[Systematic review and Meta analysis on the effectiveness and safety of tuina in treatment of functional constipation].

OBJECTIVE: To review systematically the effectiveness and safety of tuina (Chinese massage)in treatment of functional constipation. METHODS: The articles on functional constipation treated with tuina were collected by computer retrieval from 7 databases from the date of establishment to March 28, 2020, including Chinese biomedical literature database (SinoMed), China journal full-text database (CNKI), full-text database of Wanfang academic journals (Wanfang), VIP Chinese science and technology journal database(VIP), PubMed, Dutch medical literature database (EMbase) and the Cochrane Library. After data extraction and quality evaluation of the included articles, Meta analysis was conducted with RevMan5.3 software. RESULTS: A total of 16 articles were included, with 1424 cases involved. Meta analysis results showed: ①The total effective rate in the treatment group was higher than that in the control group (RR=1.28, 95%CI: 1.16-1.42, P<0.000 01). ②The effective rate for the symptoms of functional constipation in traditional Chinese medicine in the treatment group was higher than that in the control group (RR=1.38, 95%CI :1.25-1.52, Z=6.31, P<0.000 01). ③Adverse reactions in the treatment group in the treatment of functional constipation were less than those in the control group (RR=0.10, 95%CI: 0.02-0.49, Z=2.81, P=0.005).④The effective rate of functional constipation treated on the base of syndrome differentiation in the treatment group was higher than that of the control group (RR=1.50, 95%CI: 1.08-2.10, Z=2.39, P=0.02).⑤The improvements in fecal characteristics, defecation time and defecation frequency of the patients with functional constipation in the treatment group were better than those in the control group (P<0.05). CONCLUSION: Tuina therapy presents a certain advantages on its curative effect on functional constipation, has less adverse reactions and relieves the relevant symptoms of functional constipation. But more randomized controlled trials with high quality and large sample are required to provide further verification of its effect.

Quantitative Proteomics Identify the Possible Tumor Suppressive Role of Protease-Activated Receptor-4 in Esophageal Squamous Cell Carcinoma Cells.

Exposure to carcinogens of tobacco smoke may result in methylation of protease-activated receptors-4 (PAR4) gene and further induces the loss of PAR4 expression, which is considered to be involved in carcinogenesis of esophageal squamous cell carcinoma (ESCC). Here we employed a TMT-based quantitative proteomic approach to identify PAR4-regulated changes of proteomic profiles in ESCC cells and to identify potentially therapeutic value. A total of 33 proteins were found significantly changed with 15 up-regulated and 18 down-regulated in PAR4-activating peptide (PAR4-AP) treated ESCC cells compared with controls. Bioinformatics analysis showed that key higher expressed proteins included those associated with apoptosis and tumor suppressor (e.g. CASP9), and lower expressed proteins included those associated with anti-apoptosis, autophagy and promoting cell proliferation (e.g. CHMP1B, PURA, PARG and HIST1H2AH). Western blot verified changes in five representative proteins including CASP9, CHMP1B, PURA, PARG and HIST1H2AH. Immunohistochemistry analysis showed that CHMP1B, PURA, PARG and HIST1H2AH expression in ESCC tissues were significantly higher than those in adjacent nontumorous tissues. Our findings will be helpful in further investigations into the functions and molecular mechanisms of PAR4 in ESCC.

Activation of PAR4 Upregulates p16 through Inhibition of DNMT1 and HDAC2 Expression via MAPK Signals in Esophageal Squamous Cell Carcinoma Cells.

A previous study showed that a downexpression of protease-activated receptor 4 (PAR4) is associated with the development of esophageal squamous cell carcinoma (ESCC). In this study, we explored the relationship between PAR4 activation and the expression of p16, and elucidated the underlying mechanisms in PAR4 inducing the tumor suppressor role in ESCC. ESCC cell lines (EC109 and TE-1) were treated with PAR4-activating peptide (PAR4-AP). Immunohistochemistry for DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) was performed in 26 cases of ESCC tissues. We found that DNMT1 and HDAC2 immunoreactivities in ESCC were significantly higher than those in adjacent noncancerous tissues. PAR4 activation could suppress DNMT1 and HDAC2, as well as increase p16 expressions, whereas silencing PAR4 dramatically increased HDAC2 and DNMT1, as well as reduced p16 expressions. Importantly, the chromatin immunoprecipitation-PCR (ChIP-PCR) data indicated that treatment of ESCC cells with PAR4-AP remarkably suppressed DNMT1 and HDAC2 enrichments on the p16 promoter. Furthermore, we demonstrated that activation of PAR4 resulted in an increase of p38/ERK phosphorylation and activators for p38/ERK enhanced the effect of PAR4 activation on HDAC2, DNMT1, and p16 expressions, whereas p38/ERK inhibitors reversed these effects. Moreover, we found that activation of PAR4 in ESCC cells significantly inhibited cell proliferation and induced apoptosis. These findings suggest that PAR4 plays a potential tumor suppressor role in ESCC cells and represents a potential therapeutic target of this disease.

MiR-650 represses high-risk non-metastatic colorectal cancer progression via inhibition of AKT2/GSK3ß/E-cadherin pathway.

Although 5-year survival rate of non-metastatic colorectal cancer (CRC) is high, about 10% of patients in stage I and II still develop into metastatic CRC and eventually die after resection. Currently, there is no effective biomarker for predicting the prognosis of non-metastatic CRC in clinical practice. In this study, we identified miR-650 as a biomarker for prognosis prediction. We observed that the expression of miR-650 in tumor tissues had a positive association with overall survival. MiR-650 inhibited cell growth and invasion in vitro and in vivo. Furthermore, miR-650 targeted AKT2 and repressed the activation of the AKT pathway (AKT2/GSK3ß/E-cadherin). Thus it induced the translocation of E-cadherin and ß-catenin in cancer cells. Our results highlight the potential of miR-650 as a prognostic prediction biomarker and therapeutic target in non-metastatic CRC via inhibition of the AKT2/GSK3ß/E-cadherin pathway.

Hepatitis B virus X protein influences enrichment profiles of H3K9me3 on promoter regions in human hepatoma cell lines.

We previously showed that hepatitis B virus (HBV) X protein (HBx) could promote the trimethylation of histone H3 lysine 9 (H3K9me3) to repress tumor suppressor genes in hepatocellular carcinoma (HCC). In this work, we analyze 23,148 human promoters using ChIP-chip to determine the effects of HBx on H3K9me3 enrichments in hepatoma cells with transfection of HBx-expressing plasmid. Immunohistochemistry for HBx and H3K9me3 was performed in 21 cases of HBV-associated HCC tissues. We identified that H3K9me3 immunoreactivity was significantly correlated with HBx staining in HCC tissues. ChIP-chip data indicated that HBx remarkably altered promoter enrichments of H3K9me3 in hepatoma cells. We identified 25 gene promoters, whose H3K9me3 enrichments are significantly altered in hepatoma cells transfected HBx-expressing plasmid, including 19 gaining H3K9m3, and six losing this mark. Most of these genes have not been previously reported in HCC, and BTBD17, MIR6089, ZNF205-AS1 and ZP1 have not previously been linked to cancer; only two genes (DAB2IP and ZNF185) have been reported in HCC. Genomic analyses suggested that genes with the differential H3K9me3 enrichments function in diverse cellular pathways and many are involved in cancer development and progression.

Clonality assessment of adenomatoid tumor supports its neoplastic nature.

Adenomatoid tumor is a relatively rare disease that predominantly involves male and female internal genital tracts. Although its clinical and pathologic features are well characterized, there is still controversy regarding its nature as a true neoplasm or a variant of mesothelial hyperplasia of a reactive nature. We sought to resolve this debate by investigating the clonality of uterine adenomatoid tumor from 13 female cases. The mesothelial cells and surrounding normal myometrium were precisely harvested using laser capture microdissection, and genomic DNA was extracted for clonal analysis by assessing the patterns of X-chromosome inactivation. Fluorescent polymerase chain reaction amplification of a highly polymorphic short tandem repeat of the human androgen receptor (HUMARA) gene with and without methylation-sensitive restriction endonuclease HpaII digestion was performed on DNA extracted from mesothelial cells, using normal myometrium and male blood sample as controls. Of the 13 cases successfully amplified, all 10 informative cases showed concordant nonrandom X-chromosome inactivation pattern consistent with monoclonality. In comparison, surrounding normal myometrium showed a polyclonal pattern of X-chromosome inactivation, and male blood sample failed to be amplified after HpaII treatment. Our results demonstrate that adenomatoid tumor is a monoclonal disease favoring a neoplastic process. This neoplastic rather than reactive nature probably accounts for its frequently observed infiltrative growth pattern and the occurrence of diffuse adenomatoid tumor, especially when host immunity is compromised. Additional studies with larger sample sizes will be needed to conclusively prove our conclusion.

Chemokine Expression Profiles of Human Hepatoma Cell Lines Mediated by Hepatitis B Virus X Protein.

The hepatitis B virus X protein (HBx), which is encoded by hepatitis B virus (HBV), plays crucial roles in the tumorigenesis of HBV associated hepatocellular carcinoma (HCC). Recent studies suggest that the HBx is involved in regulation of host immune cytokines and chemokines in HBV-associated HCC patients. However, effects of the HBx on autocrine chemokine expression profiles of hepatoma cells, which were shown in modulation of tumor-immune cell interactions, have not been investigated comprehensively. In the present study, human hepatoma cell lines SMMC-7721 and HepG2 were transfected with HBx-expressing plasmid. Human chemokine antibody array 1 (RayBio®), which simultaneously detects 38 chemokine factors, was used to determine chemokine expression profiles. Real-time polymerase chain reaction (real-time PCR) was used to further confirm the differential expression of chemokines. Chemokine antibody array revealed that all 38 chomekines were found to be expressed by SMMC-7721 and HepG2 cell lines. Interleukin-8 (IL-8) was obviously up-regulated, and epithelial neutrophil-activating protein 78 (ENA78), eosinophil chemotactic protein-1 (Eotaxin-1), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and macrophage inflammatory protein-3ß (MIP-3ß) were significantly declined in both cell lines following transfection of HBx-expressing plasmid. Other chemokines showed little or no significant changes. HBx-induced differential chemokine expression levels were validated by real-time PCR. Hierarchical cluster analysis identified a distinction of chomekine expression profiles between HBX-expressing hepatoma cell lines and controls. Our findings provide new evidence that HBx is able to selectively regulate chomekines in hepatoma cells that may be involved in the regulation of tumor-immune cell interactions.

Hepatitis B virus X protein induces the histone H3 lysine 9 trimethylation on the promoter of p16 gene in hepatocarcinogenesis.

Our previous study showed hepatitis B virus X protein (HBx) suppresses the p16 expression in hepatocarcinogenesis. In this study we explored the relationship between HBx and trimethylation of H3K9 (H3K9me3), and elucidated the underlying mechanisms in HBx inducing the tumor suppressor p16 gene silence. SMMC-7721 and HepG2 hepatoma cell lines were transfected with HBx-expressing plasmid. Immunohistochemistry, Western blotting and real-time polymerase chain reaction, were performed to detect the expressions of HBx, H3K9me3, and jumonji domain-containing protein 2B (JMJd2B). H3K9me3 enrichment on the p16 promoter was measured by immunoprecipitation-PCR (ChIP-PCR) analyses, and 39 cases of hepatitis B virus (HBV) associated-hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues were also examined. We demonstrated that HBx was able to upregulate H3K9me3 and suppress JMJd2B mRNA and protein levels in SMMC-7721 and HepG2 hepatoma cell lines. JMJd2B, as a specific target of H3K9me3 for demethylation, was inversely correlated with the levels of H3K9me3 in SMMC-7721 (r=-0.666, P<0.05) and HepG2 cells (r=-0.625, P<0.05). The ChIP-PCR data indicated that HBx remarkably increased H3K9me3 on the p16 promoter region. Immunohistochemistry analysis showed that H3K9me3 expression in HBx positive HCC samples were significantly higher than that in HBx negative HCC tissues and were associated with decreased levels of JMJd2B expression. JMJd2B immunoreactivity was also remarkably inversed to that of HBx in HCC tissues (r=-0.630, P<0.05). Our results provide evidence that HBx is able to induce H3K9me3 on the p16 promoter via the decrease of demethylase JMJd2B expression and thus promote the repression of p16 gene expression to enhance hepatocarcinogenesis.