Real-time Quantitative Reverse Transcriptase-polymerase Chain Research Articles

High expression of L-type amino acid transporter 1 is associated with colorectal cancer metastasis

Objective To investigate the expression and significance of L-type amino acid transporter 1 (LAT1) in human colorectal cancer. Methods Sixty pairs of colorectal cancer tissues and paracancerous tissues were collected from sixty advanced colorectal cancer (CRC) patients for detecting LAT1 expression. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunohistochemical analysis were used to analyze the expression of LAT1 in colorectal cancer tissues and adjacent normal tissues. In addition, we also study the relationship between LAT1 expression and the clinical features of sixty patients with colorectal cancer. Results Bioinformatics analysis through GEPIA database suggested that LAT1 mRNA expression was elevated in colorectal cancer. We also observed markedly increased LAT1 mRNA and protein expression in colorectal cancer compared with adjacent normal tissues (P<0.01). In addition, we found high expression of LAT1 protein was closely related to lymphatic metastasis (P<0.05), liver metastasis (P<0.05), and TNM stage (P<0.05). Conclusion LAT1 expression is increased in colorectal cancer and asscciated with tumor metastasis. Key words: L-type amino acid transporter 1; Colorectal cancer; Lymphatic metastasis; Liver metastasis; TNM stage

Knockdown of L-type amino acid transporter 1 inhibits the proliferation and migration of colorectal cancer cell line HT29

Objective To investigate the effect of L-type amino acid transporter 1 (LAT1) on colorectal cancer cell proliferation and migration. Methods LAT1-specific small interfering RNA (siLAT1) and control small interfering RNA (siCON) were synthesized. Then, HT29 cell line was selected to generate the colorectal cancer cell model with LAT1 deficiency using small interfering RNA (siRNA) transfection. real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to confirm the efficiency of the transfection after treatment with siRNA for 48 h. After that, cell counting kit-8 (CCK-8) assay and Transwell migration assay were used to examine whether silencing LAT1 with siRNA could affect cell proliferation and migration in HT29 cells, respectively. Results Silencing LAT1 with siRNA could drastically reduce the expression of LAT1 mRNA and the Knockdown efficiency was (73.2±5.2)% (t=11.410, P<0.01). Meanwhile, Western blotting also comfirmed that siLAT1 could reduce protein expression of LAT1 (0.68±0.06 versus 0.21±0.04, t=4.492, P<0.05). Furthermore, CCK-8 assay suggested that knockdown of LAT1 significantly inhibited the viability of HT29 cells treated with siLAT1 versus those treated with siCON (P<0.05). The results of the Transwell migration assay indicated that migration of HT29 cells treated with siLAT1 was drastically reduced compared with those treated with siCON (66±11 versus 156±29, t=5.083, P<0.05). Conclusion Downregulated LAT1 suppressed the ability of colorectal cell proliferation and migration in vitro. Key words: L-type amino acid transporter 1; Colorectal cancer; Small interfering RNA; Proliferation; Migration

The mechanism of action of HOX antisense intergenic RNA in gastric cancer

Objective Investigate the mechanism of HOX antisense intergenic RNA (HOTAIR) in the pathogenesis of gastric cancer. Methods 40 patients with gastric cancer were selected, there were 29 males and 21 females with an average age of (51.7±10.8) years; Ⅰ period 20 cases, Ⅱ period 10 cases, Ⅲ period 6 cases, Ⅳ period 4 cases; 9 cases of high differentiation, 12 cases of middle differentiation and 19 cases of low differentiation. The expression levels of long non-coding RNA (lncRNA) HOTAIR in cancer tissues and adjacent tissues of gastric cancer patients were detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), andanalyzed the correlation between the expression of HOTAIR and the clinical characteristics of gastric cancer patients. lncRNA HOTAIR small interfering RNA (siRNA) were designed and synthesized, and the silencing model was established in the primary SGC-7901 gastric cancer cell line. The expression of lncRNA HOTAIR in primary SGC-7901 gastric cancer cells was detected, add the effects of lncRNA HOTAIR on the proliferation and apoptosis of cancer cellsanalyzed. The molecular regulatory mechanism was studied by Western blotting and RT-qPCR. Results The expression of lncRNA HOTAIR in cancer tissues was (4.367±0.352), higher than that in adjacent tissues (0.984±0.782) (P 0.05). After 48 h of lncRNA HOTAIR siRNA transfection, the ratio of early apoptotic cells [(22.69±3.21)% vs. (10.97±1.52)%], late apoptotic cells [(30.41±3.97)% vs. (7.40±1.71)%] and necrotic cells [(5.53±0.74)% vs. (3.44±0.42)%] was significantly higher than that of untransfected cells (t=5.715, 9.220, 4.254, P<0.05). The relative expression of Wnt3a protein in the cells after transfection was significantly lower than that in the untransfected group (0.39±0.07 vs. 0.92±0.13, t=6.217, P<0.05). Conclusion lncRNA HOTAIR is up-regulated in gastric cancer, while inhibiting HOTAIR expression can inhibit the activation of Wnt/β-catenin signaling pathway by down-regulating Wnt3a protein expression, thereby promoting the apoptosis of gastric cancer cells. Key words: Long non-coding RNA HOX antisense intergenic RNA; Gastric cancer; Apoptosis

Effect of Actinidia root extract RAE on biological behavior of hepatocellular carcinoma cells by microRNA-34-5p

Objective To study the effects of Actinidia root extract on proliferation, apoptosis, migration and invasion of Hepatocellular carcinoma cells. Methods The microRNA (miRNA, miR)-NC group (transfected miR-NC), the miR-34-5p group (transfected miR-34-5p mimics), the RAE + anti-miR-NC group (transfected anti-miR-NC), RAE + anti-miR-34-5p group (transfected anti-miR-34-5p), all were transfected into HCCLM3 cells by liposome method; the expression of miR-34-5p was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR); the protein expression of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax), E-cadherin and membrane palm glycosylated protein-2 (MPP-2) were detected by Western blotting; flow cytometry to detect apoptosis; transwell chamber to detect cell migration and invasion. Results Compared with Con group, Actinidia root extract (RAE) (1, 10, 100 mg/L) significantly inhibited the proliferation of hepatoma cells in a concentration-dependent manner (F=421.057, 625.513, 676.701, 578.243, 880.710 and 206.608, P<0.05), and the optimal concentration was 10 mg/L. It can inhibit the proliferation, migration and invasion of hepatocarcinoma cells, promote apoptosis, down-regulate bcl-2 and MPP-2, up-regulate the expression of miR-34-5p, bax and E-cadherin; overexpression of miR-34-5p there were the same effect of the extract of the Actinidia root extract on the hepatocellular carcinoma cells, inhibition of miR-34-5p can reverse the proliferation, migration, invasion inhibition and apoptosis promotion of the Actinidia root extract hepatocellular carcinoma cells (F=34.118, 286.677, 253.530, 123.103, 103.396 and 109.770, P<0.05). Conclusion Actinidia root extract can inhibit the proliferation, migration, invasion and apoptosis of hepatocellular carcinoma cells, and its mechanism is related to the up-regulation of miR-34-5p. Key words: Actinidia root extract; Hepatocellular carcinoma; MicroRNA-34-5p; Biological behavior

Alternations of Circadian Clock Genes Expression and Oscillation in Obstructive Sleep Apnea.

Circadian misalignment plays an important role in disease processes and can affect disease severity, treatment outcomes, and even survivorship. In this study, we aim to investigate whether expression and daily oscillation patterns of core circadian clock genes were disturbed in patients with obstructive sleep apnea/hypopnea (OSA) syndrome. We performed real-time quantitative reverse transcriptase-polymerase chain reactions to examine the expression of the nine core circadian clock genes in leukocytes of peripheral blood collected at 12 AM, 6 AM, 12 PM, and 6 PM from 133 patients with OSA and 11 normal controls. Daily expression patterns of the nine circadian clock genes were observed in normal controls, but three of these genes (BMAL1, CLOCK, CRY2) were disrupted in patients with OSA. The expressions of eight circadian clock genes (except PER1) at midnight were significantly downregulated in patients with severe OSA. Binary logistic regression analysis selected CRY1 and PER3 as independent factors for severe OSA and showed that the combined expressions of CRY1 and PER3 enhanced the capability of predicting severe OSA (Odds ratio, 5.800; 95% CI, 1.978 to 17.004; p = 0.001). Our results show that combined expressions of CRY1 and PER3 at midnight could be a potential predictor for severe OSA.

EFEMP2 Inhibits Breast Cancer Invasion And Metastasis In Vitro And In Vivo.

BackgroundEGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is an extracellular matrix (ECM) glycoprotein, which is regarded as potential prognostic biomarkers in some carcinoma. Little is known about the association of EFEMP2 and breast cancer.MethodsEFEMP2 expressions in normal breast tissue, benign fibroadenoma, breast cancer, the normal mammary epithelial cell line, and 4 different invasive breast cancer cell lines were evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Expression and prognostic value of EFEMP2 in breast cancer were verified by the Public databases (Oncomine and Kaplan-Meier plotter database). Lentiviral vector with EFEMP2 cDNA was constructed and used to infect breast cancer cell lines to investigate the effects of EFEMP2 on the biological behavior of breast cancer cells by functional in vitro and in vivo assays.ResultsDown-regulated EFEMP2 expression was found in breast cancer tissues and cells, and low expression of EFEMP2 was associated with poor prognosis in patients with breast cancer. Analysis by the Public database leaded to the same conclusion. Up-regulated EFEMP2 expression significantly hampered the invasion and metastasis abilities of breast cancer cells and the process of epithelial interstitial transformation (EMT) via the Wnt/β-catenin pathway.ConclusionEFEMP2 expression was lower in breast cancer and closely related to the prognosis of patients, its anti-oncogenic roles indicated the underlying therapeutic target for the future treatment of breast cancer.

WiNTRLINC1/ASCL2/c-Myc Axis Characteristics of Colon Cancer with Differentiated Histology at Young Onset and Essential for Cell Viability.

WiNTRLINC1 is a long non-coding RNA (lncRNA) that positively regulates the Wnt pathway via achaete-scute complex homolog 2 (ASCL2) in colorectal cancer. ASCL2 was recently reported to play a critical role in chemoresistance, however clinical relevance of the WiNTRLINC1/ASCL2 axis remains obscure in colon cancer. WiNTRLINC1/ASCL2 expression was investigated at messenger RNA (mRNA) level in 40 primary colon cancer tissues and the corresponding normal mucosa tissues, together with Wnt-related genes (c-Myc/PRL-3) and other lncRNAs (H19, HOTAIR, and MALAT1). Knock-down experiments of WiNTRLINC1 clarified its role in their expression and chemoresistance. Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed definite overexpression of WiNTRLINC1 mRNA in primary colon cancer compared with the corresponding normal colon mucosa tissues (p = 0.0005), such as ASCL2, c-Myc, and PRL-3 (p < 0.0001). The four gene expression signatures were tightly associated in the center of the ASCL2 gene (r = 0.72, p < 0.0001) in clinical samples. WiNTRLINC1 was not significantly associated with prognostic factors in colon cancer and other lncRNAs, while the WiNTRLINC1/ASCL2/c-Myc signatures were unique to young-onset colon cancer with differentiated histology. On the other hand, undifferentiated histology was significantly associated with H19 expression. Knockdown of the WiNTRLINC1 gene reduced the expression of ASCL2/c-Myc, but rather augmented PRL-3 at mRNA level, and robustly affected cell viability in colon cancer cell lines. The enhanced WiNTRLINC1/ASCL2/c-Myc axis involved in Wnt pathway activation is a common pathway essential for differentiated colon tumorigenesis, especially with young onset, and may be essential for a viable phenotype of colon cancer.

Analysis and significance of circular RNA expression profile in clear cell renal cell carcinoma

Objective To detect the changes of expression of circular RNA (circRNA) in clear cell renal cell carcinoma (ccRCC) and adjacent renal tissue. Methods Four pairs of speicimen of renal clear cell carcinoma and adjacent renal parenchmal tissues diagnosed by pathology were collected and divided in two groups in the Department of Urology, First Affiliated Hospital of Zhengzhou University in December 2018. High-throughput sequencing of circRNA was done. The study analysed and compared expression profiles of circRNA for the two groups, statistically significant difference (P<0.05) were identified. Then we selected four differentially expressed circRNAs randomly for real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) to verify the sequencing results. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis were done for differential circRNA in two groups. Results 20668 circRNAs were found, main of them derived from the exon region of the parental gene. 578 circRNAs (|log2FC|≥1, P 0.05). GO and KEGG analysis showed that significantly differentially expressed circRNA involved in various biological processes such as ccRCC components, molecular functions, and signaling pathways. Protein interaction network analysis suggested that protein endcoded by the parental gene of differential circRNAs played important role. Conclusion circRNA, its parental gene and related encoded protein play an important role in the occurrence and development of ccRCC. Key words: Circular RNA; Clear cell renal cell carcinoma; Expression profile; Bioinformatics analysis

Effect of macrophage capping protein expression on proliferation and migration of prostate cancer cells

Objective To investigate the expression and explore the biological function of macrophage capping protein (CapG) in prostate cancer (PCa). Methods Immunohistochemistry was used to detect the expression of CapG in 115 cases of prostate cancer and 42 cases of adjacent normal prostate in the Second Affiliated Hospital of Wenzhou Medical University from January 2013 to June 2018. The correlation between CapG expression and clinicopathological features in PCa was evaluated by statistical methods. The expression of CapG in 3 prostate cancer tissues was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. CapG expression was knocked down by small interfering RNA (siRNA), and the effect of CapG on proliferation and migration of human prostate cancer cells was detected by methyl thiazol tetrazolium (MTT) (5 g/L) and Transwell chamber experiments. Results The positive rates of CapG expression in prostate cancer and adjacent normal prostate tissues were 55.6% and 16.7%, respectively, and the difference was statistically significant (χ2=18.879, P<0.05); CapG expression was significantly correlated with prostate specific antigen (PSA) level, Gleason score, T stage and lymph node metastasis of prostate cancer (P<0.05). Down-regulation of CapG significantly inhibited the proliferation and migration of PC3 and DU145 cells (P<0.05). Conclusion CapG is highly expressed in prostate cancer and affects the proliferation and migration of prostate cancer cells. Key words: Prostate cancer; Macrophage capping protein; Proliferation; Metastasis

Knockdown of mediator complex subunit 19 suppresses the proliferation in prostate cancer LNCaP cells

Objective To study the role of mediator complex subunit 19 (Med 19) in prostate cancer by analyzing the effects of lentivirus-mediated suppression of Med 19 expression on LNCaP prostate cancer cells in vitro and explore the mechanism of proliferation. Methods The lentivirus vectors containing small interfering RNA (siRNA) targeting Med 19 gene were constructed and transfected to LNCaP cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to detect the Med 19 expression in the siRNA group (LNCaP-Med 19-siRNA cells)and the control group (LNCaP-Med 19-scRNA cells) at 72 h after the transfection. The influence of Med 19 on the proliferation of prostate cancer cells were assessed using methyl thiazol tetrazolium (MTT), Soft Agar Assay. Cell cycle was analyzed with flow cytometry assay. Results Med 19 relative mRNA level and Med19 protein expressing in the siRNA group were significantly inhibited (t=-69.1413, P<0.01). Compared with the control group, the siRNA group displayed a significant cell proliferation defect by MTT and the number of colonies (8.61±0.51) was significant decreased (t=-11.948, P<0.01; t=-98.775, P<0.01). The siRNA group displayed an increased proportion of cells (67.29±1.70)% in the G0/G1 phase compared with the control group [(56.23±1.46)%, t=-21.906, P<0.01]. Conclusion Knockdown of Med 19 gene increased the number of human prostate cancer LNCaP cells in G0/G1 phase, resulting in significantly inhibited proliferation and growth of prostate cancer. Key words: Mediator complex subunit 19; Prostate carcinoma; Proliferation

Expression and clinical significance of chemokines CXC tchemokine ligand 10 and CXC tchemokine ligand 12 in breast cancer tissues

Objective To research the expression of chemokines CXC tchemokine ligand 10 (CXCL10) and CXC tchemokine ligand 12 (CXCL12) in breast cancer tissues and the correlation with clinicopathological characteristics. Methods The expression of chemokines CXCL10/CXCL12 was detected in 122 breast cancer tissues and tissues adjacent to cancer by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunohistochemistry. The correlation between the expression difference and the clinicopathological characteristics was analyzed. Results Chemokines CXCL10 and CXCL12 mRNA expression was significantly higher in breast cancer tissues than in adjacent tissues (1.10±2.02 vs. 0.46±1.33, t=2.959, P 0.05). The expression of CXCL10 in invasive breast cancer with TNM stage of Ⅲ/Ⅳ or with lymph node metastasis was significantly increased (χ2=12.285, 9.240, P 0.05). Conclusion Chemokines CXCL10 and CXCL12 may be related to the occurrence and development of breast cancer, and CXCL10 may be closely related to lymph node metastasis and malignant progression of breast cancer. Key words: Breast cancer; Chemokines; CXC tchemokine ligand 10; CXC tchemokine ligand 12

Expression and clinical significance of long-chain non-coding RNA-SAP30L-AS1 in prostate cancer

Objective To study the expression and clinical significance of long-chain non-coding RNA-SAP30L-AS1 in prostate cancer. Methods 76 prostate cancer specimens were confirmed by pathology after operation, including 39 cases with prostate diameter ≥2.5 cm, <2.5 cm in 37 cases, 38 cases in low-risk group and 38 cases in high-risk group. The expression of long-chain non-coding RNA-SAP30L-AS1 in 76 patients with prostate cancer was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The difference of expression of long-chain non-coding RNA-SAP30L-AS1 in low-risk and high-risk prostate cancer tissues was analyzed statistically. Results Long-chain non-coding RNA-SAP30L-AS1 was associated with high grade, clinical stage and lymph node metastasis (Gleason score, χ2=4.828, P 0.05). Conclusion Long-chain non-coding RNA-SAP30L-AS1 is highly expressed in high grade and clinical stages, which indicates that it plays an important role in the occurrence and development of prostate cancer. Key words: Prostate cancer; Long-chain non-coding RNA-SAP30L-AS1

Role of P2X7 receptor in the osteogenic differentiation of bone marrow stromal cells induced by electromagnetic fields

Objective The aim of this study was to elucidate the role of P2X7 receptors in the osteogenic differentiation of bone marrow stromal cells (BMSCs) induced by electromagnetic fields (EMF), and the underlying mechanism. Methods BMSCs of 4-week-old male SD rat were isolated and cultured in vitro. The cells at passage 3 were used in this study. To select the most effective frequency EMF, mRNA and protein levels of P2X7 receptoe in BMSCs, which were induced by 1 mT, 7.5-75.0 Hz positive metaphysical EMF for 1 week, were investigated by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. BMSCs cultured in osteogenic medium with or without A740003 (a specific P2X7 antagonist, 5 μmol/L) were stimulated by selected frequency EMF for 1 week, and then expression of runt related transcription factor-2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN) and activation of protein kinase B (Akt)/glycogen synthase kinase 3β (GSK3β)/β-catenin in BMSCs were investigated by Western blotting or RT-qPCR.Measurement data were expressed as (Mean ± SD) and ANOVA analysis of variance was used. Results 1 mT, 15 Hz EMF exhibited the optimal effect on expression of P2X7 receptor, and the relative expression of P2X7 mRNA and protein was 2.0 times and 1.9 times as much as that in control group, respectively. The relative expression of RUNX2, ALP and OPN mRNA in 1 mT, 15 Hz EMF group were 2.1, 5.3 and 3.7 times of that in control group, and the relative expression of RUNX2, ALP and OPN protein was 1.3, 1.6 and 1.5 times of that in control group, respectively. When BMSCs induced by A740003 medium were stimulated by 1 mT, 15 Hz EMF, the relative expression of RUNX2, ALP and OPN mRNA decreased by 36%, 42% and 41% than that in control group, and the relative expression of RUNX2, ALP and OPN protein decreased by 28%, 30% and 26% than that in control group, respectively. Use of A740003 or phosphoinositol 3 kinase (PI3K)/Akt pathway blocker LY294002 (5 μmol/L) alone decreased the levels of phosphorylated Akt and GSK3β induced by EMF, and also decreased the expression of intracellular β-catenin. Conclusion EMF increase the expression of P2X7 of rat BMSCs, which activates Akt/GSK3β/β-catenin axis and finally promotes the osteogenesis. Key words: Electromagnetic fields; Purinergic receptor P2X7; Bone mesenchymal stem cells; Osteogenic differentiation

Serum miR-663 expression and the diagnostic value in colorectal cancer

Colorectal cancer (CRC) is one of the most common digestive cancers leading to deaths worldwide. In this study, we aimed to investigate the diagnostic value of miR-663 in CRC. The expression of miR-663 was detected by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). The association between miR-663 and clinical parameters of subjects was evaluated by chi-square test. Additionally, ROC (receiver operating characteristic) analysis was performed to evaluate the diagnostic role of miR-663 in CRC. The expression of miR-663 in CRC patients was significantly upregulated compared with benign colorectal disease patients and healthy controls (p < .01). Besides, the expression of miR-663 was significantly associated with tumour differentiation, invasion, lymph node metastasis and TNM stage (p < .05). The cutoff value of miR-663 was 1.31, and the corresponding sensitivity and specificity were 83.1% and 73.8%, respectively. In ROC analysis, the area under the curve (AUC) was 0.806, which indicated that miR-663 could act as an independent diagnostic biomarker for CRC. In conclusion, miR-663 was up-regulated in CRC patients and may be an effective biomarker for CRC diagnosis.

Study on the relationship of transforming growth factor-β1 and a disintegrin and metalloproteinase with thrombospondin motif-4 in carotid atherosclerotic plaques

Objective To investigate the relationship between the expression level of transforming growth factor-β1 (TGF-β1) and a disintegrin and metalloproteinase with thrombospondin motif-4 (ADAMTS4) in human carotid atherosclerotic (AS) plaques. Methods Plaque tissues (n=46) were collected after carotid endarterectomy and divided into stable plaque group and unstable plaque group according to the histopathological features by hematoxylin and eosin (HE) staining. Immunohistochemistry (IHC) and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to detect the expression of ADAMTS4 and TGF-β1 in plaques, respectively. Umbilical arteries were served as the control group (n=10) and statistics analysis were conducted as needed. Results IHC results showed that both of ADAMTS4 and TGF-β1 were expressed in all plaques; RT-qPCR results showed that the expression level of ADAMTS4 mRNA was significantly higher in unstable plaque group (4.43±2.02) compared to the stable plaque group (3.16±1.57) and the controls (1.02±0.22, F=17.545, P<0.05); TGF-β1 mRNA in stable plaque group (4.68±1.42) was also higher than that in the unstable plaques (3.57±1.76) and the controls (1.14±0.31), and the difference was statistically significant between groups (F=24.676, P<0.05). Pearson correlation analysis showed that there existed a positive correlation between indexes (r=0.439, P<0.05). Conclusion ADAMTS4 and TGF-β1 were increased together during the process of AS and plaque evolution, which may be related to dose or cell type dependence. Key words: Carotid atherosclerosis; Unstable plaques; Transforming growth factor-β1; A disintegrin and metalloproteinase with thrombospondin motif-4

The expression of microRNA-675 in gastric cancer and its effect on biological behavior

Objective To investigate the association between microRNA5 (miRNA, miR)-675 expression level in tumor tissues and adjacent tissues from patients with gastric cancer and prognosis. Meanwhile, to explore the influence of miR-675 in the gastric carcinoma (SGC-7901) cells. Methods The miRNA data of gastric cancer in TCGA databases were collected, and the survival time was analyzed according to the expression level of miR-675 in tumor tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-675 in 30 gastric tumor tissues and adjacent non-tumor tissues. Cell counting assay kit-8 (CCK-8) was used to analyze the effect of miR-675 on the proliferation of SGC-7901. Flow cyometry was used to analyze the role of miR-675 in the apoptosis of SGC-7901. Results The expression levels of miR-675 in tumor tissues was significantly increased in the patients with gastric carcinoma form TCGA data and clinical samples (3.78±0.10 vs. 2.66±0.23, t=3.236, P<0.01), the difference was statistically significant. More importantly, the medial survival time was lower in the patients with high expression levels of miR-675 than that of lower expression of miR-675 group (675 d vs. 1 294 d, χ2=7.711, P<0.01), the difference was statistically significant. The results of CCK-8 proliferation assay showed that cell proliferation rate was reduced in miR-675 inhibitor transfected cells compared with the control and scrambled RNA-transfected cells (t=3.875, P<0.01), the difference was statistically significant. It was also found that the apoptosis of miR-675 inhibitor transfected cells was significantly increased (18.13±3.39)%. Conclusion MiR-675 may play an important role in the development of gastric cancer. Key words: Gastric cancer; MicroRNA-675; Prognosis

Utility of G protein-coupled receptor 35 expression for predicting outcome in colon cancer

The utility of mRNA and protein determinations of G protein-coupled receptor 35, that is, GPR35a (GPR35 V1) and GPR35b (GPR35 V2/3), as indicators of outcome for colon cancer patients after curative surgery was investigated. Expression levels of V1 and V2/3 GPR35, carcinoembryonic antigen and CXCL17 mRNAs were assessed in primary tumours and regional lymph nodes of 121 colon cancer patients (stage I-IV), colon cancer cell lines and control colon epithelial cells using real-time quantitative reverse transcriptase-polymerase chain reaction. Expression of G protein-coupled receptor 35 was investigated by two-colour immunohistochemistry and immunomorphometry. GPR35 V2/3 mRNA, but not V1 mRNA, was expressed in colon cancer cell lines, primary colon tumours and control colon epithelial cells. Haematoxylin and eosin positive (H&E(+)), but not H&E(-), lymph nodes expressed high levels of GPR35 V2/3 mRNA (P<0.0001). GPR35b and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer tumour cells. Kaplan-Meier and hazard ratio analysis revealed that patients with lymph nodes expressing high levels of GPR35 V2/3 mRNA and, in particular, in the group of patients with lymph nodes also expressing carcinoembryonic antigen mRNA, had a short disease-free survival time, 67 months versus 122 months at 12-year follow-up (difference: 55 months, P = 0.001; hazard ratio: 3.6, P = 0.002). In conclusion, high level expression of G protein-coupled receptor 35 V2/3 mRNA in regional lymph nodes of colon cancer patients is a sign of poor prognosis.

Steroid 5-alpha reductase 1 is negatively regulated by Homeobox gene D10 in prostate cancer LNCaP cells

Objective To investigate the relationship between homeobox gene D10 (HOXD10) and steroid 5-alpha reductase 1 (SRD5A1) and the effect of the former on the latter in prostate cancer cells. Methods Expression of HOXD10 and SRD5A1 and their correlation was analyzed basing on Taylor and TCGA database. Then HOXD10-overexpressing prostate cancer LNCaP cells was constructed, and the effect of HOXD10 on SRD5A1 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. Immunohistochemistry was used to analyze the expression of HOXD10 and SRD5A1 in human prostate cancer tissues.Statisticalanalysis of data using the GraphPad Prism Version 6 software. Results mRNA expression of HOXD10 decreased gradually with the progression of prostate cancer (Taylor: Benign: 8.94±0.09, Primary: 7.93±0.05, Met: 7.66±0.11, F=43.72, P<0.05), while SRD5A1 increased gradually (Taylor: Benign: 7.75±0.02, Primary: 7.79±0.02, Met: 7.98±0.01, F=5.306, P<0.05), and demonstrated a significant negative correlation (TCGA: HOXD10: 80.72±1.60, SRD5A1: 231.60±2.48; r=-0.220, P<0.01). Moreover, the expression of SRD5A1 in prostate cancer cells or patients was significantly reduced in which with higher expression of HOXD10 (Immunohistochemistry: χ2=5.868, P<0.05). (RT-qPCR: overexpressing HOXD10-LNCaP SRD5A1: 0.66±0.02, vector-LNCaP SRD5A1: 0.99±0.00, t=11.31, P<0.01; Western blotting: overexpressing HOXD10-LNCaP SRD5A1: 0.58±0.00, vector-LNCaP SRD5A1: 0.82±0.02, t=11.31, P<0.01). Conclusion HOXD10 negatively regulates SRD5A1 in prostate cancer and may provide a new strategy for endocrine therapy of prostate cancer. Key words: Homeobox gene D10; Steroid 5-alpha reductase 1; Prostate cancer

Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors’ expression and localization, in particular for CB1.

Role of serine-threonine kinase receptor-related protein in the biological function of hepatocellular carcinoma cells

Objective To detect the expression of serine-threonine kinase receptor-related protein (STRAP) in hepatocellular carcinoma (HCC) cells and normal liver tissues, and to explore its biological functions. Methods The relative expression levels of STRAP in 10 HCC cell lines and normal liver tissues were detected, and STRAP overexpression and knockdown cell lines were constructed in HepG2 and SNU449, respectively. The real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to determine the expression of STRAP after the construction of cell lines. The migration and invasion ability of HCC cells was detected by Transwell chamber and Transwell chamber containing Matrigel. Cell proliferation was examined by cell counting kit-8 (CCK-8) and cell clone formation assay, and cell apoptosis was verified by flow cytometry. Results The expression level of STRAP in HCC cell lines was significantly higher than that in normal liver tissues. After STRAP knockdown, the relative expression of mRNA in knockdown group and control group was (0.17±0.19) and (2.22±1.02), respectively. The expression level of the knockdown group was significantly lower than that in the control group (P<0.05), while overexpressing STRAP, the relative expression levels of mRNA in overexpression group and control group were (3.25±0.39) and (1.05±0.37) (P<0.01). After the STRAP gene knockdown, the number of cell clones in control group and knockdown group was (221.30±9.60) and (58.30±4.49) respectively. The number of cells passing through the membrane in control group and knockdown group was (0.76±0.04) and (0.44±0.06) in cell migration assay, and (1.11±0.05) and (0.60±0.05) in cell invasion assay, respectively. In the cell proliferation assay, the number of cell proliferation in the knockdown group decreased as compared with that in the control group at 72, 96 and 120 h. The apoptosis rate in the control group and knockdown group was (4.11±0.12) and (11.73±0.40), respectively. The colony forming ability, migration invasive ability and proliferation ability of SNU449 were significantly inhibited (P<0.05), while the apoptosis was significantly increased (P<0.01). After overexpression of SRTAP gene, the proliferation of HepG2 cells increased at 48, 72, 96 and 120 h as compared with the control group (P<0.001). The cell proliferation assay indicated that the apoptosis rate in the control group and overexpression group was (8.87±0.12) and (8.34±0.06) respectively (P<0.05). Conclusion STRAP is highly expressed in HCC cells, and promotes proliferation, enhances migration and invasion, and inhibits apoptosis of HCC cells. Key words: Carcinoma, hepatocellular; Serine-threonine kinase receptor-related protein; Proliferation; Apoptosis; Metastasis