Effects of hepatitis C virus core protein on cell cycle of HepG2 cells

AIM: To investigate the molecular mechanism of hepatitis C virus (HCV) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D1 and pRb/p130 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV-core-1b was constructed. The cDNA of HCV core protein was subcloned into pBabe-Flag-puro vector to generate pBabe-Flag-HCV-core-1b. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus-containing supernatant collected from the cell culture was used to infect HepG2 cells and subsequently the cell line that stably expressed the core protein of HCV was obtained.The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/p130 was examined by Western blotting. RESULTS: The pBabe-Flag-HCV-core-1b vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type 1b core protein impaired the cell cycle progression in G₀/G₁ phase and significantly reduced the levels of cyclin D1 and pRb/p130 in the cells. CONCLUSION: A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed, and a HepG2 cell line which stably expressed the core protein of HCV is also established. HCV gene type 1b core protein inhibits the cell cycle possibly through down-regulation of cyclin D1 and pRb/p130 proteins in the cells.     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.     

Role of translationally controlled tumor protein in PRL-3-promoted metastasis of colon cancer cells

AIM: To study the role of translationally controlled tumor protein (TCTP) in the proliferation, migration and invasion of phosphatase of regenerating liver-3(PRL-3)-promoted colon cancer cells.METHODS: The vectors pAcGFP-C3 and pAcGFP-C3-PRL-3 were constructed and transfected into the colon cancer cell line LoVo.LoVo-PRL-3 cells stably expressing PRL-3 and LoVo-control cells were established. The expression levels of PRL-3 and TCTP in both cells were detected by Western blotting and real-time PCR. The specific siRNA sequence for TCTP mRNA and control-siRNA were synthesized and transfected into the LoVo-PRL-3 cells. TCTP expression at mRNA and protein levels in LoVo-PRL-3 was detected by Western blotting and real-time PCR 24 h, 48 h and 72 h after transfection. The proliferation, migration and invasion abilities of LoVo-control cells, LoVo-PRL-3 cells, TCTP-siRNA and control-siRNA cells were detected by CCK-8 assay and the method of Transwell cell culture chambers.RESULTS: The expression of TCTP at mRNA and protein levels in LoVo cells was significantly increased after PRL-3 transfection (P<0.05). TCTP mRNA was significantly inhibited 24 h, 48 h and 72 h after transfection of TCTP-siRNA (P<0.01). TCTP protein was also significantly inhibited 48 h and 72 h after transfection (P<0.01). Compared with LoVo-control cells, the proliferation, migration and invasion abilities of LoVo-PRL-3 cells were significantly enhanced (P<0.05). However, lowering the up-regulated expression of TCTP in LoVo-PRL-3 cells inhibited the proliferation, migration and invasion abilities (P<0.05). CONCLUSION: PRL-3 promotes proliferation, migration and invasion of colon cancer cells by up-regulating the TCTP expression. siRNA targeting TCTP may be an effective method for prevention and treatment of colon cancer cell metastasis.     

Construction of lentiviral vector-mediated siRNA knockdown of ER-α36and its action on gastric cancer cell growth

AIM: To construct a lentiviral vector for stable delivery of the ER-α36gene and to detect its effect on SGC7901 cell growth. METHODS: The efficient RNAi targeting sequences identified for the ER-α36gene were screened. The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA. Then it was digested by XhoI and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector. PCR was used to screen the positive clones and sequence. The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line. Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect. 17β-estrodial at concentration of 1×10-10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined. RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors. Immunofluorescence assay demonstrated that transfection efficiency was above 80%. Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mRNA and protein levels with tetracycline (TeT) simulating as revealed by real-time PCR and Western blotting. Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silenceER-α36expression are constructed successfully and can be used to study the role of ER-α36 in gastric cancer. The ER-α36is related with many kinds of cancer cell growth, including gastric cancer cells.     

Metabolic mechanism of retinoic acid in glioma cells and its impact on cell proliferation

AIM: To investigate the mechanism for regulating the synthesis and metabolism of retinoic acid in glioma cell line SWO-Z2 and its effect on cell proliferation. METHODS: The siRNA targeting to human KLF9 mRNA was transfected into SWO-Z2 cells. The silencing efficiency was detected by real-time PCR and Western blotting. After silencing of KLF9 , the protein level of ALDH1A1 was detected by Western blotting. CCK-8 colorimetric assay was used to screen the optimal concentration of retinoic acid, and the strongest inhibitory effect of retinoic acid from 3 types of chemicals,13- cis -retinoic acid (13- cis RA),9- cis -retinoic acid (9- cis RA)and all- trans retinoic acid (ATRA), on SWO-Z2 cell growth was selected. Western blotting was also applied to explore the expression levels of cyclin D1, Bcl-2, cleaved PARP and GFAP in SWO-Z2 cells with the treatment of ATRA for 72 h. Simultaneously, the mRNA levels of retinoic acid receptors (RARs) in SWO-Z2 cells were determined by real-time PCR. RESULTS: siRNA-KLF9 knocked-down the expression of KLF9 and down-regulated the expression of aldehyde dehydrogenase 1 family member A1 (ALDH1A1) at mRNA and protein levels (P<0.05). Among the 3 retinoic acid drugs, ATRA was the most effective in inhibiting the proliferation of SWO-Z2 cells. After treated with ATRA on SWO-Z2 cells for 72 h, the expression of cleaved PARP was increased, Bcl-2 and cyclin D1 were decreased, and GFAP didn't change. The mRNA level of RARs in SWO-Z2 cells was very low. CONCLUSION: KLF9 positively regulates the expression of ALDH1A1 gene to increase the synthesis of retinoic acid. ATRA inhibits proliferation but does not induce differentiation of SWO-Z2 cells, which might result from lack of retinoic acid receptors in human glioma cells.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

RASSF1A expression mediated by lentivirus inhibits growth of small-cell lung cancer cell line H446

AIM：To explore the inhibitory effect of Ras-association domain family 1A (RASSF1A) on the small-cell lung cancer cell growth. METHODS：The lentiviral expression vector containing RASSF1A gene was constructed and used to infect the small-cell lung cell line H446. The growth curve and cell cycle were detected by MTT assay and flow cytometry. The mRNA and protein levels of cell cycle-associated proteins were determined by real-time PCR and Western blotting. RESULTS：We obtained the H446 cells in which RASSF1A was stably expressed (named RASSF1A-H446). Compared with normal cell group and negative cell group, RASSF1A inhibited the proliferation of H446 cells, and arrested H446 cells in G1 phase. The expression of p21 and p27 was significantly increased, and E2F1 was significantly decreased in RASSF1A-H446 cells. CONCLUSION：RASSF1A inhibits the H446 cell growth by increasing the expressions of p21 and p27, and decreasing the expression of E2F1.     

Apoptosis of the prostate cancer cell line PC-3M induced by E2F decoy DNA

AIM：To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS：E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry (FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS：The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION：These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.     

Effects of interferon-inducible protein 16 on proliferation and migration of human brain vascular adventitial fibroblasts

AIM：To investigate the effects of interferon-inducible protein 16 (IFI16) on the proliferation and migration of human brain vascular adventitial fibroblasts (HBVAFs). METHODS：The siRNA of IFI16 gene was transfected into HBVAFs. Forty-eight hours after transfection, the cells were exposed to 2×106 U/L interferon alpha (IFN-α) for 24 h. Cell cycle was analyzed by flow cytometry. Cell migration was determined by scratch assay and transwell method. The mRNA and protein levels of IFI16, p53 and p21 were measured by real-time PCR and Western blotting, respectively. RESULTS：After transfection with IFI16 siRNA, the expression of IFI16, p53 and p21 at mRNA and protein levels was decreased in HBVAFs, and the cell cycle at G1/S transition was promoted. Meanwhile, stimulated with IFN-α up-regulated the expression of IFI16, p53 and p21 at mRNA and protein levels, and inhibited the cell cycle transition at G1/S and cell migration in HBVAFs. Such effect was restrained by transfection with IFI16 siRNA into HBVAFs. CONCLUSION：The expression of IFI16 inhibits the proliferation and migration of HBVAFs, which may be related to the activation of p53 and p21 expression.     

Effect of over-expression of transcription factor CDX2 on proliferation and cell cycle of human gastric cancer cell line SGC-7901

AIM: To study the effect and the molecular mechanism of CDX2 over-expression on the proliferation, growth and cell cycle of human gastric cancer cell line SGC-7901. METHODS: The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were transfected with the negative control lentiviral vector for the negative control, and the cells in blank control group were without any treatment. The cell proliferation was detected by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting. RESULTS: Compared with LV-GFP group and blank control group, the proliferation activity of the SGC-7901 cells was significantly lower (P<0.05), the G0/G1 phase proportion increased (P<0.05), the mRNA and protein levels of Bcl-2, cyclin D1 and survivin were reduced (P<0.05), and the mRNA and protein levels of Bax were up-regulated (P<0.05) in LV-CDX2-GFP group. No statistically significant difference of the above indexes was observed (P>0.05) between LV-GFP group and blank control group. CONCLUSION: Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax.     

Antisense nucleic acid of K-ras inhibits growth of lung adenocarcinoma cancer cell A549

AIM: To explore the mechanism of inhibiting lung adenocarcinoma cancer mediated by antisense nucleic acid of K-ras.METHODS: The expression of K-ras was detected in A549 cells and 6 lung adenocarinoma samples. The antisense expression vector of K-ras was successfully constucted (named antisense- K-ras-pcDNA3.1). After transfection, the growth curve and Apoptosis were determined by MTT assay and Annexin V staining, respectively. Cyclin A, cyclin D1, cyclin E, CDK2, CDK4, P53, Rb and caspase-3 were detected by Western blotting. RESULTS: K-ras was highly expressed in 4 samples of lung adenocarcinoma and A549 cells. In A549 cells transfected with antisense nucleic acid of K-ras, the cell growth was significantly inhibited and the apoptosis was visible.The expression levels of cyclin A, cyclin D1, cyclin E, CDK2 and CDK4 were significantly decreased, and the expression levels of P53, Rb and caspase-3 were significantly increased. CONCLUSION: The growth inhibition in A549 cells mediated by antisense nucleic acid of K-ras is related to the decreases in the expression of cyclin A, cyclin D1, cyclin E, CDK2 and CKD4 , and the increases in the expression of P53, Rb and caspase-3.     

Dominant negative human epidermal growth factor receptor induces G0/G1 arrest in human gastric cancer cells

AIM：To explore the effect of dominant negative epidermal growth factor receptor (DNEGFR) on the cell cycle of human gastric cancer cells. METHODS：Two human gastric cancer cell lines were used in the study. The cells were divided into 6 groups, including untreated SGC-7901 cells (US group), SGC-7901 cells stably transfected with pEGFP-N1 (ES group), SGC-7901 cells stably transfected with pEGFPN1-DNEGFR (DS group), untreated NCI-N87 cells (UN group), NCI-N87 cells stably transfected with pEGFP-N1 (EN group), and NCI-N87 cells stably transfected with pEGFPN1-DNEGFR (DN group). The cell cycle was determined by flow cytometry. The protein levels of cyclin-dependent kinase 2 (CDK2), cyclin D1, phosphorylated glycogen synthase kinase 3 beta at Ser9 ［p-GSK-3β (Ser9)］, p21 and p27 were detected by Western blotting. RESULTS：Transfection of the human gastric cancer cells with pEGFPN1-DNEGFR led to G0/G1 arrest, and down-regulated CDK2, cyclin D1, p-GSK-3β (Ser9) and up-regulated p21 and p27 as well. CONCLUSION：DNEGFR down-regulates cyclin D1 by activating GSK-3β, down-regulates CDK2, and up-regulates p21 and p27, which induce G0/G1 arrest in human gastric cancer cells in the end.     

Effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells

AIM:To investigate the effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells. METHODS:Transient transfection of NANOG targeting siRNA into HepG2 cells was performed. The expression of NANOG and cyclin D1 at mRNA and protein levels was detected by real-time PCR and Western blotting. Cell proliferation was examined by CCK-8 assay and colony formation assay, and cell cycle was tested by flow cytometry. RESULTS:After transfection with NANOG-targeting siRNA, the inhibition of NANOG expression was observed. Compared with mock group, the mRNA and protein expression levels of NANOG and cyclin D1 were decreased (P<005). In addition, knockdown of NANOG expression inhibited the cell proliferation and increased the proportion of G 0/G 1-phase cells (P<0.05). CONCLUSION:Silencing of NANOG expression in HepG2 cells causes down-regulation of cyclin D1 expression and decreases the cell proliferation ability.     

Establishment of nasopharyngeal carcinoma (NPC) cell lines stable expressing NPC-derived LMP1

AIM: To establish nasopharyngeal carcinoma(NPC) cell lines stable expressing NPC-derived latent membrane protein 1(LMP1) gene.METHODS:General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed by using recombinant techniques, then transfected these vectors into a poor differentiated NPC cell line named CNE-2 ,integration and expression of N-LMP1 in CNE-2 cells were detected by PCR,RT-PCR and Western blot. RESULTS:(1) General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed successfully.(2) It showed that N-LMP1 gene expressed in CNE-2 cells correctly.CONCLUSION: The first NPC cell lines which stable express NPC-LMP1 were established. The cell lines obtained will provide important basis for exploring the role of NPC-LMP1 in nasopharynx carcinogenesis.     

Effects of RUNX3 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of RUNX3 gene on the growth and drug sensitivity of SH-SY5Y cells.METHODS: The siRNA plasmid of RUNX3 was constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting. The growth curve, cell cycle distribution, drug sensitivity assay and accumulation of adriamycin in cells were detected by MTT assay and flow cytometry. The expressions of cyclin D1, CDK4, CDK6, p21, p27, Bcl-2, Bax, P-gp and MRP were analyzed by Western blotting. RESULTS: mU6pro-RUNX3 siRNA was successfully constructed and transfected into SH-SY5Y cells. Down-regulation of RUNX3 significantly promoted the cellular proliferation, inhibit the drug sensitivity and intracellular adriamycin accumulation of cells, compared with that in the controls (P<0.05). The expressions of P-gp, Bcl-2 and cyclin D1 in transfected cells were increased, while p21 decreased.CONCLUSION: RUNX3 might play important roles in the development of neuroblastoma.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45

AIM: To investigate the effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45. METHODS: The protein levels of CADM1 in 3 human gastric carcinoma cell lines were detected by Western blotting. Eukaryotic expression vector pcDNA-CADM1 was constructed and transfected into MKN-45 cells. The MKN-45 cells stably expressing CADM1 were selected by G418 and identified by Western blotting. Furthermore, CCK-8 assay and Boyden chamber were used to analyze the effects of CADM1 overexpression on the prolife ration and invasion of gastric carcinoma cells. Western blotting was also utilized to detect the levels of cell proliferation- and invasion-related proteins. RESULTS: Relative level of CADM1 protein in MKN-45 cells was significantly lower than that in MKN-28 cells and SGC-7901 cells. Additionally, eukaryotic expression vector pcDNA-CADM1 was successfully constructed and MKN-45 cells stably expressing CADM1 were obtained. Compared with non-treatment and pcDNA3.1 groups, the proliferation of MKN-45 cells was obviously inhibited in pcDNA-CADM1 group. The result of Boyden chamber showed that the migrated cell numbers in pcDNA-CADM1 group (52.35±3.89) were significantly lower than that in untreated group (101.53±6.89) and pcDNA3.1 group (98.77±7.03). Compared with non-treatment and pcDNA3.1 groups, the protein level of p21 was significantly up-regulated and protein expression of MMP-2 and MMP-9 was obviously down-regulated. CONCLUSION: Overexpression of CADM1 may markedly inhibit cell proliferation and reduce invasion ability, and thus may be a novel target for treating gastric carcinoma.     

MicroPNA-21 promotes proliferation of rat Schwann cells following nerve injury

AIM: To investigate the molecular mechanism of microRNA-21 (miR-21) in the regulation of Schwann cell proliferation following nerve injury. METHODS: The expression of miR-2l was detected by real-time PCR. Synthetic miR-21 mimic and its control were transfected into rat Schwann cells. CCK-8 assay was performed to evaluate the influence of miR-21 on the proliferation of Schwann cells. The cell cycle distribution was determined by flow cytometry. The expression of transforming growth factor β-induced protein (TGFBI) and cyclin D1 were detected by Western blotting. RESULTS: The expression of miR-21 in model group was 7.87±0.75 and 7.75±0.80 times higher than that in sham operation group and blank group respectively. After transfected with miR-21 mimic, the expression of miR-21 in experimental group was 2.21±0.14 and 2.29±0.21 times higher than that in negative control group and blank group respectively. Moreover, the A₄₅₀ value of CCK-8 assay in experimental group at 48 h was higher than that in negative control group and blank group. The proliferation index in experimental group was higher than that in negative control group and blank group. At the same time, the expression of TGFBI obviously decreased and the cyclin D1 increased in the Schwann cells 48 h after transfection with miR-21. CONCLUSION: miR-21 promotes the proliferation activity of Schwann cells by down-regulating TGFBI expression.