Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation.

OBJECTIVE: Protein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. METHODS: GC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. RESULTS: The expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. CONCLUSION: PDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.

Early-stage CCNG1+ HR+ HER2+ Invasive Breast Carcinoma in Older Women: Current Treatment and Future Perspectives for DeltaRex-G, a CCNG1 Inhibitor.

Women with HR+HER2+ early-stage breast cancer are disadvantaged by the lack of clinical trials focused on women ≥70 years of age. In the past years, there has been increasing controversy on the use of toxic chemotherapy as standard of care treatment for early- stage HR+ HER2+ breast carcinoma in older women. With precision medicine coming of age, molecular profiling of tumors and circulating tumor DNA has identified target oncogenes that could be used in designing an optimal treatment for this group of women. This article reviews the current treatment of early-stage triple receptor positive breast cancer, the risks of chemotherapy in older women, and CCNG1, a novel biomarker in development for the use of DeltaRex-G, a CCNG1 inhibitor. Further, future perspectives for DeltaRex-G in older women with early stage CCNG1+ HR+ HER2+ breast cancer are discussed.

Abnormal expression of miRNA-122 in cerebral infarction and related mechanism of regulating vascular endothelial cell proliferation and apoptosis by targeting CCNG1.

OBJECTIVE: To analyze the value of serum miRNA-122 expression in the diagnosis, severity, and prognosis of Acute Cerebral Infarction (ACI) and the correlation mechanism of serum miRNA-122 on the proliferation and apoptosis of vascular endothelial cells in ACI. METHOD: A total of 60 patients with ACI who were admitted to the emergency department of the Taizhou People's Hospital from January 1, 2019, to December 30, 2019, and 30 healthy controls during the same period were selected. General clinical data of all patients at admission were collected. Including age, sex, medical history, and inflammatory factors (C-Reactive Protein [CRP], Interleukin-6 [IL-6], Procalcitonin [PCT], Neutrophil Gelatinase-Associated Lipid carrier protein [NGAL]). The National Institutes of Health Stroke Scale (NIHSS) score at admission and short-term prognosis (the Modified Rankin Score [mRS]) score at 3 months after onset were recorded. The expression level of miRNA-122 in the serum of patients with ACI and normal controls was detected by reverse-transcription quantitative Real-Time Polymerase Chain Reaction (RT-QPCR), and the correlation between the expression level of miRNA-122 in the serum of patients with ACI and the level of inflammatory factors, NIHSS and mRS scores were analyzed. The expression levels of miRNA-122 in the serum of patients with ACI, normal people, and Human Umbilical cord Endothelial Cells (HUVECs) cultured in a blank control group were detected by RT-QPCR and statistically analyzed. MTT and flow cytometry was used to compare the proliferation and apoptosis of vascular endothelial cells in the miRNA-122 mimics and inhibitors transfection groups and the corresponding negative control group. The mRNA and protein levels of apoptosis-related factors Bax, Bcl-2, Caspase-3, and angiogenesis-related proteins Hes1, Notch1, Vascular Endothelial Growth Factors (VEGF), and CCNG1 were detected by RT-QPCR and Western blot. Bioinformatics methods predicted CCNG1 to be the target of miRNA-122, and the direct targeting relationship between CCNG1 and miRNA-122 was verified by a dual-luciferase reporting assay. RESULT: Serum miRNA-122 expression in patients with ACI was significantly higher than that in healthy controls, with an area under the receiver operating characteristic curve of 0.929, 95% Confidence Interval of 0.875‒0.983, and an optimal cut-off value of 1.397. The expression levels of CRP, IL-6, and NGAL in patients with ACI were higher than those in healthy control groups, p < 0.05; miRNA-122 was positively correlated with CPR, IL-6, NIHSS score, and mRS score. At 48h and 72h, the proliferation rate of HUVECs cells in the miRNA-122 mimics group decreased and the apoptosis rate increased. Cell proliferation rate increased, and apoptosis rate decreased significantly in the groups transfected with miRNA-122 inhibitors. The mRNA and protein levels of pro-apoptotic factors Bax and caspase-3 were significantly increased in the miRNA-122 mimics transfection group, while those of anti-apoptotic factor Bcl-2 were significantly decreased compared to those of the control group. The expression of Bax and Caspase-3 decreased, and the expression of anti-apoptotic factor Bcl-2 increased in the transfected miRNA-122 inhibitors group. mRNA expression levels of Hes1, Notch1, VEGF, and CCNG1 in the miRNA-122 mimic transfected group were significantly decreased, while mRNA expression levels in the miRNA-122 inhibitors transfected group were significantly increased. Bioinformatics showed that there was a miRNA-122 binding site in the 3'UTR region of CCNG1, and dual luciferase assay confirmed that CCNG1 was the target of miRNA-122. CONCLUSION: Serum miRNA-122 increased significantly after ACI, which may be a diagnostic marker of ACI. miRNA-122 may be involved in the pathological process of ACI and is related to the degree of neurological impairment and short-term prognosis in patients with ACI. miRNA-122 may play a regulatory role in ACI by inhibiting cell proliferation, increasing apoptosis, and inhibiting vascular endothelial cell regeneration through the CCNG1 channel.

PRMT6 functionally associates with PRMT5 to promote colorectal cancer progression through epigenetically repressing the expression of CDKN2B and CCNG1.

BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) is a type I arginine methyltransferase that asymmetrically dimethylates histone H3 arginine 2 (H3R2me2a). However, the biological roles and underlying molecular mechanisms of PRMT6 in colorectal cancer (CRC) remain unclear. METHODS: PRMT6 expression in CRC tissue was examined using immunohistochemistry. The effect of PRMT6 on CRC cells was investigated in vitro and in vivo. Mass spectrometry, co-immunoprecipitation and GST pulldown assays were performed to identify interaction partners of PRMT6. RNA-seq, chromatin immunoprecipitation, Western blot and qRT-PCR assays were used to investigate the mechanism of PRMT6 in gene regulation. RESULTS: PRMT6 is significantly upregulated in CRC tissues and facilitates cell proliferation of CRC cells in vitro and in vivo. Through RNA-seq analysis, CDKN2B (p15INK4b) and CCNG1 were identified as new transcriptional targets of PRMT6. PRMT6-dependent H3R2me2a mark was predominantly deposited at the promoters of CDKN2B and CCNG1 in CRC cells. Furthermore, PRMT5 was firstly characterized as an interaction partner of PRMT6. Notably, H3R2me2a coincides with PRMT5-mediated H4R3me2s and H3R8me2s marks at the promoters of CDKN2B and CCNG1 genes, thus leading to transcriptional repression of these genes. CONCLUSIONS: PRMT6 functionally associates with PRMT5 to promote CRC progression through epigenetically repressing the expression of CDKN2B and CCNG1. These insights raise the possibility that combinational intervention of PRMT6 and PRMT5 may be a promising strategy for CRC therapy.

Phase I-II study using DeltaRex-G, a tumor-targeted retrovector encoding a cyclin G1 inhibitor for metastatic carcinoma of breast.

Background: Metastatic breast cancer is associated with a poor prognosis and therefore, innovative therapies are urgently needed. Here, we report on the results of a Phase I-II study using DeltaRex-G for chemotherapy resistant metastatic carcinoma of breast. Patients and Methods: Endpoints: Dose limiting toxicity; Antitumor activity. Eligibility: ≥18 years of age, pathologic diagnosis of breast carcinoma, adequate hematologic and organ function. Treatment: Dose escalation of DeltaRex-G 1-4 x 1011cfu intravenously thrice weekly x 4 weeks with 2-week rest period. Treatment cycles repeated if there is ≤ Grade 1 toxicity until disease progression or unacceptable toxicity. Safety: NCI CTCAE v3 for adverse events reporting, vector related testing. Efficacy: RECIST v1.0, International PET criteria and Choi criteria for response, progression free and overall survival. Results: Twenty patients received escalating doses of DeltaRex-G from 1 × 1011 cfu to 4 × 1011 cfu thrice weekly for 4 weeks with a 2-week rest period. Safety: ≥ Grade 3 treatment-related adverse event: pruritic rash (n = 1), no dose limiting toxicity, no replication-competent retrovirus, nor vector-neutralizing antibodies detected. No vector DNA integration was observed in peripheral blood lymphocytes evaluated. Efficacy: by RECIST v1.0: 13 stable disease, 4 progressive disease; tumor control rate 76%; by PET and Choi Criteria: 3 partial responses, 11 stable disease, 3 progressive disease; tumor control rate 82%. Combined median progression free survival by RECIST v1.0, 3.0 months; combined median overall survival, 20 months; 1-year overall survival rate 83% for Dose Level IV. Biopsy of residual tumor in a participant showed abundant CD8+ killer T-cells and CD45+ macrophages suggesting an innate immune response. Two patients with pure bone metastases had >12-month progression free survival and overall survival and are alive 12 years from the start of DeltaRex-G therapy. These patients further received DeltaRex-G + DeltaVax for 6 months. Conclusion: Taken together, these data indicate that 1) DeltaRex-G has a distinctively high level of safety and exhibits anti-cancer activity, 2) PET/Choi provide a higher level of sensitivity in detecting early signs of tumor response to DeltaRex-G, 3) DeltaRex-G induced 12- year survival in 2 patients with pure bone metastases who subsequently received DeltaVax immunotherapy, and 4) DeltaRex-G may prove to be a biochemical and/or immune modulator when combined with other cancer therapy/immunotherapy.

The advent of a pan-collagenous CLOVIS POINT for pathotropic targeting and cancer gene therapy, a retrospective.

The 'Clovis Point'-an enabling prehistoric gain-of-function in stone-age tool technologies which empowered the Paleoindian-Americans to hunt, to strike-deep, and to kill designated target megafauna more efficiently-was created biochemically by molecular-genetic bio-engineering. This Biomedical "Clovis Point" was crafted by adapting a broad-spectrum Pan-Collagen Binding Domain (Pan-Coll/CBD) found within the immature pre-pro-peptide segment of Von Willebrand Factor into a constructive series of advanced medical applications. Developed experimentally, preclinically, and clinically into a cutting-edge Biotechnology Platform, the Clovis Point is suitable for 1) solid-state binding of growth factors on collagenous scaffolds for improved orthopedic wound healing, 2) promoting regeneration of injured/diseased tissues; and 3) autologous stem cell capture, expansion, and gene-based therapies. Subsequent adaptations of the high-affinity Pan-Coll/CBD (exposed-collagen-seeking/surveillance function) for intravenous administration in humans, enabled the physiological delivery, aka Pathotropic Targeting to diseased tissues via the modified envelopes of gene vectors; enabling 4) precision tumor-targeting for cancer gene therapy and 5) adoptive/localized immunotherapies, demonstrating improved long-term survival value-thus pioneering a proximal and accessible cell cycle control point for cancer management-empowering modern medical oncologists to address persistent problems of chemotherapy resistance, recurrence, and occult progression of metastatic disease. Recent engineering adaptations have advanced the clinical utility to include the targeted delivery of small molecule APIs: including taxanes, mAbs, and RNA-based therapeutics.

Abnormal expression of miRNA-122 in cerebral infarction and related mechanism of regulating vascular endothelial cell proliferation and apoptosis by targeting CCNG1

Abstract Objective: To analyze the value of serum miRNA-122 expression in the diagnosis, severity, and prognosis of Acute Cerebral Infarction (ACI) and the correlation mechanism of serum miRNA-122 on the proliferation and apoptosis of vascular endothelial cells in ACI. Method: A total of 60 patients with ACI who were admitted to the emergency department of the Taizhou People's Hospital from January 1, 2019, to December 30, 2019, and 30 healthy controls during the same period were selected. General clinical data of all patients at admission were collected. Including age, sex, medical history, and inflammatory factors (C-Reactive Protein [CRP], Interleukin-6 [IL-6], Procalcitonin [PCT], Neutrophil Gelatinase-Associated Lipid carrier protein [NGAL]). The National Institutes of Health Stroke Scale (NIHSS) score at admission and short-term prognosis (the Modified Rankin Score [mRS]) score at 3 months after onset were recorded. The expression level of miRNA-122 in the serum of patients with ACI and normal controls was detected by reverse-transcription quantitative Real-Time Polymerase Chain Reaction (RT-QPCR), and the correlation between the expression level of miRNA-122 in the serum of patients with ACI and the level of inflammatory factors, NIHSS and mRS scores were analyzed. The expression levels of miRNA-122 in the serum of patients with ACI, normal people, and Human Umbilical cord Endothelial Cells (HUVECs) cultured in a blank control group were detected by RT-QPCR and statistically analyzed. MTT and flow cytometry was used to compare the proliferation and apoptosis of vascular endothelial cells in the miRNA-122 mimics and inhibitors transfection groups and the corresponding negative control group. The mRNA and protein levels of apoptosis-related factors Bax, Bcl-2, Caspase-3, and angiogenesis-related proteins Hes1, Notch1, Vascular Endothelial Growth Factors (VEGF), and CCNG1 were detected by RT-QPCR and Western blot. Bioinformatics methods predicted CCNG1 to be the target of miRNA-122, and the direct targeting relationship between CCNG1 and miRNA-122 was verified by a dual-luciferase reporting assay. Result: Serum miRNA-122 expression in patients with ACI was significantly higher than that in healthy controls, with an area under the receiver operating characteristic curve of 0.929, 95% Confidence Interval of 0.875‒0.983, and an optimal cut-off value of 1.397. The expression levels of CRP, IL-6, and NGAL in patients with ACI were higher than those in healthy control groups, p < 0.05; miRNA-122 was positively correlated with CPR, IL-6, NIHSS score, and mRS score. At 48h and 72h, the proliferation rate of HUVECs cells in the miRNA-122 mimics group decreased and the apoptosis rate increased. Cell proliferation rate increased, and apoptosis rate decreased significantly in the groups transfected with miRNA-122 inhibitors. The mRNA and protein levels of pro-apoptotic factors Bax and caspase-3 were significantly increased in the miRNA-122 mimics transfection group, while those of anti-apoptotic factor Bcl-2 were significantly decreased compared to those of the control group. The expression of Bax and Caspase-3 decreased, and the expression of anti-apoptotic factor Bcl-2 increased in the transfected miRNA-122 inhibitors group. mRNA expression levels of Hes1, Notch1, VEGF, and CCNG1 in the miRNA-122 mimic transfected group were significantly decreased, while mRNA expression levels in the miRNA-122 inhibitors transfected group were significantly increased. Bioinformatics showed that there was a miRNA-122 binding site in the 3′UTR region of CCNG1, and dual luciferase assay confirmed that CCNG1 was the target of miRNA-122.

The stimulative function of long noncoding RNA CDKN2B-AS1 in osteosarcoma by targeting the microRNA-122/CCNG1 axis.

Osteosarcoma (OS), a prevalent aggressive malignancy in the bone, has limited therapeutic targets and diagnostic biomarkers. In the current investigation, RT-qPCR showed that CDKN2B-AS1 was enhanced in OS samples and cells. This research was set to examine the modulation of CDKN2B-AS1 in OS. The expression of CDKN2B-AS1 and downstream molecules was analyzed by RT-qPCR method. CCK8, EdU staining along with Transwell assays were applied to evaluate cell proliferation and invasion. Those in vitro investigations specified that silencing of CDKN2B-AS1 with shRNAs obviously impeded the proliferation and invasion of MG63 cells. To authenticate the relationships between CDKN2B-AS1 and microRNA-122-5p (miR-122-5p) or cyclin G1 (CCNG1) and miR-122-5p, we next employed luciferase reporter assay. We displayed that CDKN2B-AS1 repressed miR-122-5p to restore CCNG1 expression. All in all, our findings substantiated the indispensable function of CDKN2B-AS1 in OS progression and the possible molecular mechanism.

Differential expression of cyclins CCNB1 and CCNG1 is involved in the chondrocyte damage of kashin-beck disease.

The purpose of this study was clarify the relationship between the differential expression of cyclins CCNB1 and CCNG1 and chondrocyte damage in Kashin-Beck disease. Systematic review and high-throughput sequencing of chondrocytes derived from Kashin-Beck disease patients were combined to identify the differentially expressed cyclins and cyclin-dependent kinase genes. In parallel, weaned SD rats were treated with low selenium for 4 weeks and then T-2 toxin for 4 weeks. Knee cartilage was collected to harvest chondrocytes for gene expression profiling. Finally, the protein expression levels of CCNB1 and CCNG1 were verified in knee cartilage tissue of Kashin-Beck disease patients and normal controls by immunohistochemical staining. The systematic review found 52 cartilage disease-related cyclins and cyclin-dependent kinase genes, 23 of which were coexpressed in Kashin-Beck disease, including 15 upregulated and 8 downregulated genes. Under the intervention of a low selenium diet and T-2 toxin exposure, CCNB1 (FC = 0.36) and CCNG1 (FC = 0.73) showed a downward expression trend in rat articular cartilage. Furthermore, compared to normal controls, CCNB1 protein in Kashin-Beck disease articular cartilage was 71.98% and 66.27% downregulated in the superficial and middle zones, respectively, and 12.06% upregulated in the deep zone. CCNG1 protein was 45.66% downregulated in the superficial zone and 12.19% and 9.13% upregulated in the middle and deep zones, respectively. The differential expression of cyclins CCNB1 and CCNG1 may be related to articular cartilage damage in Kashin-Beck disease.

Three year results of Blessed: Expanded access for DeltaRex-G for an intermediate size population with advanced pancreatic cancer and sarcoma (NCT04091295) and individual patient use of DeltaRex-G for solid malignancies (IND# 19130).

Background: Innovative treatments are urgently needed for metastatic cancer. DeltaRex-G, a tumor-targeted retrovector encoding a dominant-negative/cytocidal cyclin G1 (CCNG1 gene) inhibitor construct-has been tested in over 280 cancer patients worldwide in phase 1, phase 2 studies and compassionate use studies, demonstrating long term (>10 years) survivorship in patients with advanced cancers, including pancreatic cancer, osteosarcoma, malignant peripheral nerve sheath tumor, breast cancer, and B-cell lymphoma. Patient and Methods: Endpoints: Survival, response, treatment-related adverse events. Study one is entitled "Blessed: Expanded Access for DeltaRex-G for Advanced Pancreatic Cancer and Sarcoma (NCT04091295)". Study two is entitled "Individual Patient Use of DeltaRex-G for Solid Malignancies (Investigational New Drug#19130). In both studies, patients will receive DeltaRex-G at 1-3 x 10e11 cfu i.v. over 30-45 min, three x a week until significant disease progression or unacceptable toxicity or death occurs. Results: Seventeen patients were enrolled, nine sarcoma, two pancreatic adenocarcinoma, one non-small cell lung cancer, two breast carcinoma, one prostate cancer, one cholangiocarcinoma and one basal cell carcinoma and actinic keratosis. Three patients were enrolled in Study 1 and 14 patients were enrolled in Study 2. Twelve of 17 enrolled patients were treated with DeltaRex-G monotherapy or in combination with United States Food and Drug Administration-approved cancer therapies. Five patients died before receiving DeltaRex-G. Efficacy Analysis: Of the 12 treated patients, 5 (42%) are alive 15-36 months from DeltaRex-G treatment initiation. Two patients with early-stage HR + HER2+ positive or triple receptor negative invasive breast cancer who received DeltaRex-G as adjuvant/first line therapy are alive in complete remission 23 and 16 months after DeltaRex-G treatment initiation respectively; three patients with metastatic chordoma, chondrosarcoma and advanced basal cell carcinoma are alive 36, 31, and 15 months after DeltaRex-G treatment initiation respectively. Safety Analysis: There were no treatment-related adverse events reported. Conclusion: Taken together, the data suggest that 1) DeltaRex-G may evoke tumor growth stabilization after failing standard chemotherapy, 2) DeltaRex-G may act synergistically with standard chemotherapy/targeted therapies, and 3) Adjuvant/first line therapy with DeltaRex-G for early-stage invasive carcinoma of breast may be authorized by the USFDA when patients refuse to receive toxic chemotherapy.

Tumor protein p53 mutation in archived tumor samples from a 12-year survivor of stage 4 pancreatic ductal adenocarcinoma may predict long-term survival with DeltaRex-G: A case report and literature review.

DeltaRex-G is a replication-incompetent amphotropic murine leukemia virus-based retroviral vector that displays a collagen-matrix-targeting decapeptide on its surface envelope protein, gp70, and encodes a cytocidal 'dominant negative', i.e. a truncated construct of the executive cyclin G1 (CCNG1) oncogene. DeltaRex-G inhibits the CCNG1 function of promoting cell competence and survival through the commanding CCNG1/cyclin-dependent kinase (CDK)/Myc/mouse double minute 2 homolog (Mdm2)/p53 axis. In 2009, DeltaRex-G was granted Fast Track designation from the US Food and Drug Administration for the treatment of pancreatic cancer. In 2019, the results of a phase 1/2 study that used DeltaRex-G as monotherapy for stage 4 chemotherapy-resistant pancreatic ductal adenocarcinoma (PDAC) were published. A unique participant of the aforementioned phase 1/2 study is now an 84-year-old Caucasian woman with chemoresistant PDAC who was treated with DeltaRex-G, 3x1011 colony forming units (cfu)/dose, 3 times/week for 4 weeks with a 2-week rest period, for 1.5 years. During the treatment period, the patient's tumors in the liver, lymph node and peritoneum exhibited progressive decreases in size, which were accompanied by a reduction and normalization of serum carbohydrate antigen 19-9 levels, and the patient achieved complete remission after 8 months of DeltaRex-G therapy with minimal side effects (grade 2 fatigue). Henceforth, the patient has been in remission for 12 years with no evidence of disease, no late therapy-related adverse events, and no further cancer therapy following DeltaRex-G treatment. The present study reports a mutation of tumor protein p53 (TP53) (G199V) found retrospectively in the patient's archived tumor samples. TP53 is a well-characterized tumor suppressor gene, and a critical regulatory component of the executive CCNG1/CDK/Myc/Mdm2/p53 axis, which regulates proliferative cell competence, DNA fidelity and survival. Studies are underway to determine whether TP53 mutations in pancreatic cancer can help identify a subset of patients with advanced metastatic cancer with an otherwise poor prognosis who would respond favorably to DeltaRex-G, which would broaden the treatment options for patients with otherwise lethal PDAC.

FAM225A promotes sorafenib resistance in hepatocarcinoma cells through modulating miR-130a-5p-CCNG1 interaction network.

Chemotherapy resistance is still a key hurdle in current hepatocellular carcinoma (HCC) treatment. Therefore, clarifying the molecular mechanisms contributing to this acquired resistance is urgent for the effective treatment of liver cancer. In this research, we observed that lncRNA FAM225A expression is dramatically upregulated not only in hepatocellular carcinoma tissues and cell lines but also in sorafenib-resistant HepG2/SOR cells. Moreover, FAM225A knockdown significantly weakened HepG2/SOR cells resistance to sorafenib treatment by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Similar results were obtained from the tumor xenograft model in mice. Further mechanistic researches revealed that the direct interaction between FAM225A and miR-130a-5p, while miR-130a-5p negatively modulated CCNG1 expression by targeting 3'UTR of CCNG1. MiR-130a-5p inhibition or CCNG1 overexpression could partially offset FAM225A knockdown-induced increased viability of HepG2/SOR cells in response to sorafenib challenge. Collectively, our findings provide evidence that FAM225A/miR-130a-5p/CCNG1 interaction network regulates the resistance of HCC cells to sorafenib treatment and could supply a possible strategy for restoring sorafenib sensitivity in HCC therapy.

MicroRNA-122-5p inhibits cell proliferation, migration and invasion by targeting CCNG1 in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal human malignancy, and previous researches support the contribution of microRNA (miRNA) to cancer progression. MiR-122-5p is reported to participate in the regulation of various cancers, while the function of miR-122-5p in PDAC remains unclear. In this study, we investigated the precise mechanism of miR-122-5p involved in PDAC pathogenesis. METHODS: The expression levels of miR-122-5p were detected in human PDAC tissues and cell lines by miRNA RT-PCR. The effects of miR-122-5p on cell proliferation were explored by MTT assays, colony formation assays and flow cytometry assays. The ability of migration and invasion was determined by transwell assays. Dual Luciferase reporter assay was performed to validate the direct interaction between miR-122-5p and its target gene. The related molecules of cell cycle, apoptosis and epithelial-mesenchymal transition (EMT) were examined with qRT-PCR and western blot. In addition, xenograft mouse models were applied to explore the effects of miR-122-5p in vivo. RESULTS: MiR-122-5p was underexpressed, while CCNG1 was highly expressed in PDAC tissues and cells. MiR-122-5p was negatively correlated with TNM stage, tumor size and lymph node metastasis in PDAC patients. Overexpression of miR-122-5p suppressed the proliferation, migration and invasion in vitro and inhibited tumorigenesis in vivo. Furthermore, CCNG1 was a direct target of miR-122-5p. Upregulated CCNG1 could partially reverse the effects caused by miR-122-5p. Moreover, miR-122-5p inhibited EMT through downregulation of CCNG1. CONCLUSION: Overexpression of miR-122-5p could inhibit cell proliferation, migration, invasion, and EMT by downregulating CCNG1 in PDAC, suggesting a potential therapeutic target for PDAC.

Silencing CCNG1 protects MPC-5 cells from high glucose-induced proliferation-inhibition and apoptosis-promotion via MDM2/p53 signaling pathway.

PURPOSE: Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus and one of the most important causes of end-stage renal disease, but its pathogenesis has not been elucidated so far, and there is no effective treatment. METHODS: DN models of rats and MPC-5 cells were established with streptozotocin (STZ) and high glucose (HG) in vivo and in vitro, respectively. Cell markers desmin and nephrin in foot kidney tissue were detected by Western blot. CCNG1 level in vitro was analyzed by Western blot and immunohistochemistry. CCK-8 assay and flow cytometry were conducted to analyze the effect of CCNG1 on HG-treated MPC-5 cells. Apoptosis-related proteins (Bcl-2, Bax and p53), CCNG1, and MDM2 were determined by RT-qPCR and Western blot. RESULTS: The level of nephrin was decreased, while desmin was increased in STZ-induced DN rats and CCNG1 level was also enhanced by STZ. In vitro experiments indicated that MPC-5 cell viability was inhibited and apoptosis was induced by HG and we also found that CCNG1 expression was up-regulated by HG and negatively correlated with MDM2 level. The effects of HG on MPC-5 cell viability, apoptosis, and cell cycle were reversed by silencing CCNG1, but further deteriorated by overexpression of CCNG1. Furthermore, overexpression of MDM2 inhibited HG-induced MPC-5 cell injury and CCNG1 expression. CONCLUSIONS: These findings revealed that down-regulation of CCNG1 has protection effects in DN that is mechanistically linked to MDM2-p53 pathways.

lncRNA LINC01494 Promotes Proliferation, Migration And Invasion In Glioma Through miR-122-5p/CCNG1 Axis.

BACKGROUND: Long noncoding RNAs (lncRNAs) are recognized as key effectors in tumor, including glioma. LINC01494 is an uncharacterized novel lncRNA. In this research, we aimed to investigate the function of LINC01494 in glioma. METHODS: Gene relative expression was analyzed by qRT-PCR method. CCK8, colony formation and Transwell assay was used to determine cell proliferation, migration and invasion. Bioinformatics analyses were used to predict the target of LINC01494 and miR-122-5p. Luciferase reporter assay was utilized to validate the interactions between LINC01494 and miR-122-5p or CCNG1 and miR-122-5p. RESULTS: LINC01494 was identified as a significantly upregulated lncRNA in glioma through bioinformatics analysis. Furthermore, LINC01494 upregulation indicated poor prognosis. Meanwhile, in vitro investigation indicated that silencing LINC01494 with siRNAs obviously inhibited the proliferation, cell cycle, migration and invasion of glioma cells. Besides, it is found that LINC01494 expression was negatively correlated with miR-122-5p. We demonstrated that LINC01494 inhibited miR-122-5p to upregulate CCNG1 expression through direct interaction. Rescue assay further demonstrated that LINC01494/miR-122-5p/CCNG1 signaling cascade plays a critical role in regulating glioma cell proliferation, migration and invasion. CONCLUSION: Taken together, our findings demonstrated the essential function and molecular mechanism of LINC01494 in glioma progression.

A Phase I-II Study Using Rexin-G Tumor-Targeted Retrovector Encoding a Dominant-Negative Cyclin G1 Inhibitor for Advanced Pancreatic Cancer.

Rexin-G is a replication-incompetent retroviral vector displaying a cryptic SIG-binding peptide for targeting abnormal Signature (SIG) proteins in tumors and encoding a dominant-negative human cyclin G1 construct. Herein we report on the safety and antitumor activity of escalating doses of Rexin-G in gemcitabine-refractory pancreatic adenocarcinoma, with one 10-year survivor. For the safety analysis (n = 20), treatment-related grade 1 adverse events included fatigue (n = 6), chills (n = 2), and headache (n = 1), with no organ damage and no DLT. No patient tested positive for vector-neutralizing antibodies, antibodies to gp70, replication-competent retrovirus (RCR), or vector integration into genomic DNA of peripheral blood lymphocytes (PBLs). For the efficacy analysis (n = 15), one patient achieved a complete response (CR), two patients had a partial response (PR), and 12 had stable disease (SD). Median progression-free survival (PFS) was 2.7, 4.0, and 5.6 months at doses 0-I, II, and III, respectively. Median overall survival (OS) and 1-year OS rate at dose 0-I were 4.3 months and 0%, and at dose II-III they were 9.2 months and 33.3%. To date, one patient is still alive with no evidence of cancer 10 years after the start of Rexin-G treatment. Taken together, these data suggest that Rexin-G, the first targeted gene delivery system, is uniquely safe and exhibits significant antitumor activity, for which the FDA granted fast-track designation.

MicroRNA-23b and microRNA-27b plus flutamide treatment enhances apoptosis rate and decreases CCNG1 expression in a castration-resistant prostate cancer cell line.

The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.

miR-23b suppresses lung carcinoma cell proliferation through CCNG1.

Lung carcinoma with high incidence rate could be divided into four subtypes, including small cell carcinoma, squamous cell carcinoma, adenocarcinoma and large cell carcinoma. miR-23b has been reported to have a low expression and play major roles in abundant tumors, however there is little research in lung carcinoma and hence the purpose of this study was to explore the impact of miR-23b in lung carcinoma. The RNA level of miR-23b and cyclin G1 (CCNG1) was measured by reverse transcription quantitative PCR. Luciferase activity reporter assay was used to verify that CCNG1 is a target of miR-23b. MTT and Transwell assays were utilized to test the functional studies of miR-23b in lung cancer cells. In lung carcinoma and lung cancer cells miR-23b expression is low compared with that in paracancerous tissues and normal lung cells. Low miR-23b expression inhibited lung cancer cell proliferation measured by MTT assay. We applied luciferase reporter to determine whether CCNG1 is a target of miR-23b and there was a negative correlation between them. Moreover, interference with CCNG1 reduced the cell proliferation ability, which partially reversed function of miR-23b. miR-23b inhibited cell proliferation of lung cancer by directly targeting CCNG1. It is suggested that miR-23b/CCNG1 axis may present a new target for the treatment of lung cancer.

miR-516b functions as a tumor suppressor by directly modulating CCNG1 expression in esophageal squamous cell carcinoma.

BACKGROUND: miR-516b, as a tumor suppressor in several tumors, its regulatory role in esophageal squamous cell carcinoma (ESCC) hasn't been previously reported. OBJECTIVE: This study was to investigate the potential role of miR-516b in ESCC. METHODS: miR-516b expression was measured in ESCC tumor specimens and matched adjacent non-cancerous tissues from 80 ESCC patients. The association between miR-516b and clinicopathological features of these patients was analyzed. The effect of miR-516b was evaluated by cell proliferation, migration, invasion and apoptosis assays in ESCC cell line EC9706 and TE-9. The role of miR-516b in vivo was further studied by constructing ESCC xenograft mice model. The direct target of miR-516b was predicted by public miRNA database and confirmed by luciferase reporter assay. The regulation of miR-516b on the target gene was further confirmed in vitro and in vivo. The expressions of proteins related to cell cycle and apoptosis were analyzed by western blot analysis, and cell migration and invasion were assessed by transwell assays. RESULTS: miR-516b expression was reduced in ESCC tissues and cells, and correlated with advanced TNM stage, depth of invasion, lymphatic metastasis and poorer overall survival in ESCC patients. miR-516b was upregulated by miR-516b mimics repressing cell proliferation, and inducing G1 cell cycle arrest and apoptosis. miR-516b upregulation also suppressed the growth of ESCC xenograft tumor in nude mice and the invasion of ESCC cells via regulating the epithelial-mesenchymal transition pathway. CCNG1 was identified as a direct downstream target of miR-516b. CONCLUSION: The results demonstrated miR-516b functions as a tumor suppressor by directly modulating CCNG1 expression in ESCC cells, and may be a novel therapeutic and prognostic biomarker for ESCC.

Cell cycle checkpoint control: The cyclin G1/Mdm2/p53 axis emerges as a strategic target for broad-spectrum cancer gene therapy - A review of molecular mechanisms for oncologists.

Basic research in genetics, biochemistry and cell biology has identified the executive enzymes and protein kinase activities that regulate the cell division cycle of all eukaryotic organisms, thereby elucidating the importance of site-specific protein phosphorylation events that govern cell cycle progression. Research in cancer genomics and virology has provided meaningful links to mammalian checkpoint control elements with the characterization of growth-promoting proto-oncogenes encoding c-Myc, Mdm2, cyclins A, D1 and G1, and opposing tumor suppressor proteins, such as p53, pRb, p16INK4A and p21WAF1, which are commonly dysregulated in cancer. While progress has been made in identifying numerous enzymes and molecular interactions associated with cell cycle checkpoint control, the marked complexity, particularly the functional redundancy, of these cell cycle control enzymes in mammalian systems, presents a major challenge in discerning an optimal locus for therapeutic intervention in the clinical management of cancer. Recent advances in genetic engineering, functional genomics and clinical oncology converged in identifying cyclin G1 (CCNG1 gene) as a pivotal component of a commanding cyclin G1/Mdm2/p53 axis and a strategic locus for re-establishing cell cycle control by means of therapeutic gene transfer. The purpose of the present study is to provide a focused review of cycle checkpoint control as a practicum for clinical oncologists with an interest in applied molecular medicine. The aim is to present a unifying model that: i) clarifies the function of cyclin G1 in establishing proliferative competence, overriding p53 checkpoints and advancing cell cycle progression; ii) is supported by studies of inhibitory microRNAs linking CCNG1 expression to the mechanisms of carcinogenesis and viral subversion; and iii) provides a mechanistic basis for understanding the broad-spectrum anticancer activity and single-agent efficacy observed with dominant-negative cyclin G1, whose cytocidal mechanism of action triggers programmed cell death. Clinically, the utility of companion diagnostics for cyclin G1 pathways is anticipated in the staging, prognosis and treatment of cancers, including the potential for rational combinatorial therapies.