MiRNA-153-3p promotes gefitinib-sensitivity in non-small cell lung cancer by inhibiting ATG5 expression and autophagy.

OBJECTIVE: To clarify the function of miRNA-153-3p in gefitinib-sensitive non-small cell lung cancer (NSCLC) and the underlying mechanism. PATIENTS AND METHODS: The expressions of miRNA-153-3p, LC3B and ATG5 in gefitinib-resistant and gefitinib-sensitive NSCLC tissues were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miRNA-153-3p to LC3B or ATG5 was analyzed. We evaluated autophagy level in gefitinib-resistant NSCLC cells by calculating the percentage of PC-9/GR and HCC827/GR cells with positive GFP-LC3, as well as determining autophagy-related gene levels. The potential binding between ATG5 and miRNA-153-3p were verified by Dual-Luciferase reporter gene assay. The regulatory effects of miRNA-153-3p/ATG5 on gefitinib-sensitivity and apoptosis were finally examined by cytotoxicity assay and Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) staining, respectively. RESULTS: MiRNA-153-3p was lowly expressed in gefitinib-resistant NSCLC relative to the gefitinib-sensitive ones. MiRNA-153-3p was negatively correlated with autophagy activity marker LC3B in gefitinib-resistant NSCLC patients. Compared with parental cells, gefitinib-resistant NSCLC cell lines PC-9/GR and HCC827/GR presented a lower level of miRNA-153-3p and a higher level of autophagy. The overexpression of miRNA-153-3p greatly inhibited autophagy level. ATG5 could directly bind to miRNA-153-3p, and ATG5 was highly expressed in gefitinib-resistant NSCLC. The correlation analysis found a negative correlation between ATG5 and miRNA-153-3p and a positive correlation between ATG5 and LC3B in gefitinib-resistant NSCLC. More importantly, ATG5 reversed the regulatory effects of miRNA-153-3p on autophagy, gefitinib-sensitivity and apoptosis of PC-9/GR and HCC827/GR cells. CONCLUSIONS: MiRNA-153-3p is lowly expressed in gefitinib-resistant NSCLC patients. The overexpression of miRNA-153-3p enhances gefitinib-sensitivity in NSCLC by inhibiting autophagy via downregulating ATG5.

[Clinical analysis of the double-wing flap for treatment of toe syndactyly].

Objective: To explore the clinical effect of double-wing flap for the treatment of toe syndactyly. Methods: Retrospective analysis of 47 patients (60 syndactyly toes) who underwent double-wing flap to reconstruct toe web space in orthopedics department of the Third Affiliated Hospital of Zhengzhou University from February 2010 to October 2017.There were 21 males and 26 females, with an average age of 18.9 months (range: 10-48 months). All patients were treated with zigzag incisions to separate the toe syndactylys without skin grafts.The condition of wound healing and appearance of toes were observed. Results: The average follow-up time was 62.3 months (range: 6 to 80 months). There were no complications such as hematoma, infection, flap necrosis and no flexion contracture and obvious scar hyperplasia in all the 47 cases, 4 of the 60 webs developed web creep.All webs had good appearance with 45 degrees inclination from the dorsal to the metatarsal side and had good flexion and abduction function. Conclusions: The double-wing flap is a simple and safe operation for toe syndactyly which has a good clinical effect.

Long non-coding RNA SNHG15 indicates poor prognosis of non-small cell lung cancer and promotes cell proliferation and invasion.

OBJECTIVE: To investigate the expression of human long non-coding ribonucleic acid (RNA) small nucleolar RNA host gene 15 (SNHG15) in non-small cell lung cancer (NSCLC) tissues and its prognostic significance, and to study the influencing mechanism of SNHG15 on biological functions in lung cancer cell lines. PATIENTS AND METHODS: The expression levels of SNHG15 in 49 pairs of lung cancer tissues and para-carcinoma tissues were detected via quantitative real-time polymerase chain reaction (qRT-PCR). The lung cancer cells were transiently transfected with small-interfering (si)-SNHG15 using RNA interference technique. The effect of si-SNHG15 on the proliferation of lung cancer cells was observed via methyl thiazolyl tetrazolium (MTT) assay, its effect on apoptosis of A549 cells was detected via Hoechst 33342 staining and flow cytometry, and its effects on invasion and migration of A549 cells were studied via wound healing assay and transwell assay. RESULTS: Results of qRT-PCR showed that the expression of SNHG15 in cancer tissues was increased compared with that in para-carcinoma tissues. Results of cell counting kit-8 (CCK-8) assay showed that knocking down SNHG15 could significantly inhibit the proliferation of lung cancer A549 cells. Hoechst 33342 staining and flow cytometry revealed that knocking down SNHG15 could significantly promote apoptosis of A549 cells. Wound healing assay and transwell assay revealed that knocking down SNHG15 could significantly inhibit the invasion and metastasis capacities of lung cancer A549 cells. Results of Western blotting showed that knocking down SNHG15 could inhibit the invasion and metastasis of A549 cells through inhibiting the expressions of epithelial-mesenchymal transition (EMT), matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. CONCLUSIONS: The expression of SNHG15 in lung cancer tissues is significantly higher than that in para-carcinoma tissues, the prognosis of patients accompanied with a high expression of SNHG15 is poor, and knockdown of SNHG15 in A549 cells can inhibit cell proliferation, invasion, and metastasis, and promote apoptosis.

Effects of miR-143 overexpression on proliferation, apoptosis, EGFR and downstream signaling pathways in PC9/GR cell line.

OBJECTIVE: The functions of microRNAs in the regulation of apoptosis in non-small cell lung cancer (NSCLC) and the application in the therapeutical treatments were intensively studied. However, whether overexpression of miR-143 in lung cancer cells will affect the cell behaviors, such as proliferation or some underlying pathway, is largely unknown. This study aimed to examine the effect of miR-143 in PC9/GR cell line on the proliferation, apoptosis, EGFR and downstream signal pathways. MATERIALS AND METHODS: The non-small cell lung cancer (PC9/GR) cells were treated with concentration-increased gefitinib to acquire gefitinib resistance. Then, the acquired gefitinib-resistance cells were divided into 3 groups, blank control group (BC group), negative control group (NC group), and miR-143 transfected group (miR-143 group). miR-143 mRNA was detected by quantitative PCR. The proliferation was detected by CCK-8. The cell apoptosis was determined by flow cytometry. The expression of EGFR and downstream signal pathway factors of p-EGFR, AKT, p-AKT, ERK1/2 and p-ERK1/2 were detected by Western blot. RESULTS: The cell proliferation in miR-143 transfected group was significantly suppressed compared with BC and NC group, while the apoptosis was dramatically increased. The p-EGFR, p-AKT, p-ERK1/2 protein expression was significantly inhibited. CONCLUSIONS: These results demonstrated that overexpression of miR-143 downregulated cell proliferation, promoted the apoptosis, and suppressed the phosphorylation of EGFR, AKT and ERK1/2; thus, miR-143 may play a role in treatment of NSCLC to enhance therapeutic efficacy.

Analysis of allopolyploidy-induced rapid genetic and epigenetic changes and their relationship in wheat.

We used the conventional and methylation-sensitive randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses to assess genome-wide changes and explore the relationships between genetic and epigenetic variations among individuals of a newly synthesized allohexaploid wheat line whose genomic constitution is identical to that of the natural common wheat, compared with its parent plants and a natural counterpart named Chinese Spring. We found rapid, extensive, and predominantly consistent non-Mendelian changes in the form of genetic and DNA methylation variations in the allohexaploid individuals. Specifically, at least 30-40% of the epigenetic component was truly independent of genetic changes, which answered a critical question, i.e. its autonomy in relation to the genetic context. Striking correlations were detected between genetic and epigenetic changes. Interestingly, as previously reported, the paternally donated nuclear genomes showed more genetic changes than the maternally donated ones; the loss of paternal bands was significantly correlated with the hypomethylation of CG or CHG sequences, suggesting an unknown link between genetic instability and hypomethylation. Sequence analysis indicated that most variations occurred in the cellular genes and sequences related to transposable elements. Based on these findings, the possible mechanisms and effects of the genomic changes in allopolyploid speciation and evolution were discussed.

Effect of exemestane on the invasive growth of endometrial carcinoma HHUA cells.

OBJECTIVE: To investigate the effect of exemestane on HHUA human endometrial carcinoma cells. MATERIALS AND METHODS: The HHUA human endometrial carcinoma cells were treated with various concentrations of exemestane, and its effects on cell growth and apoptosis were investigated in vitro. The cell apoptosis was analyzed by flow cytometry and RT-PCR was used to investigate the expression of CD44s. The invasion ability of HHUA human endometrial carcinoma cells which treated with exemestane were assessed using transwell chamber model. RESULTS: At increasing doses of exemestane, a simultaneous increase in apoptotic subpopulations was detected when compared with group A (p < 0.05); the CD44s expression was found to be suppressed after the exemestane treatment. The decrease was a dose-dependent with exemestane treatment. CONCLUSION: 6x108 mol/L exemestane is an optimal dose to inhibit the expression of CD44s mRNA and inhibit the invasive growth of the endometrial carcinoma HHUA cells.

Single nucleotide polymorphism in the RECQL5 gene increased osteosarcoma susceptibility in a Chinese Han population.

In this study, we investigated the association between a RECQL genetic polymorphism and osteosarcoma in a Chinese population. We selected rs820196 in the RECQL5 gene and genotyped 185 patients with osteosarcoma and 201 age- and gender-matched non-cancer controls. We found that the CC genotype was more frequent in the osteosarcoma group compared to the control group (P = 0.011). We also found that the C allele was more common in osteosarcoma patients than that in control subjects (P = 0.004). Our results suggested that the RECQL5 genetic polymorphism was associated with osteosarcoma in a Chinese population.

Association of vitamin D receptor BsmI gene polymorphism with risk of low bone mineral density in post-menopausal women: a meta-analysis.

The vitamin D receptor BsmI gene polymorphism is reportedly associated with low bone mineral density (BMD) in postmenopausal women, but results from previous studies are conflicting. In the present study, we investigated the association between this polymorphism and the risk of low BMD through a meta-analysis of published studies. A literature search of the Pubmed, Embase, and CNKI databases from inception through July 2013 was conducted. The meta-analysis was performed using the STATA 12.0 software. Crude odds ratios with 95% confidence intervals were used to assess the strength of any association. Eleven case-control studies were included for a total of 1468 low BMD cases and 2177 healthy controls. No significant variation in low BMD risk was detected in any of the genetic models. Further stratified analyses were performed to examine the effect of ethnicity. In the subgroup analysis, no significant association was found in Caucasians and in Asians. The meta-analysis results suggest that the BsmI polymorphism is not associated with low BMD risk in postmenopausal women.

Transpositional activation of mPing in an asymmetric nuclear somatic cell hybrid of rice and Zizania latifolia was accompanied by massive element loss.

We have reported previously that the most active miniature inverted terminal repeat transposable element (MITE) of rice, mPing, was transpositionally mobilized in several rice recombinant inbred lines (RILs) derived from an introgressive hybridization between rice and wild rice (Zizania latifolia Griseb.). To further study the phenomenon of hybridization-induced mPing activity, we undertook the present study to investigate the element's behavior in a highly asymmetric somatic nuclear hybrid (SH6) of rice and Z. latifolia, which is similar in genomic composition to that of the RILs, though probably contains more introgressed alien chromatins from the donor species than the RILs. We found that mPing, together with its transposase-donor, Pong, underwent rampant transpositional activation in the somatic hybrid (SH6). Because possible effects of protoplast isolation and cell culture can be ruled out, we attribute the transpositional activation of mPing and Pong in SH6 to the process of asymmetric somatic hybridization, namely, one-step introgression of multiple chromatin segments of the donor species Z. latifolia into the recipient rice genome. A salient feature of mPing transposition in the somatic hybrid is that the element's activation was accompanied by massive loss of its original copies, i.e., abortive transpositions, which was not observed in previously reported cases of mPing activity. These data not only corroborated our earlier finding that wide hybridization and introgression may trigger transpositional activation of otherwise quiescent transposable elements, but also suggest that transpositional mobilization of a MITE like mPing can be accompanied by dramatic reduction of its original copy numbers under certain conditions, thus provide novel insights into the dynamics of MITEs in the course of genome evolution.

Allopolyploidy in wheat induces rapid and heritable alterations in DNA methylation patterns of cellular genes and mobile elements.

Whereas accumulating recent evidences indicate that allopolyploid formation in plants is accompanied by rapid and non-Mendelian genomic changes, some other works showed genomic stasis in both nascent and natural allopolyploids. To further study the issue, we performed global DNA fingerprinting of a newly synthesized allohexaploid wheat and its natural counterpart, the common wheat, by AFLP analysis. It was found that ca. 20% bands showed deviation from parental additivity in both synthetic and the natural common wheat. Sequence analysis indicates that a majority of the changed bands represent known-function genes and transposable elements. DNA gel blot analysis showed that the main type of changes in the amphiploid is epigenetic in nature, i.e., alteration in DNA methylation patterns. Two types of alterations in methylation, random and non-random, were detected, and both types were stably inherited. Possible causes and implications of the epigenetic changes in allopolyploid genome evolution and speciation are discussed.

Activation of a rice endogenous retrotransposon Tos17 in tissue culture is accompanied by cytosine demethylation and causes heritable alteration in methylation pattern of flanking genomic regions.

Tos17 is a copia-like, cryptic retrotransposon of rice, but can be activated by tissue culture. To study possible epigenetic mechanism controlling activity of Tos17, we subjected three rice lines (the parental line cv. Matsumae and two introgression lines, RZ2 and RZ35) that harbor different copies of the element to tissue culture. For each line, we investigated transcription and transposition of Tos17 in seed plants, calli and regenerated plants, cytosine-methylation status at CG and CNG positions within Tos17, effect of 5-azacytidine on methylation status and activity of Tos17, and cytosine-methylation states in genomic regions flanking original and some newly transposed copies of Tos17 in calli and regenerated plants. We found that only in introgression line RZ35 was Tos17 transcriptionally activated and temporarily mobilized by tissue culture, which was followed by repression before or upon plant regeneration. The activity and inactivity of Tos17 in calli and regenerated plants of RZ35 are accompanied by hypo- and hyper-CG methylation and hemi- and full CNG methylation, respectively, within the element, whereas immobilization of the element in the other two lines is concomitant with near-constant, full hypermethylation. Treatment with 5-azacytidine induced both CG and CNG partial hypomethylation of Tos17 in two lines (Matsumae and RZ35), which, however, was not accompanied by activation of Tos17 in any line. Heritable alteration in cytosine-methylation patterns occurred in three of seven genomic regions flanking Tos17 in calli and regenerated plants of RZ35, but in none of the five regions flanking dormant Tos17 in the other two lines.

Immunoglobulin-A and -G responses against virus-like particles (VLP) of human papillomavirus type 16 in women with cervical cancer and cervical intra-epithelial lesions.

Immunoglobulin-A and -G (IgA and IgG) responses against HPV-16-like particles (VLP) were tested by ELISA in 104 women with cervical abnormalities, 26 atypical cells of undetermined significance (ASCUS) and 14 cytologically normal women with HPV DNA. As controls, 130 age-matched cytologically normal women with no HPV DNA were selected from the population in which the cases were generated. The existence of HPV DNA in cervical samples was tested by a PCR-based method. The normal women positive with HPV-16 DNA were followed up at 4- to 7-month intervals for 16 to 24 months. IgA and IgG antibodies against HPV-16 VLP were frequently detected in these women repeatedly positive with HPV-16 DNA, suggesting that persistent HPV infection is crucial for effective antibody responses against the viruses. IgA response appears earlier and persists longer than IgG response. Women with HPV DNA of types 16, 31/33/35, 58 and unknown types showed significantly higher seropositivity for both IgA and IgG antibodies than the controls (p < 0.05 for both). No significant seropositivity for IgA or IgG was detected in the HPV-18/45-DNA-positive group. HPV 31/33/35, 58 appear to be types close to HPV 16, whereas HPV 18/45 appears to be distinct from HPV 16 in antigenicity. IgA and IgG responses against HPV-16 VLP were more frequently observed in women with normal cervices with HPV DNA, ASCUS, HSIL and cervical cancer than in the controls. Strong IgA and IgG responses depended on HPV-16 infection in HSIL and cervical cancer, but there was no correlation between the serological responses and the status of HPV DNA in ASCUS and LSIL. Antibody positivity reflects persistent viral infection that may increase the risk for malignant progression of the cervix. This serological assay using HPV-16 VLP may therefore be useful as a new diagnostic tool supplementing cervical cytological tests.

Erythrocyte membrane lipid composition fluidity in patients with essential hypertension.

The lipid composition and fluidity of erythrocyte membrane in 36 patients with essential hypertension were examined. The results showed that either cholesterol/phospholipid molar ratio or lipid peroxides content was significantly increased (P < 0.01 or 0.001), and the superoxide dismutase activity, contents of four classes of phospholipids, i.e., phosphatidyl ethanolamine, phosphatidylcholine, phosphatidylserine and sphingomyelin of erythrocyte membrane, and lipid fluidity were significantly decreased (P < 0.05, < 0.01 or < 0.001) as compared with those in 35 normotensive control subjects. These results suggested that the changes of lipid composition and fluidity might associate with the decreased activities of cation transport systems in cell membranes and play an important role in the pathogenesis of essential hypertension.