Advances in the study of exosome-derived miRNAs in the pathogenesis, diagnosis, and treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an inflammatory disease caused by autoantibodies, with high morbidity and mortality. It involves multiple systems, particularly the renal, which can lead to lupus nephritis (LN); its multi-system effects have a significant impact on the physical and mental health of patients. Exosomes are vesicles that are secreted during cell activity and carry a variety of nucleic acids, proteins, and lipids. They are distributed through body fluids for cellular communication. MicroRNAs (miRNAs) are nucleic acids that are packaged within the exosome that are taken up and released in response to changes in plasma membrane structure. MiRNAs are potential participants in immune and inflammatory responses, which are transported to target cells and can inhibit gene expression in receptor cells. It has been suggested that exosomal miRNA can regulate the pathogenesis of SLE and, as such, they are of value in diagnosis and treatment. In this paper, we focus on the research progress into exosomal miRNA in SLE and inspire new directions for SLE related research.

Association of rs1137101 with hypertension and type 2 diabetes mellitus of Mongolian and Han Chinese.

BACKGROUND: Hypertension (HTN) and type 2 diabetes mellitus (T2DM) are often coincident, and each condition is considered a risk factor for the other. Both occur frequently in the Inner Mongolia region of China. The reasons for differences in risk between Han and Mongolian ethnic groups are not known. The LEPR gene and its polymorphism, rs1137101 (Gln223Arg), are both considered risk factors for HTN and T2DM, but any role of rs1137101 in the occurrence of HTN + T2DM remains unclear for Mongolian and Han populations in the Inner Mongolia region. AIM: To investigate the relationship between rs1137101 and the occurrence of HTN with T2DM in Mongolian and Han populations in Inner Mongolia. METHODS: A total of 2652 subjects of Han and Mongolian ethnic origins were enrolled in the current study, including 908 healthy controls, 1061 HTN patients and 683 HTN patients with T2DM. RESULTS: The association between the rs1137101 polymorphism and HTN with T2DM was analyzed, and differences between Han and Mongolian individuals assessed. There was a significant correlation between rs1137101 and HTN (co-dominant, dominant, over-dominant and log-additive models) and HTN + T2DM (co-dominant, dominant, over-dominant and log-additive models) after adjustment for sex and age in individuals of Mongolian origin. rs1137101 was significantly associated with HTN (co-dominant, recessive and log-additive models) and HTN + T2DM (co-dominant, dominant, over-dominant and log-additive models) in the Han Chinese population. CONCLUSION: Mongolian and Han subjects from Inner Mongolia with HTN who had rs1137101 were protected against the development of T2DM. Allele A has the opposite impact on the occurrence of HTN in Mongolian and Han Chinese populations.

Epithelial-mesenchymal transition and metastasis of colon cancer cells induced by the FAK pathway in cancer-associated fibroblasts.

OBJECTIVE: The role and mechanism of tetrathiomolybdate (TM) in cancer-associated fibroblasts (CAFs) in colon cancer using three-dimensional (3D) culture were investigated, and the associations between the focal adhesion kinase (FAK) pathway and epithelial-mesenchymal transition (EMT) in CAFs were explored. METHODS: A 3D co-culture model of colon cancer LOVO cells with CAFs and normal fibroblasts (NFs) was established using Matrigel as a scaffold material. The differential expression of LOXL2 (lysyl oxidase-like 2) in the supernatant of CAFs and NFs was determined using ELISA, and expression levels of EMT-related proteins and FAK signaling pathway-related proteins were determined using western blot. RESULTS: LOXL2 levels secreted by CAFs were higher compared with that secreted by NFs. In the CAF + LOVO group, compared with the LOVO group, E-cadherin expression decreased significantly, while N-cadherin and F-PAK expression increased significantly. TM results were opposite compared with the above results. CONCLUSIONS: CAFs stimulate EMT in human colon cancer LOVO cells by secreting LOXL2 to activate the FAK signaling pathway, thereby promoting tumor metastasis. TM inhibited the occurrence of EMT in the CAF-induced colon cancer LOVO cell line, thereby reducing the invasion and metastasis of colon cancer cells.

Transcriptional Regulation of Latency-Associated Transcripts (LATs) of Herpes Simplex Viruses.

Herpes simplex viruses (HSVs) cause cold sores and genital herpes and can establish lifelong latent infection in neurons. An engineered oncolytic HSV (oHSV) has recently been approved to treat tumors in clinics. HSV latency-associated transcripts (LATs) are associated with the latent infection, but LAT transcriptional regulation was seldom reported. For a better treatment of HSV infection and tumors, here we sequenced the LAT encoding DNA and LAT transcription regulatory region of our recently isolated new strain HSV-1-LXMW and did comparative analysis of the sequences together with those of other four HSV-1 and two HSV-2 strains. Phylogenetic analysis of LATs revealed that HSV-1-LXMW is evolutionarily close to HSV-1-17 from MRC University, Glasgow, UK. For the first time, Using a weight matrix-based program Match and multi-sequences alignment of the 6 HSV strains, we identified HSV LAT transcription regulatory sequences that bind to 9 transcription factors: AP-1, C-REL, Comp1, E2F, Hairy, HFH-3, Kr, TCF11/MAFG, v-Myb. Interestingly, these transcription regulatory sequences and factors are either conserved or unique among LATs of HSV-1 and HSV-2, suggesting they are potentially functional. Furthermore, literature analysis found that the transcription factors v-myb and AP-1 family member JunD are functional in regulating HSV gene transcription, including LAT transcription. For the first time, we discovered seven novel transcription factors and their corresponding transcription regulatory sequences of HSV LATs. Based on our findings and other reports, we proposed potential mechanisms of the initiation and maintenance of HSV latent infection. Our findings may have significant implication in our understanding of HSV latency and engineering of better oncolytic HSVs.

Anticancer effect of ursolic acid via mitochondria-dependent pathways.

Ursolic acid is a plant-derived pentacyclic triterpenoid found in various medicinal herbs and fruits. It has generated clinical interest due to its anti-inflammatory, antioxidative, antiapoptotic and anticarcinogenic effects. An increasing amount of evidence supports the anticancer effect of ursolic acid in various cancer cells. One of the hallmarks of malignant transformation is metabolic reprogramming that sustains macromolecule synthesis, bioenergetic demand and tumor cell survival. Mitochondria are important regulators of tumorigenes is as well as a major site of the metabolic reactions that facilitate this reprogramming and adaption to cellular and environmental changes. The current review explored the close association between the anticancer effect of ursolic acid and the activation of mitochondrial-dependent signaling pathways.

miR-1915-3p inhibits Bcl-2 expression in the development of gastric cancer.

Many gene expressions changed during the development of gastric cancer, and non-coding RNAs including microRNAs (miRNAs) have been found to regulate cancer progression by participating in the process of tumor cell growth, migration, invasion and apoptosis. Our previous study has identified 29 miRNAs that are highly expressed in gastric cancer stem cells. One of these miRNAs, miR-1915-3p, has shown great potential as a diagnostic and prognostic biomarker for the cancers in liver, colon and thyroid, as well as in immune and kidney diseases. Herein, we found that miR-1915-3p exhibited low expression level in differentiated gastric cancer cell lines and gastric cancer tissues. It was found that the miR-1915-3p inhibited the growth of gastric cancer cells and thus promoted cell apoptosis. We discovered that the expressions of miR-1915-3p were significantly correlated to the lymph node metastasis and overall survival of patients with gastric cancer. Further study showed that there was a negative correlation between miR-1915-3p and Bcl-2 (B cell lymphoma/leukemia-2) expression, suggesting that Bcl-2 was a target gene of miR-1915-3p. Hence, miR-1915-3p possibly contributes to the development and progression of gastric cancer by inhibiting the anti-apoptotic protein Bcl-2. The finding provides a potential therapeutic strategy for gastric cancer.

Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway.

AIM: We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms. METHODS: Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 µg/mL, ip) for 10 days. RESULTS: Treatment of HCT116 cells with ACBPs (35 µg/mL) for 4-6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 µg/mL) for 6-12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues. CONCLUSION: Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway.

MicroRNAs May Serve as Emerging Molecular Biomarkers for Diagnosis and Prognostic Assessment or as Targets for Therapy in Gastric Cancer.

Gastric cancer (GC) is one of the most common cancers, with high incidences in East Asia countries. Most GC patients have been reported with low early diagnosis rate and show extremely poor prognosis. Therefore, it is necessary to develop novel and more sensitive biomarkers to improve early diagnosis and therapy in order to provide longer survival and better quality of life for gastric cancer patients. MicroRNAs (miRNAs) play crucial roles in GC development and progression. miRNAs have emerged as a novel molecular biomarker for cancer diagnosis, prognosis and therapy with surprising stability in tissues, serum or other body fluids. This review summarizes major advances in our current knowledge about potential miRNA biomarkers for GC that have been reported in the past two years.

[Quercetin enhances the anti-leukemic effect of adriamycin].

This study was purposed to explore the anti-leukemic mechanism of quercetin (Que) in vivo and it enhancing chemotherapeutic effect of adriamycin (ADR) by establishing the quercetin-treated P388 transplanted nude mouse model. The P388 leukemic cells in logarithmic growth phase were taken and injected subcutaneously into BALB/c nude mice so as to establish the leukemia-transplanted nude mouse model. The model mice were treated by quercetin, ADR and their combination, and the survival changes of model mice were observed, the hemogram and peripheral blood cell count examination were performed regularly; the cell cycle was detected and the influence of quercetin on cell proliferation was analyzed by flow cytometry; the caspase-3 protein expression level was detected by ELISA; the mRNA and protein changes of NF-κB, BCL-2, BAX were measured by real-time quantitative flourascence PCR and Western blot respectively. The results indicated that the quercetin and adriamycin could significantly prolong the survival of P388 leukemia nude mice, and their combination displayed significantly prolonged effect. Quercetin and adriamycin alone or in their combination could reduce the ratio of G0/G1 phase in mice, the cell ratio in S phase and G2/M phase increased, and the effects of the combination group were more significant than that of the single agent groups. Quercetin could activate caspase-3 and promote leukemic cell apoptosis. Meanwhile, quercetin could down-regulate the expression of BCL-2 and NF-κB gene, and up-regulate the expression of BAX gene. It is concluded that through modulating the expression of apoptosis-related genes, the quercetin can inhibit leukemia cell proliferation, promote apoptosis, and enhance the chemotherapeutic effects of adriamycin. These results provide some valuable data for further research and development of quercetin as a new and effective anti-leukemic drug.

Association of miR-193b down-regulation and miR-196a up-regulation with clinicopathological features and prognosis in gastric cancer.

Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumor development, progression, and carcinogenesis. However, their clinical implications for gastric cancer remain elusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissues from those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNA expression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRT- PCR) was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationship between altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Among a total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues from gastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193b and miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Lauren type, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression of miR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regression analysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjusted OR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patients with a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; median survival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; median survival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR- 196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-change of up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a may be applied as novel and promising prognostic markers in gastric cancer.

Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells.

AIM: To investigate the effects of osteopontin (OPN) gene expression knockdown on colon cancer Lovo cells in vitro. METHODS: Four candidate small interfering RNA (siRNA) constructs targeting the OPN gene and a scrambled control sequence (NC-siRNA) were synthesized and inserted into a pGPU6/GFP/Neo expression vector. After confirmation by restriction enzyme digestion and DNA sequencing, the recombinant plasmids were subsequently transfected into a human colon cancer cell line (Lovo) using a liposome transfection method. Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1, -2, -3, -4, and Lovo-NC cells. Knockdown efficiency of each of the four siRNA constructs was determined by real-time reverse transcription polymerase chain reaction assays and western blotting, and the construct with the most effective silencing was used for subsequent experiments. Cell proliferation, adhesion, and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells. The levels of four angiogenic factors, namely vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays (ELISA). RESULTS: Recombinant vectors containing OPN-specific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells. Compared with the control Lovo and Lovo-NC cells, the levels of OPN mRNA and protein expression in Lovo-OPN-1, -2, -3, and -4 were significantly reduced (all P < 0.05), with the most efficient reduction observed in Lovo-OPN-4 cells (P < 0.05). Relative to untransfected Lovo cells, OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008 ± 0.067 and 0.160 ± 0.023, respectively. The relative OPN protein expression levels in Lovo, Lovo-NC, and Lovo-OPN-4 cells were 3.024 ± 0.211, 2.974 ± 0.630, and 0.121 ± 0.008, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of OPN. After 24, 48, 72, and 96 h of cultivation, absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210 ± 0.017, 0.247 ± 0.024, 0.314 ± 0.037, and 0.359 ± 0.043, respectively, which were significantly lower than those of Lovo (0.244 ± 0.031, 0.313 ± 0.024, 0.513 ± 0.048 and 0.783 ± 0.051) and Lovo-NC cells (0.241 ± 0.029, 0.309 ± 0.022, 0.563 ± 0.023, and 0.735 ± 0.067) (all P < 0.05). The absorption values at 595 nm, which were measured in a cell adhesion assay, showed that adhesion of Lovo-OPN-4 cells (0.215 ± 0.036) was significantly decreased compared to Lovo (0.490 ± 0.037) and Lovo-NC cells (0.462 ± 0.043) (P < 0.05). The number of invasive Lovo-OPN-4 cells (16.1 ± 1.9) was also significantly decreased compared to Lovo (49.9 ± 5.4) and Lovo-NC cells (48.8 ± 4.5) (P < 0.05). ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF (1687.85 ± 167.84 ng/L vs 2348.54 ± 143.80 ng/L and 2284.39 ± 138.62 ng/L, respectively), MMP-2 (2966.07 ± 177.36 µg/L vs 4084.74 ± 349.54 µg/L and 4011.41 ± 424.48 µg/L, respectively), MMP-9 (3782.89 ± 300.64 µg/L vs 5062.90 ± 303.02 µg/L and 4986.38 ± 300.75 µg/L, respectively) and uPA (1152.69 ± 120.79 µg/L vs 1380.90 ± 147.25 µg/L and 1449.80 ± 189.92 µg/L, respectively) (all P < 0.05). CONCLUSION: Knockdown of OPN gene expression suppresses colon cancer cell growth, adherence, invasion, and expression of angiogenic factors.

Anticancer bioactive peptide (ACBP) inhibits gastric cancer cells by upregulating growth arrest and DNA damage-inducible gene 45A (GADD45A).

Recently, we reported that anticancer bioactive peptide (ACBP), purified from goat spleens immunized with human gastric cancer extracts, significantly inhibited gastric cancer cells in vitro and gastric tumors in vivo via repressing cell growth and promoting apoptosis, making it a promising potential biological anticancer drug. However, it is not known what genes are functionally required for the ACBP effects. Here, we first found that two tumor suppressor genes, cyclin-dependent kinase inhibitor 2B (CDKN2B) and growth arrest and DNA damage-inducible alpha (GADD45A), were upregulated significantly in the cells with ACBP treatment by microarray screening and the findings were validated by real-time RT-PCR. Next, GADD45A mRNA and protein expressions were downregulated in the gastric cancer cells by lentivirus-mediated RNAi; then, cell viability, cell cycle, and apoptosis were assayed by MTT and flow cytometry. Interestingly, our results indicated that cell viability was not dependent on GADD45A without ACBP treatment; however, cell sensitivity to ACBP was significantly decreased in ACBP-treated gastric cancer cells with GADD45A downregulation. Therefore, we demonstrate that GADD45A was functionally required for ACBP to inhibit gastric cancer cells, suggesting that GADD45A may become a biomarker for ACBP sensitivity. Our findings have significant implications on the molecular mechanism understanding, biomarker development, and anticancer drug development of ACBP.

[Effect of human colon carcinoma-associated fibroblasts on biological behavior of colon carcinoma Lovo cells].

OBJECTIVE: To investigate the effects of human colon carcinoma-associated fibroblasts (CAFs) on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells. METHODS: The co-culture models among colon CAFs, NFs and Lovo cell were established by conditioned medium (CM) of human colon CAFs and colon normal fibroblasts (NFs). Lovo cells in the blank control group was treated with serum-free culture medium. The effects of human colon CAFs on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells were detected by cell proliferation assay, adhesion assay, migration assay and Transwell invasion assay. RESULTS: After co-culture with colon CAFs, the absorbance (A) value of Lovo cells was (0.667±0.059) in 48 h and (0.709±0.030) in 72 h. The A value of Lovo cells adhesion to fibronectin was (0.588±0.067). The cell mobility rates were (35.2±8.7)% in 12 h and (64.6±7.1)% in 24 h. The number of invasive cell was (56.2±4.8). All the above parameters were increased compared with those in the blank control group and NFs group (all P<0.01). CONCLUSION: Human colon CAFs can promote the proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells.

Comprehensive study on associations between nine SNPs and glioma risk.

AIM: Glioma cancer is the most common type of adult brain tumor. Recent genome-wide association studies (GWAS) have identified various new susceptibility regions and here we conducted an extensive analysis of associations between 12 single nucleotide polymorphisms (SNPs) and glioma risk. METHODS: A total of 197 glioma cases and 197 health controls were selected, and 9 SNPs in 8 genes were analyzed using the Sequenom MassARRAY platform and Sequenom Assay Design 3.1 software. RESULTS: We found the MAF among selected controls were consistent with the MAF from the NCBI SNP database. Among 9 SNPs in 8 genes, we identified four significant SNP genotypes associated with the risk of glioma, C/C genotype at rs730437 and T/T genotype at rs1468727 in ERGF were protective against glioma, whereas the T/T genotype at rs1799782 in XRCC1 and C/C genotype at rs861539 in XRCC3 conferred elevated risk. CONCLUSION: Our comprehensive analysis of nine SNPs in eight genes suggests that the rs730437 and rs1468727 in ERGF, rs1799782 in XRCC1 gene, and rs861539 in XRCC3 gene are associated with glioma risk. These findings indicate that genetic variants of various genes play a complex role in the development of glioma.

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca).

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 µg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.

cDNA cloning, overexpression, purification and pharmacologic evaluation for anticancer activity of ribosomal protein L23A gene (RPL23A) from the Giant Panda.

RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A) gene of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF) of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 µg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids orientation for the research and development of cancer protein drugs as well as possible anti-cancer mechanisms. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL23A responsible for its anticancer activity.

Polymorphism in pre-miR-30c contributes to gastric cancer risk in a Chinese population.

To explore the association between single nucleotide polymorphisms (SNPs) A/G in pre-miR-30c and gastric cancer risk and to assess various genotypes that affected the survival of patients, we performed a hospital-based, case-control study in Inner Mongolia autonomous region of China. A total of 240 gastric cancer patients and 240 age- and sex-matched cancer-free controls collected from 2006 to 2011 were enrolled in this study. Polymorphism rs928508 in pre-miR-30c was analyzed by TaqMan SNP genotyping assay. The expression of mature miR-30c was detected by real-time quantitative reverse transcription PCR in 48 gastric cancer specimens. Survival analysis was analyzed by the Kaplan-Meier survival curves. The genotype frequencies of pre-miR-30c A/G in gastric cancer patients were obviously different from those in the controls (P = 0.022). AA genotype carriers were associated with an increased risk of gastric cancer compared with GG genotype (adjusted odds ratio (OR) = 1.83, 95% confidence interval (CI): 1.07-3.15, P = 0.029). Moreover, the gastric cancer risk especially elevated in older individuals (aged >60 years), males, nonsmokers, and Helicobacter pylori (H. pylori)-infected individuals (adjusted OR = 2.66, 95% CI: 1.38-5.13, P = 0.004; adjusted OR = 1.90, 95% CI: 1.10-3.27, P = 0.022; adjusted OR = 1.94, 95% CI: 1.12-3.35, P = 0.018; adjusted OR = 1.83, 95% CI: 1.08-3.10, P = 0.024, respectively). Further stratified analysis indicated that AA genotype facilitated developing of gastric cancer with lymph node metastasis (adjusted OR = 2.23, 95% CI: 1.07-4.64, P = 0.032). Expression analysis detected that rs928508 AA showed a significantly increased level of mature miR-30c compared with GG or AG/GG genotype (P = 0.011 or P = 0.013). Patients with AA genotype were associated with unfavorable outcome in overall survival compared with AG/GG genotype (Log rank 5.848, P = 0.016). This study demonstrates that pre-miR-30c A/G polymorphism may be associated with an increased risk of gastric cancer in a Chinese population through altering mature miR-30c expression. However, their role as novel biomarkers for the diagnosis and prognosis of gastric diseases still needs to be systematically evaluated.

NQO1 C609T polymorphism correlated to colon cancer risk in farmers from western region of Inner Mongolia.

OBJECTIVE: To investigate the relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) C609T polymorphism and colon cancer risk in farmers from western region of Inner Mongolia. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to analyze NQO1 C609T polymorphism from 160 healthy controls and 76 colon cancer patients. RESULTS: Among the colon cancer patients, the incidence of NQO1 T allele (53.29%) was significantly higher than it in control group (33.75%, P<0.001). The individuals with NQO1 T allele had higher risk [2.239 (95% CI: 1.510-3.321) times] to develop colon cancer than individuals with NQO1 C allele. The incidence of NQO1 (T/T) (34.21%) in colon cancer patients was higher than that in control group (15.62%, P<0.001). Odds ratios (OR) analysis suggested that NQO1 (T/T) and NQO1 (T/C) genotype carriers had 3.813 (95% CI: 1.836-7.920) times and 2.080 (1.026-4.219) times risk compared with wild-type NQO1 (C/C) gene carriers in developing colon cancer. Individuals with NQO1 (T/T) genotype had 2.541 (95% CI: 0.990-6.552) times, 3.713 (95% CI: 1.542-8.935) times, and 3.471 (95% CI: 1.356-8.886) times risk than individuals with NQO1 (T/C) or NQO1 (C/C) genotype in well-differentiated, moderately-differentiated, and poorly-differentiated colon cancer patients, respectively. CONCLUSIONS: NQO1 gene C609T could be one of risk factors of colon cancer in farmers from western region of Inner Mongolia.

Cytochrome P450 2E1 RsaI/PstI and DraI Polymorphisms Are Risk Factors for Lung Cancer in Mongolian and Han Population in Inner Mongolia.

OBJECTIVE: To explore the relationship between cytochrome P450 2E1 (CYP2E1) RsaI/PstI and DraI polymorphism and lung cancer susceptibility in Mongolian and Han population in Inner Mongolia of China. METHODS: CYP2E1 RsaI/PstI and DraI polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in 64 lung cancer patients, 150 healthy Mongolian and 150 healthy Han individuals. The distribution of genotype and allele frequencies of CYP2E1 RsaI/PstI and DraI polymorphisms were studied. RESULTS: The risk of lung cancer was increased in individuals with CYP2E1 (cl/cl) and CYP2E1 (DD) with OR values of 2.431 (95%CI=1.082-5.460) and 2.778 (95%CI=1.358-5.683) respectively (P<0.05). When CYP2E1 RsaI/PstI and DraI polymorphisms were combined, the risk of lung cancer was reduced in individuals with CYP2E1 (cl/c2+c2/c2 and DD+CC) with OR values of 0.233 (95%CI=0.088-0.615, P<0.05). In smokers, the susceptibility to lung cancer was higher in the individuals with CYP2E1 (c1/c1) and CYP2E1 (DD) than in the individuals with c2 and C allele (P<0.05, OR=2.643 and 4.308 respectively). There was no significant difference in distribution of CYP2E1 genotype frequency between healthy Mongolian, Han population and lung cancer patients, healthy controls in Inner Mongolia. CONCLUSION: CYP2E1 (c1/c1) and CYP2E1 (DD) are predisposing factors of lung cancer in population in Inner Mongolia. CYP2E1 (c2ï¹¢C) co-mutation may decrease the risk of lung cancer. Smoking exerts synergetic effect with CYP2E1 (c1/c1) and CYP2E1 (DD) on the occurrence of lung cancer.

Identification of prognosis-related proteins in advanced gastric cancer by mass spectrometry-based comparative proteomics.

PURPOSE: The objective of this study was to identify differentially expressed proteins of advanced gastric cancer from patients with different prognosis using NanoLC-MS/MS (LTQ) (nanoflow liquid chromatography system interfaced with a linear ion trap LTQ mass spectrometer). METHODS: Eight gastric cancer patients with relatively early TNM stage and survival time >34 months were identified as good survival (group G), while the other eight with late stage and survival time <15 months as poor survival (group P). The total protein of the tissue samples from each group was extracted and pooled together respectively. The resulting two protein mixtures were trypsin-digested and analyzed using NanoLC-MS/MS (LTQ). Database searches were done against NCBI non-redundant database and SWISS-PROT database and the identified proteins were classified through an online Web Gene Ontology Annotation Plot tool. Immunohistochemistry was used to verify candidate prognosis-related proteins. RESULTS: There were 284 and 213 proteins identified for group G and group P respectively. And 117 proteins were detected exclusively in group G and 46 proteins exclusively in group P. These protein markers function in calcium ion signaling pathway, cellular metabolism, cytoskeleton formation, stress reaction, etc. Among those, the down-regulated expression of S100P was verified to claim a poor clinical outcome of gastric cancer patients (P = 0.0375). CONCLUSION: The MS-based proteomics approach is efficient in identifying differentially expressed proteins in relation to prognosis of advanced gastric cancer patients. These differentially expressed proteins could be potential prognosis-related cancer markers and deserve further validation and functional study.