.

Effects on human leukocyte antigen-G on proliferation and invasion of JEG-3 cell line

To investigate the effect of human leukocyte antigen-G (HLA-G) on the growth and invasion of JEG-3 cell line and the role of HLA-G in the onset and development of pre-eclampsia. The experiment was composed of three groups: groups of transfection, negative control and blank control, which corresponded to groups of HLA-G siRNA transfection, negative siRNA transfection and no transfection. HLA-G overexpressed choriocarcinoma cell line JEG-3 was used. The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA (siRNA). Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and western blot analysis. Changes of cell cycle, apoptosis, proliferation and invasion were respectively detected by methyl thiazolyl tetrazolium (MTT), flow cytometry assay and transwell test. (1) The mRNA and protein levels of HLA-G control group and blank control group were 0.0013 +/- 0.0014, 0.0163 +/- 0.0007 and 0.1923 +/- 0.0384, 0.2184 +/- 0.0153, respectively, which were both significantly different (P<0.05); the number of negative transfection group was 0.1606 +/- 0.0133 and 0.2020 +/- 0.0132, which had no significant difference compared with blank control group (P>0.05). (2) The integral absorbance (IA) values of the HLA-G transfection group and blank control group were 0.44 +/- 0.04 and 0.75 +/- 0.13 respectively, which was significantly different (P<0.01); the IA value of negative control group was 0.69 +/- 0.10, which was not significantly different compared with blank group (P>0.05). (3) The ratios of G2/M and S phase cells in transfection group were (10.9 +/- 2.2)% and (58.6 +/- 0.8)% respectively, significantly different compared with the blank control group [(15.4 +/- 1.9)% and (52.9 +/- 2.3)% respectively; P<0.01]. (4) The ratio of early apoptosis cells in transfection group [(14.5 +/- 2.7)%] was significantly increased compared with negative [(5.3 +/- 1.1)%] and blank control group [(4.7 +/- 0.6)% ; P<0.01]. (5) The invasion number of transfection group and blank control group were 121+/- 12 and 452 +/- 17 respectively, with a significant difference between them (P<0.01). HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.