Relationship between tumor metastasis suppressor gene KAI1 and the invasive and metastatic characteristics of osteosarcoma

AIM:To study the expression of metastasis suppressor gene KAI1 mRNA in osteosarcoma tissue and osteosarcoma cell lines,and the relationship between it and the biological behavior of the tumor cells.METHODS:RT-PCR was used to detect KAI1 mRNA in 18 cases of resected fresh osteosarcoma samples and three cultured osteosarcoma cell lines.The proliferative rate,the adhesive and invasive abilities of the 3 cell lines were detected.The results were treated by analysis system of images and analyzed with t test.RESULTS:The relative amount of KAI1 mRNA in osteosarcomas with lung metastasis was 0.80±0.50,while that was 1.48±0.64 in osteosarcomas without lung metastasis,the former was significantly lower than the latter (P<0.05).However,KAI1 mRNA had no corelation with the recurrence of osteosarcoma.The expression of KAI1 mRNA in U2-OS was highest (P<0.05),while the proliferation rate,the adhesive and invasive ability of U2-OS were the lowest among the 3 cell lines (P<0.01).CONCLUSION:The metastasis suppressor gene KAI1 might take part in influencing the lung metastasis of osteosarcoma,which might be caused by inhibiting the tumor cell proliferation,adhesion and invasion.     

Let-7a-3p inhibits activity of cancer stem cells in human lung cancer A549 cells by targeting IGF1R

AIM:To study the effect of let-7a-3p on the activity of cancer stem cells in human lung cancer A549 cells and its molecular biological mechanism. METHODS:The exepression levels of let-7a-3p in lung cancer cell lines A549, NCI-H1299, SPC-A1, H1650 and HCC-827, and human normal bronchial epithilial cell line BEAS-2B were compared by RT-qPCR. The lung cancer A549 cells were transfected with let-7a-3p mimic and negative control mimic, as let-7a-3p group and negative control group, respectively, and non-transfected control group was also set up. The content of let-7a-3p in each group was detected by RT-qPCR. Tumor sphere formation assay was used to detect the tumor sphere formation ability in 3 groups of the cancer stem cells. The proportion of cancer stem cells was detected by flow cytometry. The protein levels of NANOG, OCT4 and insulin-like growth factor 1 receptor (IGF1R) were determined by Western blot. The target gene of let-7a-3p was predicted by the bioinformatic method. The relationship between let-7a-3p and IGF1R was analyzed by double luciferase assay. Western blot was used to detect whether IGF1R over-expression antagonized the inhibitory effect of let-7a-3p on the activity of cancer stem cells. A subcutaneous transplantation tumor model was also established and the effect of let-7a-3p in vivo was observed. RESULTS:The expression level of let-7a-3p in the lung cancer cell lines was significantly lower than that in the normal bronchial epithelial cell line (P<0.01). The expression level of let-7a-3p in the A549 cells of let-7a-3p group was significantly up-regulated compared with non-transfected group (P<0.01). The number of tumor spheres in let-7a-3p group was significantly lower than that in non-transfected group. The percentage of CD133⁺ cells in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). The protein expression of NANOG and OCT4 in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). Bioinformatic prediction showed that let-7a-3p complementarily matched the 3'-UTR of IGF1R, and IGF1R might be the target gene of let-7a-3p. Luciferase assay confirmed that IGF1R was the direct downstream target gene of let-7a-3p. The protein expression of IGF1R in let-7a-3p group was significantly decreased (P<0.01). Subcutaneously transplantated tumor in let-7a-3p group was significantly smaller than that in non-transfected group. CONCLUSION:Let-7a-3p may affect the expression of lung cancer stem cell-related proteins and inhibit the potential of lung cancer stem cells by down-regulating its downstream target gene IGF1R.     

Expression of Wip1 in non small cell lung cancer tissue and cells

AIM: To detect the expression level of wip1 in lung cancer tissue and three lung cancer cell lines, and to explore the relations between the expression level of Wip1 in lung cancer and various clinical and pathological features. METHODS: Real-time PCR was employed to detect the expression of Wip1 mRNA in 44 specimens of non small cell lung cancer tissues and normal tissues. The relations between the expression of Wip1 mRNA and various clinical and pathological features were analyzed. Real-time PCR was also employed to detect the expression of Wip1 mRNA in A549, NCI-1299, NCI-H460 and HBE for relative quantitative analysis.RESULTS: In the 44 specimens, the expressions of Wip1 mRNA in both cancer tissues and normal lung tissues were positive. Wip1 gene was over-expressed in 17 specimens in 44 non small cell lung cancer specimens. The rate was 38.6%. The relative level of Wip1 mRNA in NSCLC tissues was significantly higher than that in normal lung tissues (ratio=2.1644±1.3940, P<0.01). The expression of Wip1 mRNA was also correlated with pathological grading (P<0.05). The relative level of Wip1 mRNA in three kinds of lung cancer cells was significantly higher than that in HBE cells. The difference was statistically significant (P<0.05).CONCLUSION: The Wip1 mRNA is over-expressed in non small cell lung cancer, indicating that Wip1 is related to the tumorigenesis and may become the new target of non small cell lung cancer gene therapy. The expression of Wip1 mRNA is related to tumor cell differentiation and may use for the molecular biological reference index to estimate the malignant degree of cancer.     

Expression and clinical significance of PAK4 in non-small cell lung cancer

AIM: To investigate the expression and clinical significance of PAK4 in the cell lines and tissues of non-small cell lung cancer (NSCLC). METHODS: PAK4 expression in human bronchial epithelial (HBE) cells, NSCLC cell lines, NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR and Western blot. Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression. RESULTS: PAK4 was over-expressed in the NSCLC cell lines at both mRNA and protein levels compared with HBE cells (P<0.05). PAK4 was over-expressed in the NSCLC tissues at both mRNA and protein levels compared with adjacent non-tumor tissues (P<0.05). PAK4 was over-expressed in the metastatic NSCLC tissues compared with the primary NSCLC tissues (P<0.05). Higher PAK4 staining scores were positively correlated with differentiation, lymph node metastasis, distant metastasis, and clinical stage. Kaplan-Meier analysis and log-rank test showed that overall survival was significantly different between the patients with up-regulated PAK4 and the patients with down-regulated PAK4(P<0.05). PAK4 over-expression was associated with NSCLC progression.CONCLUSION: Increased PAK4 expression was associated with tumor invasion, metastasis and prognosis in the patients with NSCLC. PAK4 is an important prognostic marker and potential therapeutic target in NSCLC.     

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

AIM: To observe the changes of proliferation and cell cycle after PRL-2 gene effectively expressed in human hepatocellular cell line.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the stable expression clones were screened by G418.The expression of PRL-2 mRNA was detected by real-time PCR.The expressive protein was identified by Western blotting.The subcellular localization was demonstrated by immunochemistry.The cell cycle was measured by flow cytometry.The population doubling time (TD) was analyzed by MTT assay.The expressions of cyclin A,cyclin D1,cyclin E,p16,p21Waf1 and p27Kip1 were detected by Western blotting.The p21Waf1 mRNA was determined by real- time PCR.RESULTS: The full length ORF of PRL-2 gene was inserted into the vector pcDNA3.1 (+),transfected into CL1 cells,and expressed successfully.Real-time PCR showed stable expression of PRL-2 mRNA.Western blotting confirmed the overexpression of PRL-2 protein.The subcellular localization of PRL-2 was in the plasmid.The proportion of cells in S-phase was increased.The population doubling time was reduced (P<0.01),a significant decrease was observed both in the mRNA and the protein expression of the p21Waf1 in comparison with untransfected or vector- transfected control cells (P<0.05).The expressions of cyclin D1,cyclin E,cyclinA,p16 and p27Kip1 were not appreciably different between the control and PRL-transfected cell lines.CONCLUSION: Eukaryocytic expression vector of PRL-2 has been successfully constructed,which shows stable and effective expression in CL1 cell line.PRL -2 increases cell proliferation by stimulating progression from G1 into S phase,which is primarily associated with decreased p21Waf1.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

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.     

Up-regulation of microRNA-187* inhibits proliferation of human colon cancer cell line HCT116

AIM:To investigate the expression of microRNA-187* (miR-187*) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187* up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS:The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187* level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), the possible target of miR-187*, was also detected. Synthetic miR-187* mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187* and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS:miR-187* was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187* in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187* expression was remarkably increased after transfection with miR-187* mimics, and ectopic expression of miR-187* significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187* mimics group, and proliferating cell nuclear antigen (PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187* mimics group were significantly increased. CONCLUSION: miR-187* is down-regulated in human colon cancer cell lines. Up-regulation of miR-187* not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.     

Silencing IDH-2 gene by siRNA-IDH-2 inhibits human small cell lung carcinoma growth

AIM：To investigate the effect of silencing isocitrate dehydrogenase 2 (IDH-2) gene by small interfering RNA (siRNA) on the biological characteristics of human small cell lung cancer cell line NCI-H446. METHODS：IDH-2 expression was knocked down in human small cell lung cancer cell line NCI-H446 by siRNA-IDH-2. The expression level of IDH-2 was determined by real-time PCR and Western blotting. The cell proliferation was measured by CCK-8 assay, the protein expression of MAPK p42 was detected by Western blotting, and the cell cycle was analyzed by flow cytometry. The migration was observed using Transwell cell migration system. BALB/c nude mice were subcutaneously injected on the back with NCI-H446 cells transfected with siRNA-IDH-2/negative control siRNA or non-transfected cells to study the tumor growth. RESULTS：siRNA-IDH-2 remarkably down-regulated the expression of IDH-2 and MAPK p42 in the NCI-H446 cells. siRNA-IDH-2 inhibited both the proliferation and migration abilities of NCI-H446 cells, and the cell cycle was arrested in S phase as compared with negative control group. Additionally, the volume of xenograft tumors in siRNA-IDH-2 group was significantly decreased as compared with control group. CONCLUSION：siRNA-IDH-2 down-regulates the expression of IDH-2 in NCI-H446 cells, reduces the cell migration efficiency and inhibits the tumor growth in vitro and in vivo.     

Expression of CD44s in lung cancer and its significance

AIM: To investigate expression of CD44s in lung cancer and it's clinical significance. METHODS: A total of 117 primary lung cancer from patients were examined for CD44s expression by immunohistochemical staining. RESULTS: CD44s mostly expressed in non-small cell lung cancer (NSCLC) but not in small ecll lung cancer (SCLC), and squamous cell carcinoma(SCC) showed much stronger expression of CD44s than adenocarcinoma(ADC)(P＜0.05). In comparison of the lung cancer with/ without lymph node metastasis, the latter showed stronger expression of CD44s(P＜0.01). According to TNM, there was a distinct statistic difference between early stage and advanced stage(P＜0.05). CONCLUSION: CD44s might be a better indicant in histological classification of lung cancer, lymph node metastasis, clinical stage and prognosis.     

Differential proteomic analysis of human lung adenocarcinoma cell line and bronchial epithelium cell line

AIM: The comparative analysis of total proteins expressed differentially between adenocarcinoma cell line A549 (a human lung adenocarcinoma) and normal cell line HBE (a human lung bronchial epithelium) was conducted to search the proteins involved in tumorigenesis and potential biomarkers of diagnosis or prognosis. METHODS: The proteins of dramatic differential expression were screened in adenocarcinoma cell Line A549 and normal cell line HBE by using immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. Furthermore, the differential expressed proteins were confirmed by the method of Western blotting. RESULTS: Compared with the two cell lines, 21 differential expressed proteins were found and their functions involves in cell metabolism, protein modification, cell motility, protein trafficking and signal transduction. The up-regulation of heat shock protein beta-1 (HSPB1) in A549 cells was identified and confirmed. CONCLUSION: These results suggest that dramatic differential proteomic expression exists between the two cell lines. The high level of HSPB1 might play an important role in the process of tumorigenesis.     

Relationship between FRAS1 protein and brain metastases of NSCLC

AIM: To explore the relationship between FRAS1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS: The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR. The protein expression of FRAS1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry. The protein expression of FRAS1 in NSCLC primary tumor tissues with or without brain metastases was also determined.RESULTS: The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues, and there was significant difference between the 2 groups (P<0.05). FRAS1 protein was expressed in the NSCLC primary tumor tissues, but was not found in the normal tissues adjacent to primary tumor tissues. The protein expression of FRAS1 in the NSCLC with brain metastases was significantly higher than that without brain metastases (P<0.01).CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC. The over-expression of FRAS1 protein may be related to brain metastases with NSCLC.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

Effect of miR-375 on viability,cell cycle and apoptosis of colon cancer HCT116 cells

AIM: To investigate the effect of microRNA-375 (miR-375) on the viability, cell cycle and apoptosis of HCT116 cells.METHODS: The expression of miR-375 in different colorectal cancer cell lines was detected by real-time PCR. The miR-375 mimics was transfected into HCT116 cells by Lipofectamine^TM 2000. The mRNA expression of miR-375 and AEG-1 was detected by real-time PCR. The HCT116 cell viability was detected by MTT assay. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry.RESULTS: Real-time PCR showed that miR-375 expression was the lowest in HCT116 among 4 colorectal cancer cell lines. The expression level of miR-375 significantly increased in miR-375 mimics group compared with that in the negative control group. The high expression level of miR-375 significantly inhibited the mRNA expression of AEG-1. After transfection with miR-375 mimics, the cell viability was inhibited, the apoptotic rate was increased, the proportion of G₁-stage cells was increased, and the proportion of S-stage cells was decreased.CONCLUSION: miR-375 inhibits the viability, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells. miR-375 may act as a tumor suppressor in colorectal cancer by inhibiting AEG-1.     

PAK6 signaling attenuates migratory and invasive abilities of non-small cell lung cancer cells

AIM：To investigate the roles of p21-activated kinase 6 (PAK6) and its target miRNA on the migratory and invasive abilities of non-small cell lung cancer cells. METHODS：miRNA candidates targeting PAK6 were predicted by a target prediction program. The expression of PAK6 was measured by real-time PCR and Western blotting after A549 cells were transfected with miR-23a mimics or inhibitory oligonucleotides. Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a. The abilities of cell migration and invasion were detected by Matrigel invasion assay and Transwell migration assay. The expression of PAK6 and matrix metalloproteinase 9 (MMP-9) was analyzed by Western blotting after A549 cells were transfected with siPAK6 or miR-23a mimics. RESULTS：miR-23a was identified by a target prediction program. Exogenetic over-expression of miR-23a resulted in a remarkable decrease in PAK6 expression (69%), whereas miR-23a inhibitory oligonucleotides induced pronounced increase in PAK6 expression (52%). The luciferase activity was significantly inhibited by 52% in wild-type PAK6 group, while there was no significant difference in the mutation group. The mRNA level of PAK6 had no change as detected by real-time PCR. Matrigel invasion assay and Transwell migration assay demonstrated there exogenetic over-expression of miR-23a markedly reduced the migration and invasion of PC-3 cells (73% and 59%, respectively). The MMP-9 expression remarkably decreased by 85％ and 76％ in the A549 cells transfected with siPAK6 and miR-23a mimics, respectively. CONCLUSION：miR-23a inhibits the migration and invasion of non-small cell lung cancer cells by repressing PAK6-MMP-9 signaling pathway.     

Mechanism of microRNA-138-5p inhibiting proliferation,migration and invasion of lung cancer cells

AIM: To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation, migration and invasion abilities of lung cancer cells.METHODS: The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group). The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p, forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR. The protein expression of FOXC1, vimentin, E-cadherin, N-cadherin and β-catenin was determined by Western blot. MTS method and colony formation assay were used to detect cell viability and proliferation ability. Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS: Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P<0.05). The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated. The proliferation, migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION: miR-138-5p inhibits the proliferation, migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin. It may be a potential target for lung cancer gene therapy.