Expression and clinical significance of SCNM1 in hepatitis B-related hepatocellular carcinoma

AIM: To investigate the expression of sodium channel modifier 1 (SCNM1) in hepatitis B-related hepatocellular carcinoma (HCC) and its relationship with clinicopathological features and prognosis.METHODS: The specimens were collected from 108 patients with hepatitis B-related HCC who were treated in the Third Affiliated Hospital of Sun Yat-sen University from January 2013 to December 2015. All patients signed the informed consent and met the requirements of medical ethics. The mRNA expression level of SCNM1 in hepatitis B-related HCC tissues and tumor-adjacent tissues was detected by RT-qPCR, and the relationship between the mRNA expression of SCNM1 and the clinicopathological characteristics of hepatocellular carcinoma was analyzed. The relationship between SCNM1 expression and the prognosis of the patients was analyzed by Kaplan-Meier plotter.RESULTS: The data from TCGA database, Human Protein Atlas database and Oncomine database showed that the expression of SCNM1 in hepatocellular carcinoma tissues was significantly higher than that in the normal liver tissues (P<0.01). SCNM1 was mainly distributed in the nucleus. The results of RT-qPCR showed that the median mRNA expression of SCNM1 in hepatitis B-related HCC tissues was significantly higher than that in the matched tumor-adjacent tissues (t=8.082, P<0.01). The mRNA expression of SCNM1 was correlated with cirrhosis, alanine aminotransferase and tumor size (P<0.05), but not with sex, age and tumor envelope. The total survi-val time of the HCC patients with high expression of SCNM1 was shorter than that of the patients with low expression of SCNM1 (HR=1.53, P=0.016), and that of the patients with hepatitis B-related HCC was even shorter (HR=2.41, P=0.015).CONCLUSION: SCNM1 is highly expressed in hepatitis B-related HCC and may play an important role in the development of hepatitis B-related HCC.     

Expression of TLR2 and TLR4 in hepatocellular carcinoma

AIM: To investigate the expression of TLR2 and TLR4 in hepatocellular carcinoma (HCC), and to analyze their correlations to clinicopathologic features of HCC. METHODS: The protein and mRNA levels of TLR2 and TLR4 in HCC and para-tumor tissue were determined by immunohistochemistry and real-time fluorescence quantitative PCR (RFQ-PCR). RESULTS: The protein and mRNA levels of TLR2 and TLR4 in HCC were lower than those in para-tumor tissue (P<0.01). At protein level, the intensities of TLR2 and TLR4 expression in HCC and para-tumor tissue were significantly higher than those in normal liver tissue (P<0.01 and P<0.05). Furthermore, the expressions of TLR2 and TLR4 in HCC with cancer embolism in portal vein were weaker than those in HCC without cancer embolism (2= 9.458,P<0.05). CONCLUSION: The intensities of TLR2 and TLR4 expression are lower in HCC than those in para-tumor tissue, whereas higher than those in normal liver tissue. TLR2 and TLR4 signals might take part in the course of HCC.     

miR-15a-5p inhibits proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells

AIM: To observe the influence of high expression of miR-15a-5p on the proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells.METHODS: The miR-15a-5p oligonucleotide, which was reconstructed with additional restriction sites of EcoR Ⅰ and Hind Ⅲ, was chemically synthesized and confirmed by sequencing. The miR-15a-5p eukaryotic expression system was constructed by pcDNA6.2-GW/Em-GFP-pre-miR-15a-5p plasmid. The miR-15a-5p was transfected into the SMMC-7721 cells transiently by plasmid, and quantified by quantitative real-time PCR at the mRNA level. The cell viability was measured by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The migration ability of the SMMC-7721 cells with high expression of miR-15a-5p was detected by wound healing test.RESULTS: The sequence of miR-15a-5p oligonucleotide 100% matched the designed sequence. Compared with control group, the miR-15a-5p expression was increased significantly (P<0.05). The viability, the living cell number and the migration ability of the SMMC-7721 cells were decreased in high expression of miR-15a-5p group with statistically significant difference (P<0.05).CONCLUSION: The abilities of proliferation and migration in human hepatocellular carcinoma SMMC-7721 cells are decreased by high expression of miR-15a-5p.     

Effect of ZNF281 on proliferation of hepatocellular carcinoma cells

AIM:To investigate the role of zinc finger protein 281 (ZNF281) in the proliferation of hepatocellular carcinoma (HCC) cells. METHODS:The mRNA expression levels of ZNF281 in peripheral blood mononuclear cells from 80 cases of healthy people and 206 cases of HCC patients were determined by real-time PCR. Statistical analysis were used to illustrate the relationship between the mRNA expression levels of ZNF281 in the peripheral blood mononuclear cells and the clinicopathologic parameters of HCC patients. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of ZNF281 in hepatoma cell lines and immortalized hepatocytes. The silencing of ZNF281 was conducted by transfection of small interfering RNA targeting ZNF281, and then the proliferation of HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was tested by Cell-Light^TM EdU Apollo^®488 In Vitro Imaging Kit. The ability of colony formation of the HCC cells was measured by colony formation assay, and the ability of anchorage-indepen-dent growth was detected by soft agar test. RESULTS:The mRNA expression level of ZNF281 in the peripheral blood mononuclear cells from HCC patients was significantly increased compared with the healthy people, and the high expression level was positively correlated with tumor size, tumor stage and tumor vascular invasion. Concurrently, the expression level of ZNF281 in hepatoma cell lines was significantly higher than that in immortalized hepatocytes. More importantly, the silencing of ZNF281 inhibited the proliferation, DNA synthesis, colony formation and anchorage-independent growth of the HCC cells. CONCLUSION:ZNF281 promotes the proliferation of HCC cells.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

Expression of human leukocyte antigen E in hepatocellular carcinoma cell lines

AIM: To investigate the human leukcyte antigen E (HLA-E) expression in hepatocellular carcinoma cell lines. METHODS: The techniques of real-time PCR and Western blotting were used to study the HLA-E expression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real-time PCR showed that no statistical difference of HLA-E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA-E mRNA expression in Hep3B2.1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA-E protein levels between L02 cells and the 5 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2.1-7), and no HLA-E protein in Hep3B2.1-7 cells was detectable. CONCLUSION: Asynchronization of HLA-E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.     

Effect of low expression of miR-21 on apoptosis of HepG2 cells induced by matrine

AIM: To explore whether miR-21 low expression enhances the effect of matrine (MAT) on the apoptosis of hepatocellular carcinoma cells.METHODS: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-21 in the HepG2 cells treated with different concentrations of MAT. The effect of miR-21 on MAT-induced HepG2 cell apoptosis was analyzed by flow cytometry. The mRNA and protein expression of Bcl-2 and Bax in the HepG2 cells treated with MAT was determined by RT-qPCR and Western blot.RESULTS: The expression of miR-21 increased with the increasing concentration of MAT. Low expression of miR-21 promoted MAT-induced apoptosis, and enhanced the expression of Bax at mRNA and protein levels (P<0.05), while inhibited the expression of Bcl-2 at mRNA and protein levels (P<0.05).CONCLUSION: Low expression of miR-21 enhances MAT-induced HepG2 cell apoptosis by inhibiting the expression of Bcl-2 and promoting Bax expression.     

Role of IGF-Ⅱ in proliferation and migration of hepatocellular carcinoma Huh7 cells

AIM: To observe the effects of insulin-like growth factorⅡ (IGF-Ⅱ) at different expression levels on hepatocellular carcinoma Huh7 cell proliferation and migration, and to explore the role of IGF-Ⅱ in the development of HCC. METHODS: The Huh7 cells were transfected with the over-expression plasmid pcDNA3.1(+)- IGF-Ⅱ or RNA interference plasmid pLVX-shRNA-IGF-Ⅱ by Lipofectamine 2000. The quantitative real-time PCR and Western blotting were used to verify the expression of IGF-II. The biological behaviors of the Huh7 cells were analyzed by CCK-8 assay, plate clone formation assay, cell scratch test and Transwell chamber experiment.RESULTS: Over-expression of IGF-II promoted the growth and migration of hepatocellular carcinoma cells (P < 0.05), and the cell proliferation was significantly inhibited in the Huh7 cells with low IGF-II expression (P < 0.05).CONCLUSION: IGF-II is involved in the regulation of biological behavior of hepatocellular carcinoma Huh7 cells in vitro, which may play a promoting role in the development of hepatocellular carcinoma.     

Expression of eEF1A2 in hepatocellular carcinoma

AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G₀/G₁ phase were significantly decreased with increased percentage of the cells in S and G₂/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G₀/G₁ phase to S phase and G₂/M phases.     

Expression of Cdc25a in cross-species hepatocellular carcinoma tissues and its significance

AIM：To study the expression of cell division cycle 25a protein (Cdc25a) in hepatocellular carcinoma (HCC) tissues of different species, including human, rat and tree shrew, and to verify the feasibility of the strategy of cross-species oncogenomics screening. METHODS：Real-time fluorescence quantitative PCR and Western blotting were applied to detect the expression of Cdc25a at mRNA and protein levels in the HCC tissues, corresponding HCC-adjacent liver tissues and normal liver tissues collected from humans, rats and tree shrews. RESULTS：The mRNA expression of Cdc25a in the HCC tissues of humans, rats and tree shrews was higher than that in normal liver tissues (P<0.05). The mRNA levels of Cdc25a in the HCC tissues of humans and rats were higher than those in the corresponding HCC-adjacent liver tissues (P<0.05). The mRNA expression of Cdc25a in the HCC tissues was significantly correlated with the portal vein tumor thrombus, the extrahepatic metastasis and the clinical stage, but was not correlated with the recurrence of tumor, the diameter of tumor, the number of tumor, the level of serum alpha-fetoprotein and the differentiation of tumor. The protein levels of Cdc25a in the HCC tissues of humans, rats and tree shrews were higher than those in the corresponding HCC-adjacent liver tissues and the normal liver tissues. CONCLUSION：Cdc25a may be a particularly crucial molecule for hepatocarcinogenesis. The cross-species oncogenomics screening may represent a feasible and convenient way for identifying key molecules of human HCC.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Expression of microRNA-1284 in gastric cancer and underlying mechanism

AIM: To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism. METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer (GC) tissue samples and 63 non-malignant adjacent tissue samples. The correlation between miR-1284 and the clinicopathological feature of GC was analyzed. Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells. After transfection, the expression of miR-1284 was examined by real-time PCR. The cell activity was evaluated by CCK-8 assay. The cell cycle and apoptosis were determined by flow cytometry. The ability of cell migration was measured by wound-healing assay. The potential target gene of miR-1284 was predicted by online bioinformatic softwares. The expression of JAG1 mRNA was examined by real-time PCR. The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting. RESULTS: Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples (P<0.05). The expression level of miR-1284 was not significantly associated with age and gender of the patients, tumor size, TNM staging and lymph node metastases (P>0.05), but significantly associated with histologic grading (P<0.05). Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly increased (P<0.05), the percentages of apoptotic cells and the cells in G₀/G₁ phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05). CONCLUSION: The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

Changes of miR-155-5p in serum of cervical cancer patients and effects of miR-155-5p on cervical cancer cells

AIM:To detect the serum level of miR-155-5p in the patients with different cervical diseases, and to analyze its effects on the proliferation, cell cycle and apoptosis of cervical cancer cells. METHODS:SYBR Green I real-time quantitative PCR was used to detect the level of miR-155-5p in the serum of the patients with different cervical diseases. miR-155-5p mimic or inhibitor was used to increase or decrease the expression of miR-155-5p in cervical cancer cells. The proliferation, cell cycle and apoptosis were measured by CCK-8 assay and flow cytometry. RESULTS:The serum level of miR-155-5p in cervical cancer group was higher than that in cervicitis group and healthy group. No statistical difference of the serum miR-155-5p level between cervical intraepithelial neoplasia group and cervical cancer group was observed. Compared with blank group, liposome group and negative control group, the proportion of S-phase cells increased and apoptotic cells decreased in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p mimic. The proportion of G2/M-phase cells increased significantly in SiHa cells transfected with 100 nmol/L and 200 nmol/L miR-155-5p inhibitor. CONCLUSION: Compared with healthy controls, the serum level of miR-155-5p in the cervical cancer patients increases, and may act as a novel tumor molecular marker for diagnosis of cervical cancer. miR-155-5p has no significant effect on the proliferation, cell cycle and apoptosis of HeLa cell. miR-155-5p may promote SiHa cells to enter S phase and inhibit the apoptosis of SiHa cells.     

Analysis of gene network regulated by microRNA-375 in HCC

AIM: To investigate the expression of microRNA-375 (miR-375) in hepatocellular carcinoma (HCC) and to analyze the target genes and signaling pathways regulated by miR-375. METHODS: The expression of miR-375 was examined at tissue microarray of HCC by in situ hybridization. The whole human genome chip and bioinformatics analysis were applied to screen out the differential expression genes and signaling pathways in 4 HCC cell lines transfected with miR-375 mimic. RESULTS: In situ hybridization showed the expression of miR-375 in HCC tissues were obviously higher than that in tumor-adjacent tissues (P < 0.05). There were 20 co-upregulated genes and 17 co-downregulated genes in all 4 cell lines. Bioinformatic analysis showed that there were 54 signaling pathways related to up-regulated genes and 48 signaling pathways related to down-regulated genes in all 4 cell lines. CONCLUSION: miR-375 may play a key role in the pathological process of HCC. The bioinformatic analysis is able to screen the target genes and signaling pathways regulated by miR-375 and to provide an explicit direction for further mechanism research on HCC.     

miR-509 regulates growth,invasion and migration of human hepatocellular carcinoma LM3 cells and survival of nude mice

AIM: To investigate the effect of microRNA-509 (miR-509) on the growth, invasion and migration of human hepatocellular carcinoma (HCC) LM3 cells and survival of tumor-bearing nude mice. METHODS: LM3 cells were transferred with miR-509 mimic and pcDNA Ras-related C3 botulinum toxin substrate 1 (pcRac1), and the expression of Rac1 was measured by Western blot. The relationship between miR-509 and Rac1 was determined by luciferase reporter assay. The invasion ability was determined by Transwell assay, and the migration ability was measured by wound healing assay. Xenograft model of HCC was established by subcutaneous injection with LM3 cells into nude mice. The survival rate of the mice were recorded and the protein level of Rac1 was determined by Western blot. RESULTS: miR-509 mimic inhibited the expression of Rac1 in the LM3 cells (P<0.05). pcRac1 attenuated the effect of miR-509 on Rac1. miR-509 also alleviated luciferase activity of wild Rac1 (P<0.05). Meanwhile, miR-509 mimic decreased the number of invasive LM3 cells and inhibited the migration of LM3 cells (P<0.05). In addition, over-expression of miR-509 up-regulated survival rate of model mice and decreased the protein level of Rac1 in the tumor tissue (P<0.01). CONCLUSION: miR-509 inhibits the invasion and migration of HCC cells and promotes the survival of tumor-bearing nude mice through inhibiting the expression of Rac1.     

miR-21 enhances resistance of glioma cells to carmustine by down-re-gulating PTEN

AIM:To explore the function of miR-21 in human glioma cells resistant to carmustine and to elucidate its related mechanism. METHODS:SWOZ2 cells were transfected with miR-21 mimics(SWOZ2-miR-21mimics) or miRNA mimics negative control(control group) by the method of jetPRIME. The real-time fluorescence quantitative PCR was used to detect and compare the levels of miR-21 expression between BCNU-resistant cell line SWOZ2-BCNU and BCNU-sensitive cell line SWOZ2, or between SWOZ2-miR-21 mimic group and control group. The drug sensitivity of these cells to BCNU was determined by CCK-8 assay. The protein expression of phosphatase and tensin homology deleted on chromosome 10(PTEN), phosphorylated protein kinase B(p-Akt) and P-glycoprotein(P-gp) in these cells were also detected by Western blotting. RESULTS:The expression level of miR-21 was remarkably higher in SWOZ2-BCNU cells than that in SWOZ2 cells. The expression level of miR-21 was significantly higher in SWOZ2-miR-21 mimics group than that in control group. The half-maximal inhibitory concentration(IC₅₀) of BCNU was obviously higher for SWOZ2-BCNU cells than that for SWOZ2 cells. The IC₅₀ of BCNU was markedly higher in SWOZ2-miR-21 mimics group than that in control group. PTEN protein expression was remarkably lower, but p-Akt and P-gp protein expression levels were markedly higher in SWOZ2-BCNU cells than those in SWOZ2 cells. The protein level of PTEN was significantly lower, but the protein levels of p-Akt or P-gp were distinctly higher in SWOZ2-miR-21 mimics group than those in control group. CONCLUSION:miR-21 enhances the resistance of human glioma cells to BCNU by down-regulating the expression of PTEN protein.     

Expression of miR-203 in human tongue carcinoma tissues and its in-fluence on viability and invasion ability of Tca8113 cells

AIM: To study the expression of miR-203 in tongue carcinoma tissues and the effect of miR-203 over-expression on the viability and invasion ability of Tca8113 cells.METHODS: Twenty-eight pairs of tongue carcinoma tissues and adjacent nontumor tissues were collected, and the clinicopathological characters were analyzed. miR-203 was detected in the tongue tissues of 28 patients with tongue carcinoma by real-time PCR. miR-203 mimics and scramble were transfected into Tca8113 cells by Lipofectamine 2000. The expression of miR-203 was detected in Tca8113, Tca8113-miR-203 mimics and Tca8113-scramble cells by RT-qPCR. The cell viability was measured by CCK-8 assay. The cell invasion ability was determined by Transwell chamber invasion experiment.RESULTS: miR-203 expression was significantly down-regulated in the tongue carcinoma tissues compared with those in the adjacent nontumor tissues. The expression of miR-203 was associated with TNM stage and lymph node metastasis. Up-regulation of miR-203 inhibited the viability and invasion ability of Tca8113 cells.CONCLUSION: miR-203 suppresses the growth and invasion of tongue carcinoma cells. miR-203 may be a potential therapeutic target for treating human tongue cancer.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.