1.Effect of RhoC on hepatocellular carcinoma cell growth and related molecular mechanisms.
Shu-li XIE ; Ming-guang ZHU ; Guo-yue LÜ ; Guang-yi WANG
Chinese Journal of Oncology 2011;33(4):270-275
OBJECTIVETo clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.
METHODSsiRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.
RESULTSThe inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).
CONCLUSIONSRhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.
Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein
2.Repetitive magnetic stimulation promotes neural stem cells proliferation by upregulating MiR-106b in vitro.
Hua LIU ; Xiao-hua HAN ; Hong CHEN ; Cai-xia ZHENG ; Yi YANG ; Xiao-lin HUANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2015;35(5):766-772
Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (P<0.05 or 0.01 for all). The expression levels of p21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (P<0.05 or 0.01 for all). In conclusion, our findings suggested that rMS enhances the NSCs proliferation in vitro in a dose-dependent manner and miR-106b/p21/cdks/cyclins pathway was involved in the process.
Animals
;
Animals, Newborn
;
Biomarkers
;
metabolism
;
Cell Proliferation
;
genetics
;
Cyclin-Dependent Kinase 2
;
genetics
;
metabolism
;
Cyclin-Dependent Kinase 4
;
genetics
;
metabolism
;
Cyclin-Dependent Kinase Inhibitor p21
;
genetics
;
metabolism
;
Cyclin-Dependent Kinase Inhibitor p57
;
genetics
;
metabolism
;
Cyclins
;
genetics
;
metabolism
;
Gene Expression Regulation
;
Hippocampus
;
cytology
;
metabolism
;
Ki-67 Antigen
;
genetics
;
metabolism
;
Magnetic Fields
;
MicroRNAs
;
genetics
;
metabolism
;
Neural Stem Cells
;
cytology
;
metabolism
;
Primary Cell Culture
;
Rats
;
Rats, Sprague-Dawley
;
Signal Transduction
4.siRNA-mediated CDK6 knockdown suppresses nasopharyngeal carcinoma cell growth and cell cycle transition in vitro.
Xiaopeng LUO ; Qiong XIA ; Jixin QIN ; Yongzhi HUANG ; Jin LIU ; Ying WANG ; Huaifei WANG ; Jiajun CHEN
Journal of Southern Medical University 2014;34(7):1071-1074
OBJECTIVETo assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro.
METHODSQRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors.
RESULTSCompared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21.
CONCLUSIONNPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.
Carcinoma ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Nasopharyngeal Neoplasms ; pathology ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Up-Regulation
5.Transfection of pemt-2-cDNA inhibits the expression of cell cycle related proteins in rat CBRH-7919 hepatoma cells.
Cui-ping LIU ; Wei ZOU ; Liang WANG ; Zhao-chun CUI
Chinese Journal of Hepatology 2006;14(5):350-352
OBJECTIVESTo unravel the molecular mechanism of proliferation inhibition induced by transfection of pemt2-cDNA into rat CBRH-7919 hepatoma cells.
METHODSWe started with the highly expressed PEMT2 clone. Cell culture and Western blotting techniques were used to examine the expression of cyclinD1/CDK4, cyclinE/CDK2, phospho-Rb, caspase-3, c-jun and caveolins.
RESULTSOur results showed that CDK4, CDK2, phospho-Rb and c-jun were down regulated in the pemt2 highly expressed cell clone. The high expression clone of pemt2-transfected cells also showed over expression of caspase-3.
CONCLUSIONThe reductions of proliferation and apoptosis of pemt2 transfected cells could be related to the G1 phase arrest induced by down-regulation of the cell cycle-associated proteins.
Animals ; Apoptosis ; physiology ; Caspase 3 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase 2 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase 4 ; biosynthesis ; genetics ; Cyclin-Dependent Kinases ; biosynthesis ; genetics ; Down-Regulation ; Liver Neoplasms, Experimental ; genetics ; metabolism ; Phosphatidylethanolamine N-Methyltransferase ; genetics ; metabolism ; Rats ; Transfection ; Tumor Cells, Cultured
6.Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma.
Wei WANG ; Xin LI ; Ping LIU ; Ying DONG
Journal of Peking University(Health Sciences) 2023;55(2):228-233
OBJECTIVE:
To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma.
METHODS:
In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features.
RESULTS:
The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis.
CONCLUSION
Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Male
;
Female
;
Humans
;
In Situ Hybridization, Fluorescence/methods*
;
Cyclin-Dependent Kinase 4/metabolism*
;
Liposarcoma/pathology*
;
Lipoma/pathology*
;
Gene Amplification
;
Transcription Factor CHOP/genetics*
;
Proto-Oncogene Proteins c-mdm2/metabolism*
7.Effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression of rats with cerebral ischemia.
Fang LIU ; Yu-hong WANG ; Guang-xian CAI ; Yan SHE ; Le SHAO ; Xiang-yi XIA
China Journal of Chinese Materia Medica 2015;40(20):4058-4062
To evaluate the regulating effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression in hippocampus tissues of rats with cerebral ischemia, SD rats were divided into the sham-operation group, the model group, the Buyang Huanwu decoction group (ig, 3.15 g · kg⁻¹) and the simple prescription group (ig, 2.41 g · kg⁻¹). Each group was further divided into five subgroups based on time points after the administration, i. e. 1 d, 3 d, 7 d, 14 d and 28 d, respectively. CDK4/Cyclin D1 expressions of the group at different time points were examined by using immunohistochemistry and real-time qPCR. According to the results, the cerebral ischemia model group showed higher CDK4/Cyclin D1 expression than the sham-operation groups (P < 0.05), suggesting that the cell cycle signal pathway would be activated by the cerebral ischemic injury. Both Buyang Huanwu decoction and simple prescription groups showed significantly lower cyclin expression than the model group at 3 d, 7 d, 14 d, 28 d (P < 0.05), indicating both Buyang Huanwu decoction and its simple prescription could play the neuroprotective effect through the cell cycle signal pathway.
Animals
;
Brain Ischemia
;
drug therapy
;
genetics
;
metabolism
;
physiopathology
;
Cell Cycle
;
drug effects
;
Cyclin D1
;
genetics
;
metabolism
;
Cyclin-Dependent Kinase 4
;
genetics
;
metabolism
;
Drugs, Chinese Herbal
;
administration & dosage
;
Humans
;
Male
;
Rats
;
Rats, Sprague-Dawley
8.Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica.
Ke-Xia YAN ; Bing-Ci LIU ; Xiang-Lin SHI ; Bao-Rong YOU ; Ming XU
Biomedical and Environmental Sciences 2005;18(5):286-296
OBJECTIVETo study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line (2BS) induced by silica.
METHODSRecombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control.
RESULTSDuring the progress of the malignant transformation of 2BS cells induced by silica, cyclinD1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively), and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% (the growth rate of malignant transformed cells induced by silica was 100%), doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously.
CONCLUSIONCyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.
Carcinogens, Environmental ; toxicity ; Cell Line ; Cell Proliferation ; Cell Transformation, Neoplastic ; chemically induced ; Cyclin D1 ; genetics ; metabolism ; physiology ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; physiology ; Humans ; Plasmids ; RNA, Antisense ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Silicon Dioxide ; toxicity
9.Activated protein 1-cyclin D1/E2F 1 pathways involved in cell cycle changes induced by benzo (a) pyrene.
Meng YE ; Bing-ci LIU ; Xiao-wei JIA ; Ai GAO ; Shi JIAO ; Feng-mei ZHANG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2008;26(2):72-76
OBJECTIVETo investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.
METHODSCells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.
RESULTSAfter B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P < 0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.
CONCLUSIONAP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.
Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; E2F1 Transcription Factor ; metabolism ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; metabolism ; Humans ; Transcription Factor AP-1 ; genetics ; metabolism ; Transfection
10.Effects of tea polyphenols and tea pigments on cell cycle regulators in rat liver precancerous lesions.
Xudong JIA ; Chi HAN ; Junshi CHEN
Chinese Journal of Preventive Medicine 2002;36(4):225-228
OBJECTIVESThis study is to investigate the effects of tea polyphenols and tea pigments on cell cycle regulators in rat liver precancerous lesions.
METHODSThe modified Solt-Farber precancerous liver rat model was used. Rats were given water, tea polypheol solution (0.1%) or tea pigment solution (0.1%) throughout the whole experiment (56 days). Cyclin D1, P21(WAF1/CIP1), GADD45 and PCNA protein expression were detected by Western blotting and the RT-PCR method was applied to study the expression of Cdk4.
RESULTSCyclin D1, Cdk4 and PCNA expressions were significantly inhibited, and the expression of P21(WAF1/CIP1) and GADD45 were significantly induced by tea polyphenols and tea pigments treatments.
CONCLUSIONTea polyphenols and tea pigments induced cell cycle arrest and inhibited cell proliferation by regulating cell cycle regulators.
Animals ; Blotting, Western ; Cell Cycle Proteins ; drug effects ; genetics ; metabolism ; Cyclin D1 ; drug effects ; metabolism ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclin-Dependent Kinases ; genetics ; Cyclins ; drug effects ; metabolism ; Flavonoids ; Intracellular Signaling Peptides and Proteins ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Phenols ; pharmacology ; Pigments, Biological ; pharmacology ; Polymers ; pharmacology ; Polyphenols ; Precancerous Conditions ; genetics ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; drug effects ; metabolism ; Proteins ; Proto-Oncogene Proteins ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Tea ; chemistry