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Objective:To explore the role of small molecule glycoprotein Serglycin (SRGN) in chemotherapy resistance of non-small cell lung cancer (NSCLC).Methods:In NSCLC H1299 cell line, shRNA technology was used to interfere with the expression of SRGN and establish stable interfering cell line. Western blot and real time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to verify the knockdown efficiency; MTS was used to detect the knockdown cell line′s drug sensitivity to cDDP and Oxaliplatin; enzyme linked immunosorbent assay (ELISA), Western blot and qRT-PCR were used to explore the effect of transforming growth factor β (TGFβ) on SRGN and vice versa; Western blot was used to detect the effect of SRGN on epithelial-mesenchymal transition (EMT) related molecules, and online data bioinformatics was used to analyze the correlation between SRGN and EMT related molecules expression; in addition, online prognostic analysis software (kmplot) was used to analyze the correlation between SRGN, TGFβ and prognosis of lung cancer patients.Results:Comparing with the control group, the test group, knocking down SRGN can obviously improve the drug sensitivity of NSCLC cell to cDDP ( P=0.032 7) or Oxaliplatin ( P=0.014 2). TGFβ can enhance the experission of SRGN in NSCLC and SRGN also can help TGFβ secreted from cells. SRGN promotes the epithelial mesenchyme transition by modulating Snail1. By analyzing TCGA database, we found that the expression of SRGN was negatively correlated with the expression of CDH1 (coding for Ecadherin protein) ( r=-0.25) and there was a positive correlation with Snai1 expression ( r=0.37). These results suggest that SRGN can promote the change of EMT in lung cancer cells through TGF β 1 and snail 1. The overall survival time of NSCLC patients with low expression of SRGN was much longer than the patients with high expression of SRGN ( P=0.007 7). The overall survival time of NSCLC patient with low expression in both SRGN and TGFβ1 or TGFβ2 was 73months or 42.8 months longer than that with high expression in both SRGN and TGFβ1/2. Conclusions:Intercting with TGFβ1, SRGN promotes EMT of NSCLC cells, which facilitates the chemoresistence of NSCLC. The simultaneous low expression of SRGN and TGFβ1 or TGFβ2 can significantly prolong the overall survival of patients with NSCLC.
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Objective To explore the role and mechanism of long non-coding RNA (lncRNA)RP11-316M1.12 in breast cancer cells.Methods Bioinformatics analysis was performed to varify RP11-316M1.12 expression pattern in breast cancer tissues and normal tissues.Quantitative real time polymerase chain reaction (qRT-PCR) assay was used to check the expression level of RP11-316M1.12 in breast cancer cells and tissues.Further,the correlationship between RP11-316M1.12 expression and clinical paremeters of breast cancer was analysed according to RP11-316M1.12 level.RP11-316M1.12 was overexpressed in MCF-7 cells,and RP11-316M1.12 was knocked down in MDA-MB-231 cells.Transwell method was used to detect the invasive ability of these cells.Western blot was used to detect the expression of epithelialmesenchymal transition (EMT) markers in these cells.Results Gene Expression Profiling Interactive Analysis (GEPIA) database suggested that RP11-316M1.12 was highly expressed in breast cancer tissues than that in normal tissues.The similar results were got in 65 cases of breast cancer tissues and 23 cases of normal tissues by qRT-PCR assay.Meanwhile,we found that RP11-316M1.12 was enhanced in breast cancer cells than that in normal epithelial cell and RP11-316M1.12 expression is related to TNM stage and distant metastasis in breast cancer.Transwell assay demonstrated that RP11-316M1.12 significantly enhanced breast cancer cells invasion.Mechanismly,over expression of RP11-316M1.12 can remakably downregulated E-cadherin,enhanced ZEB1 and Vimentin expression in these cells.Conclusions RP11-316M1.12 was enhanced in breast cancer,and RP1 1-316M1.12 could accelerate invasion of breast cancer cells.
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Objective The method for detecting expression of human CDK14 gene with Real-time quantitative PCR was developed.Methods To establish a method for detecting expression of human CDK14 gene with Real-time quantitative PCR by designing and synthesis of the primers of CDK14 target gene andβ-Actin reference gene and extracting total RNA from different lung cancer cell lines.Then the specificity,detection range and repeatability of this method were evaluated.At last,the expression level of CDK14 gene in different cell lines,which were with or without siRNA interference,were carried out by using this method.Results The method for detecting expression of human CDK14 gene with Real-time quantitative PCR,which had good specificity,good repeatability (CV=7.3 ％) and wide detection range (Ct value range of CDK14 and β-Actin amplification curve were 22.47～32.96 and 15.14～ 27.55 respectively,r2 =0.9844),was developed and it was verified by electrophoresis analysis,melting curve,PCR product sequencing.And CDK14 gene expression level,which was detected by this method,increased in HCC827 D5,H1650 and number 1 siRNA segment was effective interference segment.Conclusion The method for detecting expression of human CDK14 gene with Real-time quantitauve PCR was established successfully.
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Objective To explore the role of insulin-like growth factor-2/insulin-like growth factor1 receptor/insulin receptor substrate-1 (IGF2/IGF1R/IRS1) signal pathway inducing the chemoresistance of epidermal growth factor receptor 2 (ErbB2) positive breast cancer cells to Herceptin.Methods Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assay were used to determine the expression levels of IGF2,IGF1 R,and IRS1.The direct targets of miR-126 were validated by dual-luciferase reporter gene assay.In SKBR3/pool2 cells,IGF1 R activity was reduced by an inhibitor of IGF1 R,and IRS1 was knocked-down by shRNAs.Furthermore,3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to evaluate the sensitivity of these treated cells to Herceptin.Results IGF2,IGF1 R,and IRS1 were significantly higher expressed in SKBR3/pool2 cell compared to that in SKBR3 cell.Western blot assay showed that IGF2/IGF1R/IRS1 was activated in SKBR3/pool2 cells.Bioinformatics analysis combined with luciferase activity suggested that miR-126 directly targeted IRS1.MTS results demonstrated that the chemosensitivity to Herceptin of SKBR3/ pool2 cells with inhibitor of IGF1R or shRNAs targeting IRS1 or overexpressing miR-126 was significantly reduced.Conclusions IGF2/IGF1R/IRS1 signal pathway confers to the chemoresistance of ErbB2 positive breast cancer cells to Herceptin.
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] Objective To explore the role and mechanisms of FGF2 in chemo?resistance in breast cancer. Methods The inhibitors for different signal pathway were used to treat two drug?resistant breast cancer cell lines MCF?7/5?Fu and T47D/5?Fu established in our lab. MTS assay was used to determine chemo?sensitivity and Hoechst stain was used to measure apoptosis. Protein activation and FGF2 protein level in cell culture medium were detected by western blot and ELISA respectively. Results Akt inhibitor MK?2206 (20 nM) and mTOR inhibitor AZD8055 (2 nM) significantly reversed the chemo?resistance of MCF?7/5?Fu and T47D/5?Fu cell lines to 5?Fu and paclitaxel, but ERK1/2 inhibitor SCH772984 showed no significant effect. Compared to parent cell lines MCF?7 and T47D, p?Akt and p?S6K (represented as mTORactivity) levels were obviously up?regulated in MCF?7/5?Fu and T47D/5?Fu cell lines, and so do the FGF2 mRNA level and FGF2 protein level from culture medium. Moreover, FGFR inhibitor AZD4547 (4 nM) markedly reversed the chemo?resistance of MCF?7/5?Fu and T47D/5?Fu cell lines to 5?Fu and paclitaxel and down?regulated activation of FGFR?Akt?mTOR signal pathway. In agreement, FGF2 protein (10ng/ml) enhanced the chemo?resistance of MCF?7 and T47D cell lines to 5?Fu and paclitaxel and up?regulated activation of FGFR?Akt?mTOR signal pathway. Conclusion Activation of FGF2?FGFR?Akt?mTOR signal pathway promoted chemo?resistance of breast cancer cells.
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Objective To explore the potential impact of low concentration of metformin on the morphology and function of mitochondria of HepG2 cells. Methods HepG2 cells in experimental group and control group were treated with or without low concentration of metformin (1mM/L), respectively. The cells were incubated for 12h in the incubator with constant temperature and humidity as well as 1% oxygen. Orange Mitoview was used to stain the mitochondria to detect the effects of the drug on its morphology and quantity. Transmission electron microscope was utilized to observe the effect of metformin on the ultrastructure of mitochondria. The mitochondrial respiratory chain complex I activity in HepG2 cell was detected by Complex I Enzyme Activity Dipstick Assay Kit (DAK). Results Orange Mitoview staining showed that low concentration of metformin had little effect on the morphology and number of mitochondria of cells in experimental group , and the difference between control and experimental group was not statistically significant (P > 0.05). In addition, the result was further determined by transmission electron microscopy. However, DAK analysis showed that complex I activity of cells in experimental group was significantly lower than that in control group. Conclusion Under Hypoxia conditions, low concentration of metformin had no significant effect on the morphology and number of mitochondria of HepG2 cells, but it significantly reduces the activity of mitochondria of HepG2 cells.
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[ ABSTRACT] AIM:To explore the expression pattern of microRNA-205 ( miR-205) in glioma tissues and its role in the invasion of glioma cells.METHODS:The expression of miR-205 and TBX18 was detected by real-time PCR and immunohistochemical observation, respectively.Transwell assay was used to examine the invasion change of U251 glioma cells after miR-205 overexpression via miR-205 mimics or decrease in miR-205 expression by miR-205 inhibitor.The target of miR-205 was searched by bioinformatics analysis combined with experimental analysis.The protein level of TBX18 was determined by Western blotting after siRNA transfection and Transwell assay was conducted.RESULTS:miR-205 expres-sion was downregulated in 82.6%of detected glioma tissues and TBX18 was significantly overexpressed in glioma tissues compared with normal tissues.miR-205 overexpression remarkably inhibited the invasion potential of U251 glioma cells with a decrease in the invasive cells (P<0.01), while inhibition of miR-205 significantly enhanced the invasion ability of U251 cells.Mechanically, miR-205 directly targeted TBX18 and downregulation of TBX18 also significantly inhibited the invasion potential of U251 cells with a decrease in the invasive cells ( P<0.01 ) .CONCLUSION: miR-205 expression is de-creased in glioma, and miR-205 inhibits glioma cell invasion via targeting TBX18.Our research contributes to the mecha-nisms responsible for glioma invasion and provides theoretical base for developing new therapeutic strategy to treat glioma.
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ObjectiveTo investigate the characteristics of EGFR gene mutations among NSCLC patients in south of China and analyze the correlation between mutations and clinical features.Methods Specimens of lung cancer tissues were collected from 185 NSCIC patients in our hospital.DNA was extracted from specimens.Exon 18,19,20 and 21 of EGFR gene were amplified by FQ-PCR to be bi-directional sequenced.ResultsEGFR gene mutations in 62 (33.5％) of 185 NSCLC patients was identified in carcinoma tissues,of which,2cases,41cases,5 cases and 14 cases respectively located at exon 18,exon 19,exon 20 and exon 21.The mutation of Del L747 → P752 (P753S) ( proportion 8.1％ ),Del E746 → A750 ( proportion 45.1％ ) at exon 19 and L858R ( proportion 22.6％ ) at exon 21 were the predominant mutation in 16 kinds of mutations.Four cases of mutation at exon 19 got the different results in bi-directional sequencing.The silent mutation 2361G→A at exon 20 was observed (28.1％ ).The mutation rate in women was significantly higher than men (46.2％ vs 24.3％,x2 =9.670,P =0.002).Non-smokers had significantly higher mutation rate than smokers (41.4％ vs 17.1％,x2 =7.380,P =0.007) ; Adenocarcinoma patients had significantly higher mutation rate than squamous cell carcinoma (38.3％ vs 6.3％,x2 =6.426,P =0.011).Clinical stage Ⅲ patients had significantly lower mutation ratethan patients with stage Ⅱ orⅣ ( 10.8％ vs 53.8％,x2 =8.026,P =0.003 ;10.8％ vs 41.3％,x2 =9.518,P =0.002).No statistically significance correlation was found between the mutation ratio and age.ConclusionsEGFR gene mutation has a close relationship with females,non-smokers and adenocarcinoma.Most mutations occur in exon 19 and 20 among patients in south of China.