Objective To investigate the effects of microRNA497 (miR-497) on the metastasis of gastric cancer and its possible molecular mechanism. Methods SGC-7901 gastric cancer parent cells were cultured in an ultra-low adhesion environment, and the anoikis resistance model of SGC-7901 cells was created after re-adhesion. Clone formation assay, flow cytometry, Transwell^TM test and scratch healing test were used to detect the differences of biological behavior compared with their parent cells. Fluorescence quantitative PCR was performed to detect the expression of miR-497. Western blot analysis was used to detect the changes of key proteins of Wnt/β-catenin signaling pathway and epithelial mesenchymal transformation (EMT) related proteins such as vimentin and E-cadherin. Parent cells and anoikis resistant SGC-7901 cells were transfected with miR-497 inhibitor or miR-497 mimic, and CCK-8 assay was used to detect the proliferation activity. Transwell^TM invasion assay was performed to detect the invasion ability of cells. Transwell^TM migration test and scratch healing assay was used to determine the migration ability. Western blot analysis was used to detect the expressions of Wnt1, β-catenin, vimentin and E-cadherin. By transfecting miR-497 mimic into the anoikis resistance SGC-7901 cells and inoculating them subcutaneously in nude mice, the changes in the volume and mass of tumor tissues were measured and recorded. Western blot analysis was used to determine the expressions of Wnt1, β-catenin, vimentin and E-cadherin of tumor tissues. Results Compared with the parent cells, the anoikis resistance SGC-7901 gastric cancer cells had faster proliferation rate, stronger colony formation, lower apoptosis rate, stronger invasion and migration ability. The expression of miR-497 was significantly decreased. After down-regulation of miR-497, the proliferation ability, invasion and migration ability were significantly enhanced. The expressions of Wnt1, β-catenin and vimentin increased significantly, while E-cadherin decreased notably. The results of up-regulation miR-497 were the opposite. The tumor growth rate, tumor volume and mass of miR-497 overexpression group were significantly lower than those of control group. The expressions of Wnt1, β-catenin and vimentin decreased significantly, while the expression of E-cadherin increased significantly. Conclusion The expression of miR-497 is low in the anoikis resistance SGC-7901 cells. miR-497 can inhibit the growth and metastasis of gastric cancer cells by blocking Wnt/β-catenin signaling pathway and EMT.
Animals ; Mice ; Humans ; beta Catenin/metabolism* ; MicroRNAs/metabolism* ; Vimentin/metabolism* ; Stomach Neoplasms/pathology* ; Anoikis/genetics* ; Wnt Signaling Pathway/genetics* ; Mice, Nude ; Cell Proliferation/genetics* ; Cadherins/genetics* ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition/genetics* ; Cell Movement/genetics*

Nine cases of mesenteric desmoid-type fibromatosis were diagnosed and treated in Taizhou Hospital, Wenzhou Medical University between January 2010 and May 2022, including 2 females and 7 males, aged 16 to 59 years. The lesions were in the mesentery of small intestine with 7 cases, ileocecal junction with 1 cases and transverse colon with 1 case. The tumors had an unclear boundary and no envelope, the section was solid, gray and tough. The mean maximum diameter was (10.7±8.5) cm (range 3.5-33.0 cm). Microscopically, fusiform fibroblasts and myofibroblasts were parallel, bunched or staggered, buried in a large amount of extracellular collagen. The cell morphology was relatively consistent, without obvious atypia, and mitosis was rare. Immunohistochemistry showed that the tumor cells were positive for vimentin (9/9), β-catenin (9/9), while smooth muscle actin (5/9) stains were focally positive. Ki-67 proliferation index was 1%-10%. Cytokeratin Pan, S-100, STAT6, CD117, DOG1, CD34, desmin and anaplastic lymphoma kinase stains were negative. Genetic analysis showed that there were 7 cases of c.121G>A(p.Thr41Ala) mutation of CTNNB1 gene, 1 case of c.121G>A(p.Thr41Ala) and 1 case of c.134C>T(p.Ser45Phe) double mutation, and 1 case of wild type. Tumors were surgically resected in all 9 cases. Eight cases had no recurrence or metastasis, 1 case had recurrence 6 months later, and no recurrence or metastasis after additional surgical resection.
Male ; Female ; Humans ; Fibromatosis, Aggressive/diagnosis* ; Immunohistochemistry ; Fibroblasts/metabolism* ; Mesentery/pathology* ; beta Catenin/analysis*

OBJECTIVE: To explore the effect of abnormal miRNA expression on the proliferation of pediatric acute lymphoblastic leukemia (ALL) cells and its related mechanism. METHODS: 15 children with ALL and 15 healthy subjects were collected from the Second Affiliated Hospital of Hainan Medical University from July 2018 to March 2021. MiRNA sequencing was performed on their bone marrow cells, and validated using qRT-PCR. MiR-1294 and miR-1294-inhibitory molecule (miR-1294-inhibitor) were transfected into Nalm-6 cells, and the proliferation of Nalm-6 cells was detected by CCK-8 and colony formation assays. Western blot and ELISA were used to detect apoptosis of Nalm-6 cells. Biological prediction of miR-1294 was performed to find the target gene, which was verified by luciferase reporter assay. Si-SOX15 was transfected into Nalm-6 cells, Western blot was used to detect the expression of Wnt signaling pathway-related proteins and to verify the effect of si-SOX15 on the proliferation and apoptosis of Nalm-6 cells. RESULTS: Compared with healthy subjects, 22 miRNAs were significantly upregulated in bone marrow cells of ALL patients, of which miR-1294 was the most significantly upregulated. In addition, the expression level of SOX15 gene was significantly reduced in bone marrow cells of ALL patients. Compared with the NC group, the miR-1294 group showed increased protein expression levels of Wnt3a and β-catenin, faster cell proliferation, and more colony-forming units, while caspase-3 protein expression level and cell apoptosis were reduced. Compared with the NC group, the miR-1294-inhibitor group showed reduced protein expression levels of Wnt3a and β-catenin, slower cell proliferation, and fewer colony-forming units, while caspase-3 protein expression level was increased and apoptosis rate was elevated. miR-1294 had a complementary base-pair with the 3'UTR region of SOX15 , and miR-1294 directly targeted SOX15 . The expression of miR-1294 was negatively correlated with SOX15 in ALL cells. Compared with the si-NC group, the si-SOX15 group showed increased protein expression levels of Wnt3a and β-catenin, accelerated cell proliferation, and decreased caspase-3 protein expression level and cell apoptosis rate. CONCLUSION MiR-1294 can target and inhibit SOX15 expression, thus activating the Wnt/β-Catenin signaling pathway to promote the proliferation of ALL cells, inhibit cell apoptosis, and ultimately affect the disease progression.
Humans ; Child ; beta Catenin/genetics* ; Wnt Signaling Pathway ; Caspase 3/metabolism* ; Cell Line, Tumor ; MicroRNAs/genetics* ; Cell Proliferation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Apoptosis ; SOX Transcription Factors/metabolism*

OBJECTIVE: To explore the proliferation inhibitory effect of quinones from Blaps rynchopetera defense secretion on colorectal tumor cell lines. METHODS: Human colorectal cancer cell HT-29, human colorectal adenocarcinoma cell Caco-2 and normal human colon epithelial cell CCD841 were chosen for the evaluation of inhibitory activity of the main quinones of B. rynchopetera defense secretion, including methyl p-benzoquinone (MBQ), ethyl p-benzoquinone (EBQ), and methyl hydroquinone (MHQ), through methyl thiazolyl tetrazolium assay. The tumor-related factors, cell cycles, related gene expressions and protein levels were detected by enzyme-linked immunosorbent assy, flow cytometry, RT-polymerase chain reaction and Western blot, respectively. RESULTS: MBQ, EBQ, and MHQ could significantly inhibit the proliferation of Caco-2, with half maximal inhibitory concentration (IC₅₀) values of 7.04 ± 0.88, 10.92 ± 0.32, 9.35 ± 0.83, HT-29, with IC₅₀ values of 14.90 ± 2.71, 20.50 ± 6.37, 13.90 ± 1.30, and CCD841, with IC₅₀ values of 11.40 ± 0.68, 7.02 ± 0.44 and 7.83 ± 0.05 µg/mL, respectively. Tested quinones can reduce the expression of tumor-related factors tumor necrosis factor α, interleukin (IL)-10, and IL-6 in HT-29 cells, selectively promote apoptosis, and regulate the cell cycle which can reduce the proportion of cells in the G₁ phase and increase the proportion of the S phase. Meanwhile, tested quinones could up-regulate mRNA and protein expression of GSK-3β and APC, while down-regulate that of β-catenin, Frizzled1, c-Myc, and CyclinD1 in the Wnt/β-catenin pathway of HT-29 cells. CONCLUSION Quinones from B. rynchopetera defense secretion could inhibit the proliferation of colorectal tumor cells and reduce the expression of related factors, which would be functioned by regulating cell cycle, selectively promoting apoptosis, and affecting Wnt/β-catenin pathway-related mRNA and protein expressions.
Humans ; beta Catenin/metabolism* ; Caco-2 Cells ; Quinones/pharmacology* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Proliferation ; Colorectal Neoplasms/metabolism* ; Cell Line, Tumor ; Apoptosis ; Benzoquinones/pharmacology* ; RNA, Messenger ; Wnt Signaling Pathway

OBJECTIVES: This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways. METHODS: PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated. RESULTS: Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). CONCLUSIONS There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.
Humans ; DNA Methylation ; Transcriptome ; beta Catenin ; Leukocytes, Mononuclear ; Ligands ; DNA ; RNA, Messenger/genetics*

OBJECTIVE: To investigate the effect of electroacupuncture on osteoarthritis in rats and explore the possible mechanism. METHODS: Thirty SD rats were randomly divided into osteoarthritis model group, electro-acupuncture group and control group (n=10), and in the former two groups, early osteoarthritis was induced using a modified DMM surgical modeling method. After successful modeling, the rats in the electro-acupuncture group were treated with electro-acupuncture at bilateral "Housanli" and "Anterior knee point". Behavioral tests of the rats were performed and scored using the LequesneMG scale. Subchondral bone degeneration was observed in each group, and serum levels of IL-1β, ADAMTS-7, MMP-3 and COMP were measured using ELISA. The mRNA and protein expressions of IL-1β, Wnt-7B, β-catenin, ADAMTS-7, and MMP-3 in the cartilage tissue of the knee joints were detected using RT-PCR and Western blotting. RESULTS: In behavioral tests, the rats in the model and electroacupuncture groups had significantly higher LequesneMG scores after modeling than those in the control group (P < 0.05). After 20 days of treatment, LequesneMG scores were significantly lowered in rats in the electroacupuncture as compared with the model rats (P < 0.05). Imaging examination revealed obvious subchondral bone damage in both the electroacupuncture group and the model group, but the damages were significantly milder with former group. Compared with the model rats, the rats receiving electroacupuncture had significantly lower serum levels of IL-1β, ADAMTS-7, MMP-3 and COMP (P < 0.05) with also lower expressions of IL-1β, Wnt-7B, β-catenin, ADAMTS-7 and MMP-3 in the cartilage tissues at both the mRNA and protein levels (P < 0.05). CONCLUSION Electroacupuncture can alleviate joint pain and improve subchondral bone damage in rats with osteoarthritis by reducing IL-1β levels in the joint cartilage tissue and serum to alleviate joint inflammation and by reducing such cytokines as ADAMTS-7 and MMP-3 via regulating the Wnt-7B/β-catenin signaling pathway.
Rats ; Animals ; Electroacupuncture ; Matrix Metalloproteinase 3/metabolism* ; Rats, Sprague-Dawley ; beta Catenin/metabolism* ; Osteoarthritis/metabolism* ; Wnt Signaling Pathway ; Cartilage, Articular ; Inflammation/metabolism*

OBJECTIVE: To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism. METHODS: GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway. RESULTS: The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01). CONCLUSION PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.
Humans ; Animals ; Mice ; Up-Regulation ; beta Catenin/metabolism* ; Mice, Nude ; Brain Neoplasms/pathology* ; Lactic Acid ; Cell Line, Tumor ; Glioma/pathology* ; Cell Proliferation ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVE: To investigate the protective effects of total saponins from Panax japonicus (TSPJ) against high-fat dietinduced testicular Sertoli cell junction damage in mice. METHODS: Forty male C57BL/6J mice were randomized into normal diet group, high-fat diet group, and low-dose (25 mg/kg) and high-dose (75 mg/kg) TSPJ treatment groups (n=10). The mice in the normal diet group were fed a normal diet, while the mice in the other groups were fed a high-fat diet. After TSPJ treatment via intragastric administration for 5 months, the testes and epididymis of the mice were collected for measurement of weight, testicular and epididymal indices and sperm parameters. HE staining was used for histological evaluation of the testicular tissues and measurement of seminiferous tubule diameter and seminiferous epithelium height. The expression levels of ZO-1, occludin, claudin11, N-cadherin, E-cadherin and β-catenin in Sertoli cells were detected with Western blot, and the localization and expression levels of ZO-1 and β-catenin in the testicular tissues were detected with immunofluorescence assay. The protein expressions of LC3B, p-AKT and p-mTOR in testicular Sertoli cells were detected using double immunofluorescence assay. RESULTS: Treatment with TSPJ significantly improved high-fat diet-induced testicular dysfunction by reducing body weight (P < 0.001), increasing testicular and epididymal indices (P < 0.05), and improving sperm concentration and sperm viability (P < 0.05). TSPJ ameliorated testicular pathologies and increased seminiferous epithelium height of the mice with high-fat diet feeding (P < 0.05) without affecting the seminiferous tubule diameter. TSPJ significantly increased the expression levels of ZO-1, occludin, N-cadherin, E-cadherin and β-catenin (P < 0.05) but did not affect claudin11 expression in the testicular tissues. Immunofluorescence assay showed that TSPJ significantly increased ZO-1 and β-catenin expression in the testicular tissues (P < 0.001), downregulated LC3B expression and upregulated p-AKT and p-mTOR expressions in testicular Sertoli cells. CONCLUSION TSPJ alleviates high-fat diet-induced damages of testicular Sertoli cell junctions and spermatogenesis possibly by activating the AKT/mTOR signaling pathway and inhibiting autophagy of testicular Sertoli cells.
Male ; Animals ; Mice ; Mice, Inbred C57BL ; Testis ; Sertoli Cells ; beta Catenin ; Diet, High-Fat ; Occludin ; Proto-Oncogene Proteins c-akt ; Seeds ; Cadherins ; Intercellular Junctions

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

OBJECTIVE: Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. METHODS: A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. RESULTS: Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin⁺/CD31⁺ ratio, rather than the number of CD31⁺ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. CONCLUSION Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proliferating Cell Nuclear Antigen/therapeutic use* ; Mice, Nude ; Glycogen Synthase Kinase 3 beta/genetics* ; beta Catenin/therapeutic use* ; Liver Neoplasms/drug therapy* ; Desmin/therapeutic use* ; Ki-67 Antigen ; Cell Line, Tumor ; Hypoxia ; RNA, Messenger/therapeutic use* ; Cell Proliferation