Clinical significance of KLF15 expression in human lung adenocarcinoma

AIM: To explore the clinical significance of Krüpple-like factor 15 (KLF15) protein expression in the patients with lung adenocarcinoma for exploring the therapeutic and prognositic biomarkers of lung cancer. METHODS: Four cases of lung adenocarcinoma tissues and matched adjacent tissues were collected from our hospital, and the expression of KLF15 protein in these tissues was analyzed by Western blot. At the same time, 72 cases of archived paraffin-embedded samples and clinical data of the patients with lung adenocarcinoma were also collected. The KLF15 protein expression in the archived paraffin-embedded lung adenocarcinoma samples was detected by immunohistochemical staining. The correlations between KLF15 protein expression and clinical characteristics of the patients including prognosis were also analyzed. In addition, the KLF15 protein was up-regulated in A549 cells, and then the effects of KLF15 protein on the viability of the cells were measured by CCK-8 assay. RESULTS: The protein expression of KLF15 in the 4 cases of lung adenocarcinoma tissues was significantly lower than that in matched paracancerous tissues. Fifty-three cases of lung adenocarcinoma specimens showed low expression or no expression of KLF15 protein in total 72 cases (73.6%). The 5-year survival rate of the patients with high expression of KLF15 protein in their specimens was higher than that of the patients with the low expression of KLF15 protein (P<0.01), and the expression of KLF15 protein was significantly correlated with the pathological staging (P<0.01) and T stage (P<0.01) of the patients with lung adenocarcinoma. Furthermore, the low expression of KLF15 protein was an important poor prognostic indicator of the patients. Up-regulation of KLF15 protein in the A549 cells significantly inhibited the growth of the cells. CONCLUSION: KLF15 inhibits the growth of lung adenocarcinoma cells. It could be used as a therapeutic target and a prognostic biomarker for the patients with lung adenocarcinoma.     

Clinical significance of 4-1BBL protein expression in human glioma tissues

AIM: To investigate the expression of 4-1BBL(CD137L) protein in human glioma tissues, and to analyze its clinical significance. METHODS: The expression levels of 4-1BBL protein in 82 human glioma specimens were measured by the method of immunohistochemistry. The correlation of 4-1BBL protein level with histopathologic features and survival time was analyzed. RESULTS: 4-1BBL protein positive cells were observed in human brain astrocytic tumors, which were not found in meningioma and normal brain tissues. In our study, the expression of 4-1BBL protein in 3 cases of normal brain tissues was negative. The expression of 4-1BBL protein in 31 cases of glioma specimens were positive, and total positive rate was 37.8% (31/82). The expression of 4-1BBL protein was related to the survival time and age of the patients, but not related to the pathological grade of glioma. Statistically, the patients with positive expression of 4-1BBL protein had longer survival time (P<0.05). CONCLUSION: Expression of 4-1BBL protein may have reference value to predict the prognosis of patients with glioma.     

Clinical significance of SUMO4 expression in papillary thyroid carcinoma

AIM: To discuss the relationship between small ubiquitin-related modifier 4 (SUMO4) expression and papillary thyroid carcinoma (PTC). METHODS: The mRNA and protein levels of SUMO4 in PTC and normal thyroid tissues were determined by the methods of real-time PCR, Western blot and immunohistochemistry. The relationship between SUMO4 expression and clinicopathologic parameters in PTC was evaluated. RESULTS: The results showed that both the mRNA and protein levels of SUMO4 expression in PTC were obviously higher than those in normal thyroid tissues (P<0.01). CONCLUSION: The high level of SUMO4 expression in PTC suggests that SUMO4 plays an important role in PTC development and it is probably a molecular mechanism of PTC.     

Prokaryotic expression and functional study of human high mobil ity group box 1 protein

AIM:To construct the prokaryotic expressio n plasmid of His-tagged human high mobility group box 1 fusion protein (hHMGB1) and to express the fusion protein in E.coli for the affinity purification.METHODS:The cDNA coding region of HMGB1 was amplified by PCR fr om pGEX4T-HMGB1 and cloned into a modified pET14b vector following the routine p rocedure.After identification by enzyme digestion,PCR and sequencing,the plas mid was transformed into BL21 (DE3) competent cells,and the His-HMGB1 fusion pro tein was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPT G),and further purified by Ni-NTA affinity chromatography.The protein was f il tered for sterilization and used to stimulate human umbilical vein endothelial c ells (HUVECs).24 hours later,the cultured supernatant of HUVECs was collected for the detection of cytokines/chemokines with LiquiChip system.RESULTS:The His-HMGB1 fusion protein expression plasmid was ide ntified by enzyme digestion and sequencing.The purified His-tagged fusion prote in was analyzed by SDS-PAGE and Western blotting with specific anti-His antibody .It was found that the production of IL-8 from HUVECs was highly induced in a d ose-dependent manner by HMGB1.CONCLUSION:The His-tagged HMGB1 fusion protein expression plasm id was successfully constructed,and purified.Recombinant HMGB1 protein has a h igh bioactivity on the induction of cytokines in HUVECs,which may significantly facilitate the future study of HMGB1 biological functions.     

Clinical significance of LN/LN-R expression in nasal and paranasal sinus malignant tumor

AIM: To study the clinical significance of laminin/laminin receptor(LN/LN-R) expression in nasal and paranasal sinus malignant tumor(NPMT).METHODS: The immunohistochemical technique was used to detect the expression of LN and LN-R in 137 cases of NPMT, 78 cases of para-cancer tissues and 37 cases of non-cancer tissues.RESULTS: The positive rate of LN in NPMT(13.87%) was significantly lower than that in para-cancer tissues(87.18%) and non-cancer tissues(72.97%), and the positive rate of LN-R in nasal inverting papilloma(76.47%) was significantly higher than that in NPMT(28.47%), para-cancer tissues(10.26%) and nasal polypus and cyst tissues(10.0%). A significant difference of LN-R expression, but not LN, between endepidermis and soft tissue of the NPMT was observed(P<0.01). CONCLUSION: Decrease in LN expression provides the essential condition for the invasion and metastasis of the malignant tumor. Determining the expression level of LN in NMPT will be helpful for evaluation of tumor development and clinical management. High expression of LN-R only in the nasal inverting papilloma indicates that LN-R may play different roles in different pathotypes of NPMT.     

Cloning of P1 and P3 promoters of human insulin-like growth factorⅡ gene

AIM:To clone P1 and P3 promoters of the human insulin-like growth factor Ⅱ(IGF-Ⅱ) gene. METHODS:According to the complete DNA sequence of IGF-Ⅱ gene, the nested primer PCR was performed for amplifying P1 and P3 promoter fragments of the gene from human L-02 cell line.These PCR products were analyzed by agarose gel electrophoresis, and cloned by using TOPO TA Cloning kit. The positive clones containing P1 and P3 fragments were selected and confirmed by sequencing.RESULTS:The DNA sequences of P1 and P3 promoters cloned were accordant with GenBank data. CONCLUSION:In this study P1 and P3 promoters of the IGF-II gene were cloned successfully.     

Expression of GATA3 in human breast cancer tissues and its clinical significance

AIM: To investigate the expression of GATA3 in human breast carcinoma and its clinical significance. METHODS: The expression level of GATA3 in breast cancer tissues from 124 patients was detected by the method of immunohistochemistry and the relationships between GATA3 expression and other clinicopathological factors were analyzed. RESULTS: Low expression of GATA3 in breast cancer tissues was associated with estrogen receptor (ER)/progesterone receptor (PR) negative, high histological tumor grade, p53 mutation and vascular invasion (P<005), but not with age, tumor size,human epidermal growth factor receptor 2 (HER-2) expression and lymph node metastasis (P>005). In all breast cancer tissues, the positive expression rate of GATA3 was 56.4%. The positive expression rate of GATA3 in luminal breast cancer is 684%, higher than that in non-luminal breast cancer (326%, P<005). In all breast cancer tissues, the expression of GATA3 in middle recurrence risk group was higher than that in high recurrence risk group (P<005). CONCLUSION: GATA3 expression in breast cancer is related to differentiation and biological characteristics of the tumor, which can be a factor for evaluation of the treatment and prognosis.     

Cholesterol depletion up-regulates expression of prohibitin in human prostate carcinoma PC-3 cells

AIM: To study how cholesterol delpetion affects prohibitin expression and the development of prostate cancer. METHODS: Human prostate carcinoma PC-3 cells were cultured in normal medium (NM) and cholesterol delpetion medium (CDM) for 48 h.The mRNA expression of prohibitin was detected by real-time PCR. Prohibitin promoter was cloned and inserted into PGL3-Basic to reconstruct plasmid. The plasmid was transiently transfected into PC-3 cells. The cells were cultured in the medium with different concentrations of cholesterol for 48 h and luciferase expression was detected.RESULTS: The results of real-time PCR showed that the mRNA level of prohibitin increased about 19 times in PC-3 cells in the presence of CDM. After transfected with prohibitin promoter plasmid for 48 h, PC-3 cells cultured in CDM had higher luciferase expression than the cells cultured in NM or in CDM with cholesterol. CONCLUSION: Cholesterol depletion up-regulates prohibitin expression in PC-3 cells, which may be one of the self-prophylactic and regulatory mechanisms to protect PC-3 cells from apoptosis caused by cholesterol insufficiency.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.     

Clinical significance of STMN1 expression in cervical cancer and effect of inhibition of its expression on viability and apoptosis of cervical cancer SiHa cells

AIM: To investigate the clinical significance of stathmin 1 (STMN1) expression in cervical cancer and the influence of its expression on the viability and apoptosis of cervical cancer cells. METHODS: Western blot was used to detect the protein expression of STMN1 in cervical cancer tissues, and the relationship between the expression and clinical characteristics of cervical cancer was analyzed. STMN1-siRNA was transfected into cervical squamous-cell carcinoma SiHa cells. The protein levels of STMN1, STAT3, p-STAT3 and survivin were determined by Western blot after transfection for 48 h. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. DCFH-DA probe was used to detect the level of reactive oxygen species (ROS). RESULTS: The protein expression of STMN1 in cervical cancer tissues was significantly higher than that in paracancerous tissues (P<0.01). The STMN1 protein expression level was not correlated with age and histological types of cervical cancer patients, but was related to clinical stage, histological differentiation and lymph node metastasis (P<0.01). Transfection with STMN1-siRNA significantly reduced the expression of STMN1 in SiHa cells. Compared with control group, the cell viability in STMN1-siRNA group was significantly decreased, the apoptotic rate and ROS content were increased, and the protein levels of p-STAT3 and survivin were down-regulated (P<0.01). However, no significant difference of the STAT3 protein level was observed between STMN1-siRNA group and control group. CONCLUSION: STMN1 is highly expressed in cervical cancer, and its expression is related to clinical stage, histological differentiation and lymph node metastasis. Inhibition of STMN1 expression reduces the viability and promotes apoptosis of cancer cells by down-regulating STAT3 signaling pathway.     

Prognostic significance of CUEDC2 expression in hepatocellular carcinoma

AIM: To investigate the expression of CUE domain-containing 2 (CUEDC2) in hepatocellular carcinoma (HCC) and to analyze its clinical prognostic significance. METHODS: Total 186 formalin-fixed paraffin-embedded tissues obtained from surgical HCC with detailed clinicopathological and follow-up data were used. The expression of CUEDC2 was detected by immunohistochemistry. The relationships between the expression of CUEDC2 and clinicopathological characteristics and prognosis were analyzed. RESULTS: The positive rate of CUEDC2 in HCC was 85.5% (159/186), among which, the low expression was 52.2% (97/186) and the high expression was 47.8% (89/186). CUEDC2 expression was correlated with serum alpha-fetal protein (AFP) level, tumor size, tumor number, tumor differentiation and TNM stage (P<0.05). Kaplan-Meier survival curves showed that the patients with high expression of CUEDC2 were associated with significantly shorter overall survival and recurrence-free survival than those with low CUEDC2 expression (P<0.05). Multivariate Cox regression analysis revealed 3 independent prognostic factors including CUEDC2 expression, serum AFP and tumor number. CONCLUSION: CUEDC2 was expressed in most HCC tissues, which was relevant to tumor growth, tumor differentiation and prognosis. CUEDC2 could be a novel valuable molecular marker to predict the HCC prognosis.     

Clinical significance of Kir2.1/KCNJ2 and MRP1/ABCC1 protein expression in small-cell lung cancer

AIM: To investigate the expression of inward rectifying potassium channel 2.1 (Kir2.1/KCNJ2) and multidrug resistance-associated protein 1 (MRP1/ABCC1) in small-cell lung cancer (SCLC), and the correlation between them. METHODS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69 and H69AR cell lines was detected by real-time PCR and Western blotting. Immunohistochemistry (EnVision) method was used to detect the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 proteins in 44 cases of SCLC tissues. The interaction of Kir2.1/KCNJ2 with MRP1/ABCC1 was tested by co-immunoprecipitation (co-IP). RESULTS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69AR cells was all significantly higher than that in H69 cells (P<005). The positive expression rates of Kir2.1/KCNJ2 and MRP1/ABCC1 in the 44 cases of SCLC tissues were 47.7% (21/44) and 52.3% (23/44), respectively. A positive correlation was found between the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 in SCLC tissues (r=0.853, P<0.01). Co-IP results showed that Kir2.1/KCNJ2 could bind MRP1/ABCC1 in H69AR cells. CONCLUSION: Kir2.1/KCNJ2 is related to the multidrug resistance of SCLC. Kir2.1/KCNJ2 and MRP1/ABCC1 may have common function roles. Therefore, Kir2.1/KCNJ2 may promote the multidrug resistance of SCLC by regulating the expression of MRP1/ABCC1.     

Expression and regulatory effect of P73 antisense RNA 1T in tumors

The discovery of abundant new long non-coding RNAs (lncRNAs) and extensive investigation of their roles in various diseases have been reported, especially in cancers. The lncRNA P73 antisense RNA 1T (TP73-AS1) is involved in dysregulation of cell signaling and closely correlated with cancer development, progression, and response to therapy. This review is a brief update of the current knowledge related to the role of TP73-AS1 in cancer-associated molecular pathways and pathophysiology, and possible determinants for TP73-AS1 to function as a biomarker, aiming to stimulate the basic investigation of TP73-AS1 as well as its translation to clinical applications.