LncRNA UNC5B-AS1 promotes proliferation and epithelial-mesenchymal transition of cervical cancer cells by regulating Toll-like receptor signaling pathway / 中国药理学通报

Aim To investigate the effects of long non-coding RNA(lncRNA)UNC5B-AS1 on the proliferation and epithelial mesenchymal transformation(EMT)of cervical cancer. Methods GEO and TCGA databases were used to download data sets and differential expression analysis was performed. qRT-PCR was used to verify the differential expression of lncRNA UNC5B-AS1 in normal and cancerous cervical tissues.The interference and overexpression of lncRNA UNC5B-AS1 were transfected into cervical cancer cell lines, and plate cloning, CCK-8 and EdU experiments were used to detect the effect of lncRNA UNC5B-AS1 on the pro-liferation of cervical cancer cells.Transwell assay was used to detect its effect on migration and invasion of cervical cancer cells.The expression levels of EMT-related genes E-Cadherin, N-Cadherin and Vimentin were detected by Western blot. Transcriptome sequencing was used to obtain the signal pathway regulated by lncRNA UNC5B-AS1, and to verify the expression level of related genes. Results RNA microarray and bioinformatics analysis showed that the expression level of lncRNA UNC5B-AS1 in cervical cancer was significantly higher than that in normal cervical tissue, and correlated with the overall survival time of patients.Compared with the negative control group, knockdown lncRNA UNC5B-AS1 could reduce the proliferation, migration and invasion of cervical cancer cells, while overexpression could promote the proliferation, migration and invasion of cervical cancer cells. Western blot showed that lncRNA UNC5B-AS1 could regulate EMT of cervical cancer cells. Transcriptome sequencing showed that lncRNA UNC5B-AS1 could regulate Toll like receptor(TLR)signaling pathway. qRT-PCR and Western blot results showed that the expression levels of TLR-related genes IL-6 and TICAM2 in the knockdown and overexpression lncRNA UNC5B-AS1 group were significantly changed(P<0.05). Conclusions LncRNA UNC5B-AS1 is highly expressed in cervical cancer. Overexpression of lncRNA UNC5B-AS1 may enhance TLR signaling pathway activity, thereby promoting proliferation and EMT of cervical cancer cells.

Promoting the formation of Pi-stacking interaction to improve CTL cells activation between modified peptide and HLA.

OBJECTIVE: This study aims to investigate the use of single residue substitution to promote the formation of pi-stacking interactions between peptides and Human leukocyte antigen (HLA)-A*2402 molecules to improve the affinity of peptides and HLA molecules, as well as the level of cytotoxic T lymphocyte (CTL) cells activated by peptides-HLA (p-HLA) complex. METHODS: Molecular docking and molecular dynamics simulation were used to simulate and analyze the interactions and binding free energies between HLA-A*2402-restricted antigen peptides and HLA molecules, before and after the single residue substitution. HLA-A*2402 restricted antigen peptides before and after the single residue replacement were loaded into dendritic cells (DCs) in vitro, and further Enzyme-Linked ImmunoSpot (ELispot) test was carried out to evaluate the effect of modified antigen peptides on the immune activation of CTL cells. RESULT: After replacing the antigen peptides with a single residue, some of them could promote the formation of pi-stacking interaction. The binding free energy between the modified antigen peptides and HLA-A*2402, as well as the level of immune activation of CTL cells were mostly higher than before, especially after the replacement of the 9th residue of the polypeptide, such as C9F and C9W. There was a significant negative correlation between the level of activated CTL cells by modified antigen peptides and the total interaction amount of hydrogen bonds and salt bridges. CONCLUSION: Promoting the formation of pi-stacking interaction between antigen peptides and HLA-A*2402 molecules could increase the total binding free energy of p-HLA complex and the level of CTL cells activation. In addition, the amount of hydrogen bonds and salt bridges between peptides and HLA could reduce the level of immune activation. All the characteristics above can improve the immunogenicities of the weak antigens.

Coexistence of submandibular epithelioid angiosarcoma and papillary thyroid carcinoma: A case report.

INTRODUCTION: Reports on the coexistence of epithelioid angiosarcoma (EA) and papillary thyroid carcinoma (PTC) are rare. Over the past 50 years, only 2 cases of coexistence of EA and PTC have been reported in English literature. Therefore, we report a rare case of coexistence of EA and PTC treated with surgery and adjuvant radiation therapy. PATIENT CONCERNS: A 64-year-old man visited our hospital with a painless mass in the left submandibular gland, with poor mobility. DIAGNOSIS: Neck ultrasonography revealed nodules in the left submandibular gland and multiple cystic-solid mixed nodules in the left thyroid gland. Pathological findings revealed coexistence of EA in the left submandibular gland area and PTC in the left thyroid gland. INTERVENTIONS: The patient underwent resection of the left submandibular gland, deep maxillofacial tumor, total thyroidectomy, left neck I, II, III, and VI regional lymph node dissection, and recurrent laryngeal nerve exploration under general anesthesia. Two months postoperatively, the patient also received adjuvant radiation therapy in the local and adjacent areas, with 4MV-X IMRT DT50GY at 2Gy/day 25 fractions. OUTCOMES: The follow-up period was 37 months. The patient recovered well without focal neurological deficits, local recurrence, or distant metastasis after surgery, except for grade I skin reaction after adjuvant radiation therapy. CONCLUSIONS: This is a rare case report of the coexistence of EA in the left submandibular gland and PTC in the left thyroid gland. Although multiple examinations were used, precise preoperative diagnosis was challenging owing to the coexistence of EA and PTC. Surgery and radiotherapy were effective treatments for the coexistence of EA and PTC in this case.

Integrating 16S sequencing and metabolomics study on anti-rheumatic mechanisms against collagen-induced arthritis of Wantong Jingu Tablet / 中国天然药物

Wantong Jingu Tablet (WJT), a mixture of traditional Chinese medicine, was reported to relieve the symptoms of rheumatoid arthritis (RA), but its pharmacological mechanism was not completely understood. The aim of this study was to investigate the therapeutic mechanisms of WJT for RA in vivo. The effects of WJT on joint pathology, as well as the levels of Bax, Bcl-2, caspase-3, cleaved-caspase-3, ERK1/2, pERK1/2, TNF-α, IL-1β, and IL-6 were measured using collagen-induced arthritis (CIA) rats. The intestinal flora composition and the metabolites alteration were analyzed by 16S rDNA sequencing and metabolomics method, respectively. We found that WJT ameliorated the severity of the CIA rats which might be mediated by inducing apoptosis, inactivating the MEK/ERK signals and reducing the production of pro-inflammatory cytokines. WJT, in part, relieved the gut microbiota dysbiosis, especially bacterial phylum Bacteroidetes, Tenericutes and Deferribacteres, as well as bacterial genus Vibrio, Macrococcus and Vagococcus. 3'-N-debenzoyl-2'-deoxytaxol, tubulysin B, and magnoline were significantly associated with the specific genera. We identified serotonin, glutathione disulfide, N-acetylneuraminic acid, naphthalene and thromboxane B2 as targeted molecules via metabolomics. Our findings contributed to the understanding of RA pathogenesis, and WJT played essential roles in gut microbiota health and metabolite modulation in the CIA rats.

The Role, Function, and Mechanism of Long Intergenic Noncoding RNA1184 (linc01184) in Colorectal Cancer.

BACKGROUND: Long intergenic noncoding RNA1184 (linc01184) has been recently discovered; however, its role in human diseases is limited to date. The present study is aimed at investigating the expression pattern and mechanism of linc01184 in colorectal cancer (CRC) tumorigenesis. METHODS: The expression of linc01184 in CRC tissues and cell lines was compared with that in normal controls. The functions of linc01184 in CRC cells were identified by overexpression and small interfering RNA (siRNA) approaches in vitro. Meanwhile, the target gene prediction software, luciferase reporter, RNA pull-down, and western blotting assays were used to analyze the oncogenic mechanism. RESULTS: We found that linc01184 was obviously upregulated in CRC tissues and cells when compared to normal controls, and its upregulation had a positive association with the CRC progression. linc01184 knockdown significantly suppressed CRC cell proliferation and invasion and promoted apoptosis. Besides, linc01184 acted as a competitive endogenous RNA (ceRNA) by directly binding to microRNA-331 (miR-331), and its overexpression resulted in notable increases of human epidermal growth factor receptor 2 (HER2), phosphorylated Ser/Thr kinases (p-Akt), and extracellular regulated protein kinase 1/2 (p-ERK1/2) at posttranscriptional levels in CRC cells, which were antagonized by miR-331. CONCLUSIONS: The findings reveal for the first time that linc01184 is an enhancer for the proliferation and invasion of CRC by functioning as a ceRNA through the linc01184-miR-331-HER2-p-Akt/ERK1/2 pathway regulatory network.

Palliative whole-brain radiotherapy and health- related quality of life for patients with brain metastasis in cancer.

OBJECTIVE: To assess the use of palliative whole-brain radiotherapy (WBRT) in the treatment of brain metastases (BMs) and to evaluate the health-related quality of life (HRQOL) of these patients. MATERIALS AND METHODS: We conducted a retrospective study of 46 patients with BMs who were treated with WBRT at the First Affiliated Hospital of Xi'an Jiaotong University between January 2013 and January 2015. External beam radiotherapy techniques were used to deliver 40 Gy in 20 fractions or 30 Gy in ten fractions with a 10 MV photon beam from a linear accelerator to the whole brain. Data were stored and analyzed using SPSS version 17.0. RESULTS: Of the 46 patients, the survival time of patients in our study was 10.8±0.55 months: 11.8±0.46 months in patients with WBRT, 11.75±1.00 in patients with WBRT + chemotherapy, and 3±0.79 months in patients with supportive care, respectively (P<0.01). The HRQOL scores of all the patients were 70±1.16 (before therapy) and 76.83±1.04 (after therapy) (P<0.01). The HRQOL scores of the patients with WBRT were 72.23±0.88 (before therapy) and 78.49±0.87 (after therapy) (P<0.01). There was no central nervous system toxicity; only two (4.3%) patients were found to have BM hemorrhage. Radiation necrosis happened in one patient (2.2%). CONCLUSION: Effective treatment options for patients with BMs are important. WBRT was evaluated to ensure survival outcomes and QOL were enhanced after therapy for patients with BMs.

Analysis of the characteristics and prognosis of advanced non-small-cell lung cancer in older patients.

OBJECTIVE: Lung cancer is still the leading cause of cancer-related deaths worldwide. However, most elderly patients with advanced non-small-cell lung cancer (NSCLC) have been undertreated and the outcome related to age is controversial. A retrospective analysis was conducted for advanced NSCLC in order to investigate the characteristics and prognosis of older patients. METHODS: Medical records were collected from 165 patients with NSCLC (stages IIIA-IIIB) who had been treated with concurrent chemoradiotherapy (CRT) or radiotherapy from January 2009 to January 2011. The cases were divided into two age groups 1) patients ≥70 years old; 2) patients <70 years old. There were 73 patients in group I, 92 in group II. Patient characteristics, treatment toxicities, and prognosis were evaluated. RESULTS: Of the 165 patients analyzed, 34 patients (34/73) in group I received concurrent CRT while 47 (47/92) in group II completed that treatment. No significant difference was observed in the reason for patients who discontinued CRT in two groups (P>0.05). In the patients with adenocarcinoma, more cases were found in group II than that in group I; the more squamous cell carcinoma and the more smokers with squamous cell carcinoma were seen in older group (P<0.05). With a median follow-up of 20.5 months, the 1-year survival for group I and II were 49.3% and 40.2% respectively (P=0.243). Two-year survival for the two groups was 20.5% and 16.3% (P=0.483); 3-year survival was 9.6% and 9.8% (P=0.967). There was no significant difference between two groups statistically in survival by univariate analysis (P>0.05). The therapy-related toxicities in group I seem to be similar to the group II (P>0.05). CONCLUSION: More adenocarcinoma patients were found in youthful lung cancer and the more smokers with squamous cell carcinoma were seen in older group. Age is not the important factor for the selection and allocation of treatment in advanced NSCLC. The same prognosis and toxicities had been shown in older and young. Age may not be an independent increased risk of death in advanced NSCLC.

Sphingosine kinase 1 as an anticancer therapeutic target.

The development of chemotherapeutic resistance is a major challenge in oncology. Elevated sphingosine kinase 1 (SK1) levels is predictive of a poor prognosis, and SK1 overexpression may confer resistance to chemotherapeutics. The SK/sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor (S1PR) signaling pathway has been implicated in the progression of various cancers and in chemotherapeutic drug resistance. Therefore, SK1 may represent an important target for cancer therapy. Targeting the SK/S1P/S1PR signaling pathway may be an effective anticancer therapeutic strategy, particularly in the context of overcoming drug resistance. This review summarizes our current understanding of the role of SK/S1P/S1PR signaling in cancer and development of SK1 inhibitors.

[Biological behaviour of colon cancer cells transfected with C-erbB2 shRNA plasmid].

AIM: To study the changes of the cell growth, cell cycle distribution and cell apoptosis of colon cancer cell, HT-29, when C-erbB2 gene was knockdown by shRNA against C-erbB2. METHODS: Cell growth, cell cycle distribution and cell apoptosis were compared among three groups including plasmid experimental group(PEG), transfected reagent control group(TRCG)and negative plasmid control group(NPCG). Cell growth was measured by MTT assay. Cell cycle distribution and cell apoptosis were detected by flow cytometry. RESULTS: The inhibition rate of cell growth of PEG, TRCG and NPCG were 39.65%, 7.23% and 8.05% respectively. The cell growth was significantly inhibited in PEG(P<0.01). The cells of G0/G1 phase were 74.93%, 67.19%, 68.05% respectively in PEG, TRCG and NPCG. The cells of G0/G1 phase in PEG were significantly more than those in TRCG and NPCG(P<0.05).While the cells of S phase were 7.81%, 14.02%, 13.70% in PEG, TRCG and NPCG respectively. The cells of S phase in PEG were significantly less than those in TRCG or NPCG(P<0.05).The cell apoptosis rate were 19.21%, 3.13%, 4.08% in PEG, TRCG and NPCG respectively. The cell apoptosis rate in PEG was significantly higher than those in TRCG or NPCG(P<0.01). CONCLUSION: Cell growth was inhibited by shRNA against C-erbB2 gene. Cell cycle was blocked in G0/G1 phase and apoptosis was induced by C-erbB2 shRNA. This indicates C-erbB2 gene plays important roles in the carcinogenesis and development of colon cancer.

RNAi-mediated ERBB2 gene knockdown sensitizes human colorectal cancer cells to radiation.

This study aimed to investigate the involvement of c-erbB-2, encoded by the receptor tyrosine kinase ERBB2 gene, in the pathogenesis of colorectal cancer and to validate its potential as an anticancer target. Immunohistochemical and histopathological analyses were applied in tissue samples derived from 80 colorectal cancer patients. ERBB2 stable small hairpin RNA (shRNA) knockdown in HT29 human colorectal cancer cells was confirmed by RT-PCR and western blotting. Cell cycle profile and apoptosis were measured using PI or Annexin V-PI dual staining. A significant correlation between ERBB2 levels and Dukes' stage of colorectal cancer, in both the primary malignancy and lymph node metastatic tissues, was observed. ERBB2-depleted HT-29 cells exhibited increased sensitivity to radiation compared to control cells, likely due to enhanced G0/G1 phase cell cycle arrest and apoptosis. ERBB2 may be involved in the malignancy and metastasis of colorectal cancer. Overexpressed ERBB2 may constitute a potential target for colorectal cancer therapy.

Sorafenib and octreotide combination therapy can inhibit proliferation of and induce apoptosis in human hepatoma cells / 中华肝脏病杂志

To investigate the effects of sorafenib and octreotide combination treatment on cellular proliferation and explore the underlying molecular mechanisms by using an in vitro cell culture system with the human hepatoma cell line, HepG2. HepG2 cells were treated with different concentrations of sorafenib and octreotide alone or in combination. Untreated HepG2 cells were used as controls. Treatment-induced cytotoxicity was determined with the cell counting kit-8 by Sigma-Aldrich, and rate of apoptosis was detected by flow cytometry. Fluorescent microscopy was used to observe rates of cell growth under the various treatments. Treatment-induced changes in protein expressions were detected by enzyme-linked immunosorbent assay (for vascular endothelial growth factor (VEGF)) and Western blotting (for the Mcl-1 apoptosis mediator and the ERK1/2 and PERK1/2 kinases). Sorafenib and octreotide, used alone or in combination, inhibited proliferation and induced apoptosis in HepG2 cells. Combination treatment was more effective than either mono-treatment (F = 200.398, P less than 0.05). Fluorescent microscopy showed that combination treatment stimulated phosphatidylserine, the marker of early apoptosis, better than either mono-treatment. VEGF expression in cultures exposed to combination treatment was also significantly lower than in mono-treatment or untreated control cultures (F = 1019.725, P less than 0.05). Western blotting showed that octreotide mono-treatment had no effect on Mcl-1 expression (vs. control group; P more than 0.05) and that combination treatment significantly lowered Mcl-1 expression (vs. mono-treatment and control groups; P less than 0.05). None of the treatments affected ERK1/2 expression (all, P more than 0.05), while all treatments significantly lowered PERK1/2 expression (vs. control group; F = 2.401, P less than 0.05) and the combination treatment lowered PERK1/2 significantly more than either mono-treatment (P less than 0.05). Sorafenib and octreotide can inhibit proliferation and induce apoptosis in the human hepatoma cell line, HepG2. Combination treatment is significantly more efficacious (P less than 0.05) and produced synergistic effects. The mechanism underlying this phenomenon may depend on synergistic inhibition of VEGF, the anti-apoptotic protein Mcl-1, and the proliferation-inducing PERK1/2.

[Construction and identification of CerbB-2 siRNA expression plasmid and its transfer into human colon cancer cell lines HT-29].

OBJECTIVE: To construct a plasmid carrying small interfering RNA (siRNA) targeting human C-erbB2 gene (pGenesil- erbB2) and test its effect on Her-2 expression at the post-transcriptional level in human colon cancer cell lines HT-29 cells that highly express erbB2. METHODS: A HT-29 cell line that highly expressed CerbB-2 was selected using immunohistochemical method. The double-stranded siRNA targeting human CerbB-2 cDNA and the negative control fragment were cloned into pGenesil-1 vector, and after identification and sequence analysis, the constructed pGenesil-erbB2 plasmid was transfected into the selected HT-29 cell line. RESULTS: The pGenesil-erbB2 plasmid was successfully constructed and stably transferred into HT-29 cells. The transfection resulted in significant inhibition of Her-2 protein expression in the HT-29 cells, as shown by Western blotting. CONCLUSION: The pGenesil-erbB plasmid we constructed can be stably transfected into HT-29 cells to inhibit the expression of Her-2 protein, and can be useful in further studies of increasing the radiosensitivity of HT-29 cell lines.

[Construction of a new vector for direct cloning of PCR products].

A new method for construction of a cloning vector (T-vector) for direct ligation with PCR products was described. The T-vector derived from pUC118 in which the unique restriction site of Eam1105 I in the region of Amp(r) gene was deleted and an artificial DNA fragment flanking two Eam1105 I was introduced at the site of BamHI. The modified vector was named as pUC118E. A T-vector with 3' over hang end of a single T can be obtained via digesting of pUC118E with Eam1105I. PCR products can be easily cloned with this T-vector.

The study of correlation between anti-cyclic citrnllinated peptide antibody and rheumatoid arthritis / 中华风湿病学杂志

Objective To explore the correlation between anti-cyclic citrullinated peptide(A-CCP) antibody and tumor necrosis factor(TNF)-?, rheumatoid factor(RF), ESR, PLT count and clinical features in patients with rheumatoid arthritis(RA), and the outcome of unclassified arthritis(arthralgia)patients after six months follow up. The value of A-CCP antibdy in the diagnosis of early RA and its pathogenetic roles is in- vestigated. Methods A-CCP antibody and TNF-?were detected by ELISA and the RF was tested by the rate scatting immunity method in 91 RA patients, 46 unclassified arthritis(arthralgia)patients and 45 other rheumatic diseases patients. Results A-CCP antibody levels in serum correlated significantly with TNF-?levels, PLT count and the degree of joint swelling in RA and unclassified arthritis(arthralgia)patients(r= 0.854, P=0.O00; r=0.882, P=0.000; r=0.318, P=0.002; r=0.486, P=0.001; r=0.291, P=0.005; r=0.731, P= 0.000 respectively). A-CCP antibody levels in serum was weakly negatively correlated with the gripping power in RA patients(r=0.228, P=0.030). And it was weakly correlated with ESR in unclassified arthritis(arthrai- gia)patients(r=0.365, P=0.013). Compared with other rheumatic diseases patients, A-CCP antibody levlels in serum increased significantly in RA and unclassified arthritis(arthralgia)patients(P=0.000). Compared with normal controls, it increased in other rheumatic diseases patients(P=0.011). Twenty-four patients had positive A-CCP antibody in 46 unclassified arthritis(arthralgia)patients. Thirty-two out of 46 unclassified arthritis(arthralgia)patients were early RA after 6 monthes follow up. 95.8%(23/24)unclassified arthritis (arthralgia)patients with positive A-CCP antibody were early RA. Conclusion A-CCP antibody reflects disease activity in certain extent. It's benefit to the diagnosis of early RA. High A-CCPantibody levels com- bined with high levels of TNF-?, ESR, PLT count and joint swelling can help the diagnosis of early RA.