Knockdown of IGF2BP3 inhibits the tumorigenesis of gallbladder cancer and modifies tumor microenvironment.

Gallbladder cancer (GBC) ranks seventh among the gastrointestinal cancers. Messenger RNAs (mRNAs) could regulate the progression of GBC. For the purpose of exploring the targets for GBC treatment, RNA sequencing was used to identify the differential expressed mRNAs between GBC and adjacent tissues. Next, CCK8 assay was used to assess the cell viability, and cell proliferation was investigated by colony formation assay. Flow cytometry was performed to evaluate the cell apoptosis. Protein and mRNA expression were analyzed by western blot and RT-qPCR, respectively. Transwell was performed to evaluate the cell metastasis. GBC-derived exosomes were isolated with ultracentrifugation. To evaluate the function of exosomes in GBC, in vivo model of GBC was constructed. The data revealed IGF2BP3 was identified to be upregulated in GBC, and IGF2BP3 silencing was able to decrease GBC cell proliferation by promoting the apoptosis. The migration and invasion of GBC cells were reduced by IGF2BP3 knockdown. Silencing of IGF2BP3 obviously suppressed the level of p-STAT3 in GBC cells. Meanwhile, GBC cell-derived exosomes notably promoted macrophage M2 polarization via carrying IGF2BP3, and then the polarized macrophages promoted the malignant behavior of GBC cells. Furthermore, exosomes markedly promoted the tumor growth of GBC via promoting macrophage M2 polarization. In summary, knockdown of IGF2BP3 suppressed the malignant behavior of GBC cells. Additionally, knockdown of IGF2BP3 modified tumor microenvironment during the progression of GBC. Thus, these findings might provide a new theoretical basis for exploring a strategies against GBC.

Knockdown of circSMAD2 inhibits the tumorigenesis of gallbladder cancer through binding with eIF4A3.

BACKGROUND: Gallbladder cancer (GBC) is the seventh most common gastrointestinal cancer worldwide. This study aimed to investigate the function of circSMAD2 in GBC. METHODS: To investigate the function of circSMAD2 in GBC, the level of circSMAD2 in GBC cells was detected by RT-qPCR. CCK-8 assay was performed to investigate the cell viability. Cell apoptosis was tested by flow cytometry. In addition, transwell assay was used to detect the cell migration and invasion. RIP and RNA pull-down were used to explore the relation among circSMAD2, eIF4A3 and SMAD2. Meanwhile, xenograft mice model was established to investigate the function of circSMAD2 in GBC. RESULTS: The data revealed that circSMAD2 was upregulated in GBC, and circSMAD2 knockdown significantly inhibited the viability of GBC cells. In addition, circSMAD2 siRNA notably induced the apoptosis in GBC cells. The migration and invasion of GBC cells were obviously suppressed in the presence of circSMAD2 siRNA. Meanwhile, circSMAD2 suppressed the binding between eukaryotic translation initiation factor 4A3 (eIF4A3) and SMAD2 through binding with eIF4A3. Knockdown of circSMAD2 notably inhibited the expression of SMAD2 in GBC cells, and SMAD2 overexpression partially reversed the anti-tumor effect of circSMAD2 knockdown. Finally, circSMAD2 siRNA significantly inhibited the tumor growth of GBC in vivo. CONCLUSION: Knockdown of circSMAD2 inhibits the tumorigenesis of gallbladder cancer through binding with eIF4A3. Thus, our study provided a new strategy for the treatment of GBC.

SNP rs12982687 affects binding capacity of lncRNA UCA1 with miR-873-5p: involvement in smoking-triggered colorectal cancer progression.

BACKGROUND: This investigation was arranged to elucidate whether single nucleotide polymorphisms (SNPs) of lncRNA UCA1 was implicated in elevating colorectal cancer (CRC) risk by interacting with environmental exposures. METHODS: LncRNASNP database was firstly adopted to predict SNPs that possibly affected binding of UCA1 with miRNAs and then the interactive effect of SNPs and environmental exposure on CRC risk was evaluated by recurring to type 2 gene-environment interactions (GEI) model. Besides, MTT assay, colony formation assay, transwell assay and wound healing assay were performed to assess the activity of CRC cell lines which carried distinct genotypes of specific SNPs. The impact of nicotine on activity of CRC cells was also appraised. RESULTS: SNP rs12982687 of UCA1 intervened in the binding capacity of UCA1 with several miRNAs, especially miR-873-5p. MiRNAs regulated by UCA1, as predicted by mirPath software, shared genes that were enriched in HIF1 signaling pathway. Moreover, homozygote TT of rs12982687 reduced CRC risk among smokers, and CRC cells that carried rs12982687 (CC) displayed strong migration and invasion. By contrast, miR-873-5p mimic, which reduced UCA1 expression, delayed metastasis of CRC cells (all P < 0.05). Additionally, nicotine not merely elevated UCA1 and HIF-1α expressions in CRC cells, but also facilitated proliferation and metastasis of CRC cells (P < 0.05). CONCLUSIONS: SNP rs12982687 was involved in smoking-triggered CRC progression, given its influence on UCA1's binding with miR-873-5p and HIF-1 signaling.

Small nucleolar RNA host gene 1 promotes development and progression of colorectal cancer through negative regulation of miR-137.

Small nucleolar RNA host gene 1 (SNHG1) is critical in the progression of cancers. However, the mechanism by which SNHG1 regulates the progression of colorectal cancer (CRC) remains unclear. Expressions of SNHG1 and miR-137 in CRC tissues and cell lines were evaluated by quantitative real-time polymerase chain reaction. A luciferase reporter gene assay was conducted to investigate miR-137 target. Additionally, RNA pull-down assay was performed to explore the physical association between miR-137, SNHG1, and RNA induced silencing complex (RISC). Cell cycling and invasion were examined by flow cytometry (FCM) and transwell assays. The in vivo carcinogenic activity of SNHG1 was examined using murine xenograft models. Expression of RICTOR, serine/threonine kinase 1 (AKT), serum and glucocorticoid-inducible kinase 1 (SGK1), p70S6K1, and LC3II/LC3I ratio was examined by Western blot analysis. SNHG1 upregulation was observed in CRC tissues and cell lines, which was associated with the lymph node metastasis, advanced TNM stage and poorer prognosis. SNHG1 increased RICTOR level in CRC via sponging miR-137. In addition, SNHG1 silencing inhibited CRC cell proliferation and migration in vitro and in vivo. SNHG1 regulated RICTOR expression by sponging miR-137 and promoted tumorgenesis in CRC.

Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

Long non-coding RNA-LET is a positive prognostic factor and exhibits tumor-suppressive activity in gallbladder cancer.

The identification of cancer-associated long non-coding RNAs (lncRNAs) and the investigation of their molecular and biological functions are vital for understanding the molecular biology and progression of cancer. The lncRNA-LET, a newly identified lncRNA, was demonstrated to be down-regulated in hepatocellular cancer. However, little is known about its role in gallbladder cancer. In the present study, an obvious down-regulation of lncRNA-LET was observed in gallbladder cancer compared to their adjacent normal tissues. Meanwhile, patients with low expression of lncRNA-LET have significantly poorer prognosis than those with high expression. We confirmed that hypoxia decreased lncRNA-LET levels in gallbladder cancer cells. Moreover, lncRNA-LET overexpression was further validated to inhibit the invasion of gallbladder cancer cells under hypoxic or normoxic conditions in vitro. We demonstrated that lncRNA-LET overexpression conferred a proliferative advantage to tumor cells under hypoxic conditions. The ectopic expression of lncRNA-LET led to the promotion of cell cycle arrest at G0/G1 phase and to the induction of apoptosis under hypoxic conditions. Ectopic expression of LncRNA-LET also suppressed gallbladder tumor growth in vivo. Our findings indicate that lncRNA-LET may represent a prognostic marker and a potential therapeutic target for gallbladder cancer.

Long non-coding RNA HOTAIR, a c-Myc activated driver of malignancy, negatively regulates miRNA-130a in gallbladder cancer.

BACKGROUND: Protein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown. METHODS: A computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays. RESULTS: We demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a. CONCLUSION: Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.

Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivo.

BACKGROUND: Gallbladder carcinoma (GBC) is highly lethal, and effective treatment will require synergistic anti-tumor management. The study is aimed at investigating the oncolytic value of myxoma virus (MYXV) infection against GBC and optimizing MYXV oncolytic efficiency. METHODS: We examined the permissiveness of GBC cell lines to MYXV infection and compared the effects of MYXV on cell viability among GBC and control permissive glioma cells in vitro and in vivo after MYXV + rapamycin (Rap) treatment, which is known to enhance cell permissiveness to MYXV by upregulating p-Akt levels. We also assessed MYXV + hyaluronan (HA) therapy efficiency by examinating Akt activation status, MMP-9 expression, cell viability, and collagen distribution. We further compared hydraulic conductivity, tumor area, and survival of tumor-bearing mice between the MYXV + Rap and MYXV + HA therapeutic regimens. RESULTS: MYXV + Rap treatment could considerably increase the oncolytic ability of MYXV against GBC cell lines in vitro but not against GBC xenografts in vivo. We found higher levels of collagen IV in GBC tumors than in glioma tumors. Diffusion analysis demonstrated that collagen IV could physically hinder MYXV intratumoral distribution. HA-CD44 interplay was found to activate the Akt signaling pathway, which increases oncolytic rates. HA was also found to enhance the MMP-9 secretion, which contributes to collagen IV degradation. CONCLUSIONS: Unlike MYXV + Rap, MYXV + HA therapy significantly enhanced the anti-tumor effects of MYXV in vivo and prolonged survival of GBC tumor-bearing mice. HA may optimize the oncolytic effects of MYXV on GBC via the HA-CD44 interaction which can promote viral infection and diffusion.

Inhibitory effects of deleted in liver cancer 1 gene on gallbladder cancer growth through induction of cell cycle arrest and apoptosis.

BACKGROUND AND AIM: The biological function of tumor suppressor deleted in liver cancer 1 (DLC1) has been investigated in several types of human cancer, but its role in gallbladder cancer (GBC) is yet to be determined. In this research, we conducted in vitro and in vivo analysis to evaluate the inhibitory activities of DLC1 gene against GBC growth. METHODS: DLC1 expression in GBC tissues and cell lines was examined by immunohistochemical staining, reverse transcription polymerase chain reaction, and Western blot assay. The in vitro and in vivo effects of ectopic DLC1 expression on cell growth were evaluated. In addition, the effects of ectopic DLC1 expression on cell cycle, apoptosis, and migration were also evaluated. The expressions of cell cycle-related and apoptosis-related proteins were examined. RESULTS: The downregulation of DLC1 expression was a common event in GBC tissues and cell lines. Restoration of DLC1 expression in GBC-SD and NOZ cells significantly reduced cell proliferation, migration in vitro, and the ability of these cells to form tumors in vivo. Restoration of DLC1 expression arrested GBC-SD and NOZ cells in G0/G1 phase through inducing p21 in a p53-independent manner. In addition, restoration of DLC1 expression induced extrinsic and intrinsic apoptotic pathway through promoting the expressions of Fas L/FADD, Bax, cytochrome c, cleaved caspase-8, -9, -3, and cleaved poly-(ADP-ribose) polymerase and suppressing bcl-2 expression in GBC-SD and NOZ cells. CONCLUSIONS: Our findings suggested that dysregulated expression of DLC1 is involved in proliferation and invasion of GBC cells and may serve as a potential therapeutic target.

A novel dammarane triterpenoid alleviates atherosclerosis by activating the LXRα pathway.

BACKGROUND: We have previously demonstrated that ginsenoside compound K can attenuate the formation of atherosclerotic lesions. Therefore, ginsenoside compound K has potential for atherosclerosis therapy. How to improve the druggability and enhance the antiatherosclerotic activity of ginsenoside compound K are the core problems in the prevention and treatment of atherosclerosis. CKN is a ginsenoside compound K derivative that was previously reported to have excellent antiatherosclerotic activity in vitro, and we have applied for international patents for it. METHODS: Male C57BL/6 ApoE-/- mice were fed a high-fat and high-choline diet to induce atherosclerosis and were subjected to in vivo studies. In vitro, the CCK-8 method was applied to evaluate cytotoxicity in macrophages. Foam cells were utilized, and cellular lipid determination was performed for in vitro studies. The area of atherosclerotic plaque and fatty infiltration of the liver were measured by image analysis. Serum lipid and liver function were determined by a seralyzer. Immunofluorescence and western blot analysis were conducted to explore the alterations in the expression levels of lipid efflux-related proteins. Molecular docking, reporter gene experiments and cellular thermal shift assays were used to verify the interaction between CKN and LXRα. RESULTS: After confirming the therapeutic effects of CKN, molecular docking, reporter gene experiments and cellular thermal shift assays were used to predict and investigate the antiatherosclerotic mechanisms of CKN. CKN exhibited the greatest potency, with a 60.9% and 48.1% reduction in en face atherosclerotic lesions on the thoracic aorta and brachiocephalic trunk, reduced plasma lipid levels and decreased foam cell levels in the vascular plaque content in HHD-fed ApoE-/- mice. Moreover, CKN in the present study may exert its antiatherosclerotic effects through activated ABCA1 by promoting LXRα nuclear translocation and reducing the adverse effects of LXRα activation. CONCLUSIONS: Our results revealed that CKN prevented the formation of atherosclerosis in ApoE-/- mice by activating the LXRα pathway.

Network Pharmacology Analysis and Experimental Verification on Antiangiogenesis Mechanism of Hedyotis diffusa Willd in Liver Cancer.

Purpose: Hedyotis diffusa Willd (HDW) is one of the most well-known herbs used in the therapy of cancer. However, the potential mechanisms of its antiangiogenic effects have not been fully explored. Here, we applied a network pharmacology approach to explore the potential mechanisms of HDW against liver cancer angiogenesis (LCA) and used a mouse orthotopic liver cancer model for experimental verification accordingly. Methods: The effective components, primary active compounds, and possible targets in the therapy of LCA were predicted using network pharmacology and bioinformatics. In vivo testing of the pharmacodynamic foundation of HDW in the treatment of LCA was performed. Hepa1-6 cells were implanted in C57BL/6 mice to establish an orthotopic liver cancer model to evaluate the antitumor and antiangiogenesis effects of the drug. Furthermore, protein levels were evaluated by western blotting, immunofluorescence, and immunohistochemistry. Results: We firstly confirmed the therapeutic effect of HDW on LCA and subsequently screened 7 active compounds from HDW according to their pharmacokinetic properties. Network analysis and enrichment analysis indicated that these compounds exhibit antiangiogenic effect by acting on multiple targets and thereby regulating multiple pathways mainly involved in Akt1, IL-6, IL-1ß, IL-17, hypoxia inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α). Importantly, we preliminarily verified the results of the network pharmacology analysis in vivo. Conclusion: Collectively, our work initially explored the therapeutic mechanism of HDW on tumor angiogenesis, which lays an experimental reference for further exploring its pharmacological action and its clinical application.

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor-2 inhibits gallbladder carcinoma growth in vitro and in vivo.

Tissue factor pathway inhibitor-2 (TFPI-2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI-2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus-mediated gene transfer of TFPI-2 (Ad5-TFPI-2) was constructed to restore the expression of TFPI-2 in GBC cell lines (GBC-SD, SGC-996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI-2 was significantly downregulated in GBC tissue specimens. Ad5-TFPI-2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5-TFPI-2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase-3 and -9 and suppressing Bcl-2 activity. These data indicated that TFPI-2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC.

[The role of tissue factor pathway inhibitor-2 gene in gallbladder cancer].

OBJECTIVE: To examine the expression of tissue factor pathway inhibitor-2 (TFPI-2) in gallbladder cancer (GBC) and to investigate the anti-cancer activities of TFPI-2 against the growth of GBC. METHODS: TFPI-2 expression in gallbladder normal tissues, gallbladder polyp (GBP) tissues and GBC tissues were examined by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemical staining. Adenovirus carrying human TFPI-2 gene (Ad5-TFPI-2) were constructed and its anti-cancer effects were investigated in xenograft tumors. Xenograft tumors were constructed by injection of GBC-SD and SGC-996 cells into the flank of nude mice and the volume of xenograft tumors was measured every 3 days until the sacrifice of mice. The apoptosis index of xenograft tumors was examined by TUNEL assay. The status of Bax, Bcl-2 and caspase-3 was examined by Western blot assay. RESULTS: TFPI-2 expression was profoundly lower in GBC tissues (87.0%) when compared to normal tissues (23.3%) and GBP tissues (52.2%; χ(2) = 21.104, P = 0.000). Ad-TFPI-2 significantly inhibited the growth of xenograft tumors in nude mice. Ad-TFPI-2 inhibited GBC-SD cell growth through the induction of apoptosis. The means of total apoptotic cells per field were much higher in Ad5-TFPI-2 group than those in PBS and Ad5-GFP groups. Ad5-TFPI-2 elevated the expression of Bax and cleaved caspase-3, while it decreased the expression of Bcl-2. CONCLUSIONS: TFPI-2 gene and protein was down-regulated in GBC and the down-regulation of TFPI-2 may play a role in the tumorigenesis of GBC. Adenovirus-mediated TFPI-2 can inhibit GBC growth through the induction of apoptosis.

Tissue factor pathway inhibitor 2 suppresses the growth of thyroid cancer cells through by induction of apoptosis.

BACKGROUND: Tissue factor pathway inhibitor 2 (TFPI-2) has been recently identified as a tumor suppressor gene in several human cancers, whereas its role in thyroid cancer has been unclear. METHODS: The TFPI-2 protein level in thyroid cancer tissues and cell lines (8305C and B-CPAP) were examined using immunohistochemistry and immunoblotting. The TFPI-2 promoter methylation was examined using methylation-specific polymerase chain reaction (MSP). Lentivirus containing TFPI-2 cDNA (Lenti-TFPI-2) was constructed to elevate TFPI-2 expression in 8305C and B-CPAP cells. The effects of Lenti-TFPI-2 on cell proliferation in vitro and in vivo were evaluated by MTT assay and mouse xenograft model. Annexin V/PI double staining assay was performed to detect the effect of Lenti-TFPI-2 on cell apoptosis. RESULTS: TFPI-2 protein level were decreased in cancer tissues and lymph node metastasis, and TFPI-2 protein level is positively associated with survival time. The promoter of TFPI-2 is hypermethylated in cancer tissues. TFPI-2 mRNA and protein levels were abundant in normal human thyroid follicular cell line Nthy-ori 3-1 cells, whereas they were decreased in 8305C and B-CPAP cells. pcDNA-TFPI-2 elevated TFPI-2 mRNA and protein in 8305C and B-CPAP cells. TFPI-2 overexpression suppressed proliferation and induced apoptosis of 8305C and B-CPAP cells. CONCLUSIONS: TFPI-2 inactivation may play a role in thyroid cancer tumorigenesis and development. TFPI-2 overexpression suppressed cell proliferation through induction of cell apoptosis, suggesting that TFPI-2 may serve as a novel and effective target for thyroid cancer therapy.

Community promotion and application of Wuqinxi combined with brief behavioral therapy for insomnia: A study protocol.

BACKGROUND: Brief behavioral therapy for insomnia (BBT-I) has been proven to be a simple and effective alternative to cognitive behavioral therapy. However, low adherence limits the application in Chinese primary medical institutions, resulting in delayed or irregular treatment for many patients. This study aimed to explore the efficacy of traditional Chinese medicine external treatments on the adherence to behavioral therapy for insomnia in Chinese primary healthcare institutions, with a particular focus on patients who live in regions with weak healthcare systems. METHODS: This randomized controlled clinical trial will be conducted in primary medical institutions and will recruit 98 adult participants with insomnia. BBT-I will be used as the base treatment. The participants will be divided into experimental (combined with Wuqinxi and other traditional Chinese medicine [TCM] external treatment n = 49) and control (combined with trazodone treatment, n = 49) groups, and each group will be treated for 4 consecutive weeks. The severity index of insomnia will be used as the main indicator of disease evaluation, with an 8-point reduction in the score considered as effective and a score <8 considered as cured. The secondary indicators of the disease evaluation will include the Pittsburgh sleep quality index, Zung's self-rating anxiety scale, Zung's self-rating depression scale, treatment adherence, and adverse event reports. All participants will be followed up at the time of enrollment, 4 weeks after treatment, and 3 months after the end of treatment. DISCUSSION: This clinical trial will provide evidence for the efficacy of traditional Chinese medicine external treatment on the adherence to behavioral therapy for insomnia in primary medical institutions. This cheap and accessible model may benefit insomnia patients in medically underserved areas. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100042845. Registered on 30 January 2021, dataset: http://www.chictr.org.cn/showproj.aspx?proj=65691. Official scientific title of the research topic: Wuqinxi and other external treatment of Chinese Medicine combined with brief behavior therapy for insomnia.

Downregulation of miR-181b-5p Inhibits the Viability, Migration, and Glycolysis of Gallbladder Cancer by Upregulating PDHX Under Hypoxia.

BACKGROUND: Gallbladder cancer (GBC) is a malignant cancer with poor prognosis. Evidences have shown that miRNAs are closely related to the occurrence of GBC; thus, we aimed to explore miRNAs, which plays an important role in the occurrence and development of GBC. METHODS: Microarray analysis was performed to investigate the differentially expressed miRNAs between five non-neoplastic gallbladder tissues (normal tissues) and five gallbladder tumor tissues (tumor tissues). RT-qPCR was performed to detect the level of miR-181b-5p in cells, and CCK-8 was performed to detect cell viability. Then, glucose assay kit or lactic acid assay kit was performed to detect the level of glucose consumption or lactate production. Next, transwell and wound healing assays were used to assess cell migration. In addition, dual-luciferase reporter assay was used to verify the relationship between miR-181b-5p and PDHX. At last, Western blotting was performed to determine the protein level of PDHX. RESULTS: Microarray analysis suggested miR-181b-5p was significantly upregulated in GBC tumor tissue. KEGG analysis for the protein targets of miR-181b-5p indicates a close relationship existed between miR-181b-5p and glycolysis. In addition, the level of miR-181b-5p was notably increased in GBC-SD or G415 cells, compared with HIBEpiC cells. GBC cell viability was significantly decreased under hypoxia, and these decreases were exacerbated by miR-181b-5p antagomir. Moreover, glucose consumption or lactate production of GBC cells was significantly upregulated under hypoxia, whereas these increases were completely revered by miR-181b-5p antagomir. Further investigation revealed that PDHX was a direct target of miR-181b-5p. CONCLUSION: In this study, downregulation of miR-181b-5p inhibits the viability, migration, and glycolysis of GBC by upregulating PDHX under hypoxia. This finding suggested that miR-181b-5p might be considered as a novel therapeutic target for the treatment of GBC.

MEX3A knockdown inhibits the tumorigenesis of colorectal cancer via modulating CDK2 expression.

Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract and a leading cause of cancer-associated mortality worldwide. Mex-3 RNA binding family member A (MEX3A) promotes the progression of multiple types of cancer, including ovarian and cervical cancer. However, to the best of our knowledge, the role of MEX3A in CRC is not completely understood. Therefore, the present study aimed to investigate the function of MEX3A in CRC. The mRNA and protein expression levels of MEX3A in CRC cells were analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Cell Counting Kit-8 assays were used to measure cell viability. Cell apoptosis and cell cycle distribution were detected via flow cytometry, and CRC cell invasion was analyzed by performing Transwell assays. Moreover, the mitochondrial membrane potential in CRC cells was measured via JC-1 staining. The results of the present study revealed that the expression levels of MEX3A were upregulated in CRC tissues compared with adjacent healthy tissues. MEX3A knockdown notably inhibited CRC cell viability, and induced apoptosis and mitochondrial injury. In addition, MEX3A knockdown markedly induced G1 phase cell cycle arrest in CRC cells via downregulating CDK2 expression. In conclusion, the findings of the present study suggested that MEX3A knockdown may inhibit the tumorigenesis of CRC cells by regulating CDK2 expression. Therefore, MEX3A may serve as a novel target for CRC treatment.

[Somatostatin enhanced anti-tumor effect of doxorubicin on gallbladder cancer cells through the regulation of intracellular drug concentration].

OBJECTIVE: To investigate the possible mechanisms by which Somatostatin (SST) enhances the anti-tumor effect of doxorubicin (DOX) on gallbladder cancer cells. METHODS: GBC-SD cells were grouped into 4 groups: SST-treated group, DOX-treated group, SST+DOX co-treated group and control group. The concentrations of SST and DOX were 75 µg/ml and 5 µg/ml based on our previous studies. In control group, cells were cultivated with phosphate buffered saline (PBS). In experimental groups, cells were cultivated with medium and the corresponding drugs. After drug treatment, cell viability was examined by MTT assay at 6, 12, 24 and 36 h respectively. Meanwhile, intracellular concentrations of doxorubicin in each group was determined by microspectrofluorimetry; Real-time polymerase chain reaction (RT-PCR) was used to determine the expressions of MDR1 mRNA in the cells at different time points and the expressions of P-gp protein, a product of MDR1 mRNA, were determined by Western blot analysis. RESULTS: SST did not exhibit significant inhibitory effect on the proliferation of GBC-SD cells as compared to that of control group (P>0.05). SST+DOX co-treatment group and DOX showed significantly inhibitory effect on the growth of GBC-SD cells at Hour 12 post-treatment. However no statistical difference was found between SST+DOX and DOX groups. Interestingly, at Hour 24 post-treatment, SST+DOX group showed more robust inhibitory effect on GBC-SD cells as compared to DOX alone group. Moreover, SST could significantly down-regulate the expressions of MDR1 mRNA and P-gp protein. SST could increase intracellular DOX concentration. And the difference of intracellular DOX concentration between SST+DOX group and DOX group at Hour 24 was statistically significant. CONCLUSIONS: In our experiment, SST decreases the expression of MDR1 mRNA and P-gp protein so as to reduce the efflux of DOX and elevate DOX concentrations in GBC-SD cells. This eventually leads to enhanced cytotoxic effects of DOX on GBC-SD cells.

miR­135b­5p enhances the sensitivity of HER­2 positive breast cancer to trastuzumab via binding to cyclin D2.

Trastuzumab has led to a marked improvement in the outcomes of patients with human epidermal growth factor receptor 2 (HER­2)­positive breast cancer. However, the effects of trastuzumab on HER­2­positive breast cancer are limited by the emergence of its cardiotoxicside effects. MicroRNA (miR)­135b­5p has been shown to inhibit tumor metastasis in breast cancer. The present study aimed to explore the effects of miR­135b­5p overexpression on the efficacy of trastuzumab in HER­2­positive breast cancer. Reverse transcription­quantitative PCR was performed to detect the levels of miR­135b­5p. Cell viability was evaluated with a Cell Counting Kit­8 assay. Annexin V/propidium iodide staining was employed to detect the number of apoptotic cells. Flow cytometry assay was performed to investigate the cell cycle. Western blotting was used to detect the expression levels of Bax, cleaved caspase­3, Bcl­2, cyclin D2, p27Kip1 and cyclin E1. Cell migration and invasion were detected by Transwell assay. Luciferase assays were conducted to identify the target gene of miR­135b­5p. In addition, an in vivo tumor xenograft model was established. miR­135b­5p agomir significantly enhanced the anti­proliferative effect of trastuzumab on HER­2­positive breast cancer cells via the induction of apoptosis, whereas the anti­metastatic effect of trastuzumab was enhanced by miR­135b­5p agomir treatment. Subsequently, luciferase assays indicated that cyclin D2 was the direct target of miR­135b­5p, whereas overexpression of the latter arrested cell cycleduring the G0/G1 phase. Moreover, miR­135b­5p agomir notably increased the antitumor effect of trastuzumab in vivo. The data demonstrated that miR­135b­5p sensitized HER­2­positive breast cancer cells to trastuzumab in vitro and in vivo by directly binding to cyclin D2. These results suggested that the combination of miR­135b­5p with trastuzumab may be a therapeutic strategy for patients with HER­2­positive breast cancer.

Downregulation of miR-575 Inhibits the Tumorigenesis of Gallbladder Cancer via Targeting p27 Kip1.

BACKGROUND: Gallbladder cancer (GBC) is the most common biliary tract malignant cancer worldwide. It has been reported that microRNA-575 (miR-575) was involved in the tumorigenesis of many cancers. However, the role of miR-575 during the progression of GBC remains largely unknown. METHODS: The expression of miR-575 in GBC cells was detected by quantitative real-time polymerase chain reaction. The proliferation of GBC cells was examined by CCK-8 assay and Ki-67 staining. Apoptosis of GBC cells was measured by flow cytometry, and cell invasion was tested by transwell assay. Moreover, protein expressions in GBC cells were evaluated using Western blot. The target gene of miR-575 was predicted using Targetscan and miRDB. Finally, xenograft tumor model was established to verify the function of miR-575 in GBC in vivo. RESULTS: Our findings indicated that miR-575 antagonist decreased the proliferation and invasion of GBC cells. In addition, miR-575 antagonist significantly induced apoptosis of GBC cells via inducing G1 arrest. Meanwhile, p27 Kip1 was found to be a direct target of miR-575 with luciferase reporter assay. Moreover, miR-575 antagonist significantly decreased the expressions of CDK1 and cyclin E1 and upregulated the levels of cleaved caspase3 and p27 Kip1 in GBC cells. Finally, miR-575 antagonist notably suppressed GBC tumor growth in vivo. CONCLUSION: Downregulation of miR-575 significantly inhibited the tumorigenesis of GBC via targeting p27 Kip1. Thus, miR-575 might be a potential novel target for the treatment of GBC.