Single-cell analysis reveals exosome-associated biomarkers for prognostic prediction and immunotherapy in lung adenocarcinoma.

BACKGROUND: Exosomes play a crucial role in tumor initiation and progression, yet the precise involvement of exosome-related genes (ERGs) in lung adenocarcinoma (LUAD) remains unclear. METHODS: We conducted a comprehensive investigation of ERGs within the tumor microenvironment (TME) of LUAD using single-cell RNA sequencing (scRNA-seq) analysis. Multiple scoring methods were employed to assess exosome activity (EA). Differences in cell communication were examined between high and low EA groups, utilizing the "CellChat" R package. Subsequently, we leveraged multiple bulk RNA-seq datasets to develop and validate exosome-associated signatures (EAS), enabling a multifaceted exploration of prognosis and immunotherapy outcomes between high- and low-risk groups. RESULTS: In the LUAD TME, epithelial cells demonstrated the highest EA, with even more elevated levels observed in advanced LUAD epithelial cells. The high-EA group exhibited enhanced intercellular interactions. EAS were established through the analysis of multiple bulk RNA-seq datasets. Patients in the high-risk group exhibited poorer overall survival (OS), reduced immune infiltration, and decreased expression of immune checkpoint genes. Finally, we experimentally validated the high expression of SEC61G in LUAD cell lines and demonstrated that knockdown of SEC61G reduced the proliferative capacity of LUAD cells using colony formation assays. CONCLUSION: The integration of single-cell and bulk RNA-seq analyses culminated in the development of the profound and significant EAS, which imparts invaluable insights for the clinical diagnosis and therapeutic management of LUAD patients.

Revealing the role of regulatory T cells in the tumor microenvironment of lung adenocarcinoma: a novel prognostic and immunotherapeutic signature.

Background: Regulatory T cells (Tregs), are a key class of cell types in the immune system. In the tumor microenvironment (TME), the presence of Tregs has important implications for immune response and tumor development. Relatively little is known about the role of Tregs in lung adenocarcinoma (LUAD). Methods: Tregs were identified using but single-cell RNA sequencing (scRNA-seq) analysis and interactions between Tregs and other cells in the TME were investigated. Next, we used multiple bulk RNA-seq datasets to construct risk models based on marker genes of Tregs and explored differences in prognosis, mutational landscape, immune cell infiltration and immunotherapy between high- and low-risk groups, and finally, qRT-PCR and cell function experiments were performed to validate the model genes. Results: The cellchat analysis showed that MIF-(CD74+CXCR4) pairs play a key role in the interaction of Tregs with other cell subpopulations, and the Tregs-associated signatures (TRAS) could well classify multiple LUAD cohorts into high- and low-risk groups. Immunotherapy may offer greater potential benefits to the low-risk group, as indicated by their superior survival, increased infiltration of immune cells, and heightened expression of immune checkpoints. Finally, the experiment verified that the model genes LTB and PTTG1 were relatively highly expressed in cancer tissues, while PTPRC was relatively highly expressed in paracancerous tissues. Colony Formation assay confirmed that knockdown of PTTG1 reduced the proliferation ability of LUAD cells. Conclusion: TRAS were constructed using scRNA-seq and bulk RNA-seq to distinguish patient risk subgroups, which may provide assistance in the clinical management of LUAD patients.

Leukoreduced PRP enhanced proliferation and ECM production yet inhibited senescence, inflammation, and multi-differentiation potential of AFSCs by downregulating HMGB1.

BACKGROUND: This study assessed the role and potential mechanism of platelet-rich plasma (PRP) in the progression of intervertebral disk degeneration (IVDD). METHODS: Annulus fibrosus (AF)-derived stem cells (AFSCs) from New Zealand white rabbits received the transfection with high mobility group box 1 (HMGB1) plasmids and the subsequent treatment with bleomycin, 10% leukoreduced PRP or leukoconcentrated PRP. Dying cells were indicated by immunocytochemistry analysis for senescence-associated ß-galactosidase (SA-ß-gal) staining. The proliferation of these cells was evaluated based on the population doubling time (PDT). The expressions of HMGB1, pro-aging and anti-aging molecules, extracellular matrix (ECM)-related catabolic/anabolic factors, and inflammatory genes at the molecular or transcriptional levels were quantified via Western blot or reverse transcription-quantitative PCR (RT-qPCR). Besides, the adipocytes, osteocytes, and chondrocytes were separately dyed by Oil Red O, Alizarin Red S, and Safranin O staining. RESULTS: Bleomycin enhanced the senescent morphological changes and increased the PDT and the expressions of SA-ß-gal, pro-aging molecules, ECM-related catabolic factors, inflammatory genes, and HMGB1 while suppressing the expressions of anti-aging and anabolic molecules. Leukoreduced PRP reversed the effects of bleomycin and inhibited the differentiation of AFSCs into adipocytes, osteocytes, and chondrocytes. Besides, HMGB1 overexpression offset the roles of leukoreduced PRP in AFSCs. CONCLUSION: Leukoreduced PRP promotes cell proliferation and ECM production of AFSCs, while inhibiting their senescence, inflammation, and multi-differentiation potentials via downregulating HMGB1 expression.

Ginkgetin Alleviates Intervertebral Disc Degeneration by Inhibiting Apoptosis, Inflammation, and Disturbance of Extracellular Matrix Synthesis and Catabolism via Inactivation of NLRP3 Inflammasome.

BACKGROUND: Apoptosis, inflammation, and the extracellular matrix (ECM) synthesis and catabolism are compromised with intervertebral disc degeneration (IDD). Ginkgetin (GK) has been demonstrated to alleviate several diseases; however, its effect on IDD remains unknown. METHODS: The nucleus pulposus cells (NPCs) were stimulated with interleukin (IL)-1ß to construct the IDD models in vitro. Rats were used for the construction of the IDD models in vivo via the fibrous ring puncture method. The effect and mechanism of GK on IDD were determined by cell counting kit-8 (CCK-8), flow cytometry, western blot, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme­linked immunosorbent assay (ELISA), hematoxylin and eosin (HE) and safranine O staining, and immunohistochemistry (IHC) assays, respectively. RESULTS: GK increased the cell viability and upregulated the expressions of anti-apoptosis and ECM synthesis markers in NPCs treated with IL-1ß. GK also decreased apoptosis rate, and downregulated the expressions of proteins related to pro-apoptosis, ECM catabolism, and inflammation in vitro. Mechanically, GK reduced the expression of nucleotide binding oligomeric domain like receptor protein 3 (NLRP3) inflammasome-related proteins. Overexpression of NLRP3 reversed the effect of GK on the proliferation, apoptosis, inflammation, and ECM degradation in IL-1ß-induced NPCs. Moreover, GK attenuated the pathological manifestations, inflammation, ECM degradation, and NLRP3 inflammasome expression in IDD rats. CONCLUSION: GK suppressed apoptosis, inflammation, and ECM degradation to alleviate IDD via the inactivation of NLRP3 inflammasome.

Experimental evidence of shikonin as a novel intervention for anti-inflammatory effects.

Shikonin is a natural product with antioxidant and anti-inflammatory activities. The biological activity of shikonin is still not fully understood, as well as its association with innate immunity and immune and inflammatory bowel disease (IBD) in humans. In this study, the toxicity of shikonin on Raw264.7 cells was assayed by MTT, and polarization of inflammatory macrophages was determined by flow cytometry. The results showed that shikonin can inhibit the polarization of macrophages towards M1 type and significantly inhibited the production of NO in the concentration range of 0.5-1 µM. In addition, after treatment with shikonin, the production of IL-1ß and TNF-α was significantly decreased. After shikonin administration, the body weight loss and decrease of colon length were significantly suppressed in DSS-treated colitis C57BL/6 mice. The pro-inflammatory cytokines TNF-α and IL-1ß in colonic homogenate were significantly decreased. Shikonin treatment resulted in a notable improvement in the histopathological manifestations in DSS-treated animals at 25/50 mg/kg. Meanwhile, we found that shikonin can regulate differentiation of T helper 17 cell (Th17)/regulatory T cell (Treg), thereby regulating the balance of Th17/Treg cells and exerting an anti-inflammatory effect in IBD animal models. In conclusion, we found that shikonin protects against DSS-induced acute colitis by, among other things, reducing immune cell infiltration, polarizing macrophages, and regulating Th17/Treg differentiation, as well as by downregulating the release of inflammatory cytokines. These findings showed that shikonin can improve inflammation by affecting macrophage polarization. Our experimental data provide experimental evidence and theory basis for research on anti-inflammatory effects for the shikonin as health or functional food.

MECHANISM OF MIR-25-3P CARRIED BY EXTRACELLULAR VESICLES DERIVED FROM PLATELET-RICH PLASMA IN IL-1ß-INDUCED NUCLEUS PULPOSUS CELL DEGENERATION VIA THE SOX4/CXCR7 AXIS.

ABSTRACT: Objectives: Nucleus pulposus (NP) cell degeneration promotes the progression of intervertebral disc (IVD) degeneration. MicroRNAs (miRs) are associated with IVD degeneration. This study expounded the mechanism of microRNA (miR)-25-3p carried by extracellular vesicles (EVs) derived from platelet-rich plasma (PRP) in interleukin (IL)-1ß-induced NP cell degeneration. Methods: Platelet-rich plasma from mouse blood was obtained, and EVs were isolated from PRP (EVs derived from PRP [PRP-EVs]) and identified. Nucleus pulposus cells were isolated from the mouse lumbar IVD and treated with IL-1ß to induce NP cell degeneration. Extracellular vesicles derived from PRP were added into NP cell culture medium. Afterward, intracellular miR-25-3p, sex determining region Y-related high-mobility-group box 4 (SOX4), and CXC chemokine receptor 7 (CXCR7) levels were examined. Nucleus pulposus cell viability, apoptosis, and inflammation were detected. Extracellular vesicles derived from PRP were labeled by PKH67 to obverse the uptake of EVs by NP cells. The binding relations between SOX4 and miR-25-3p and CXCR7 were predicted and examined. Functional rescue experiments were performed to investigate the roles of miR-25-3p, SOX4, and CXCR7 in NP cell degeneration. Results: miR-25-3p was downregulated, whereas SOX4 and CXCR7 were upregulated in IL-1ß-induced NP cells. Extracellular vesicles derived from PRP increased the cell viability, and decreased apoptosis and inflammation. miR-25-3p carried by PRP-EVs into NP cells alleviated NP cell degeneration. miR-25-3p inhibited SOX4 expression and limited CXCR7 transcription. Silencing miR-25-3p or overexpressing SOX4 or CXCR7 reversed the alleviating role of PRP-EVs in NP cell degeneration. Conclusion: miR-25-3p carried by PRP-EVs into NP cells elevated intracellular miR-25-3p expression, which suppressed SOX4 expression and further limited CXCR7 transcription, thus alleviating IL-1ß-induced NP cell degeneration. Extracellular vesicles derived from PRP containing miR-25-3p may be a new method for IVD treatment.

Radiotherapy alleviates spinal cord compression caused by solitary bone plasmacytoma: A case report.

INTRODUCTION AND IMPORTANCE: Solitary bone plasmacytoma (SBP) is a hematopoietic malignancy occurring in bone tissue, which often causes bone destruction at the site of the lesion. When the lesion occurs in the spine and causes spinal stenosis and compression of the spinal cord, surgery is performed as an adjuvant treatment before radiotherapy. CASE PRESENTATION: A 36-year-old patient suffered from neck, shoulder and upper limbs pain for 3 weeks and the symptom worsened for 3 days after exercise. CT and MRI examination of the neck after emergency admission revealed C5 vertebral pathological fracture with associated spinal stenosis and spinal cord compression. PET-CT indicated a hypermetabolic soft tissue mass in the C5-6 vertebral body. Granulomatous lesions (tuberculosis) were considered, but neoplastic lesions were not ruled out. The primary diagnosis was cervical fracture caused by tuberculosis. Finally, a needle biopsy was performed at the lesion site and a diagnosis of SBP was made. Radiotherapy was immediately followed and the spinal cord compression was relieved a month later. After 6 months of follow-up, she is now in stable condition with no neck pain or neurological impairment. CONCLUSION: For patients with SPB resulting in pathological fracture of the cervical vertebra with spinal stenosis and compression of the spinal cord, forgoing surgery and undergoing radiation therapy alone may be an option.

Upregulated Plant Homeodomain Finger Protein 6 Promotes Extracellular Matrix Degradation in Intervertebral Disc Degeneration Based on Microarray Analysis.

STUDY DESIGN: mRNA analysis. OBJECTIVE: The aim of this study was to identify differentially expressed genes (DEGs) in disc degeneration, analyze the potential biological functions of DEGs, and screen for a new target to prevent the degeneration. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration (IDD) is an irreversible process and causes long-term heavy socioeconomic burdens. Existing and therapies under development are unable to prevent disc degeneration in a safe and effective manner. Therefore, elucidating the potential mechanism underlying degeneration and the development of new targets for IDD therapy are urgently required. METHODS: Nucleus pulposus (NP) cells from mild and severe IDD (Ctrl and IDD groups) were separated, and DEGs of the two groups were identified with mRNA microarray analysis, followed by bioinformatics analysis.Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to verify the microarray results. Gene over-expression and silencing technologies were used to study the role of plant homeodomain finger protein 6 (PHF6). qRT-PCR and western blot analyses were used to detect the expressions of collagen II (COL2), matrix metalloproteinases 13 (MMP13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS4). RESULTS: The study identified 377 up- and 116 downregulated DEGs in NP cells from two groups. These DEGs were mainly involved in cellular and metabolic processes and enriched in immune system and nucleotide metabolism pathways. Upregulated PHF6, with the highest verified fold change, was significantly increased in the IDD group. Over-expressing PHF6 in Ctrl NP cells significantly inhibited the expression of COL2 and enhanced the expressions of MMP13 and ADAMTS4, whereas silencing PHF6 in IDD NP cells reversed such expression alterations. CONCLUSION: Upregulated PHF6 caused IDD by promoting extracellular matrix degradation; therefore, PHF6 could be developed as a potential novel target to prevent the degeneration. Our DEG profiling of NP cells from IDD patients provided a database to identify the key genes involved in IDD. LEVEL OF EVIDENCE: N/A.

Cross-Platform Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Non-Small Cell Lung Carcinoma Patient Plasma.

Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. Here, we have compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. Two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction (ddPCR) and a next-generation sequencing (Firefly NGS) platform were included in the comparison. Fifteen EGFR mutations across twenty NSCLC patients were identified. Firefly NGS, cobas-ARMS and ddPCR all displayed superior sensitivity while ADx-ARMS was better suited for the qualitative detection of EGFR mutations with allele frequency higher than 1% in plasma and tissue samples. We observed high coincidence between the plasma and tissue EGFR mutational profiles for three driver mutations (L858R, exon 19 deletion and G719X) that are known targets of first generation EGFR-TKI therapies among patients who relapsed. Discrepancies between tissue and plasma EGFR mutational profiles were mainly attributable to spatial and temporal tumor heterogeneity, mutation inhibition due to therapy response and drug resistance (T790M). This study illustrates the challenges associated with selection of a technology platform for EGFR ctDNA analysis in the context of treatment evaluation and drug resistance detection.

Feasibility of interlaminar lumbar discectomy through percutaneous transforaminal endoscopy and blunt perforation of the ligamentum flavum.

AIM: To determine the efficacy, safety, and clinical value of a novel surgical procedure involving the blunt perforation of the ligamentum flavum (LF) during endoscopic interlaminar lumbar discectomy. MATERIAL AND METHODS: This was a prospective study of 50 patients (27 males, 17-51 years of age) undergoing lumbar discectomy for single segment L4/L5 or L5/S1 disk herniation were grouped into the control (cutting of the LF; n=28) and test (blunt perforation; n=22) groups. Intraoperative injury to the LF was evaluated by electrophysiological monitoring. The time required for perforation, total surgical time, and proportion of epidural sac and nerve root injury were assessed. RESULTS: Among the enrolled patients, 90% showed herniation of the L4/5 segment and 10% of the L5/S1 segment. The success rate for the perforation of the LF was 93%. The intraoperative observation showed mild self-closing injury to the LF tissue. The test group showed shorter overall surgical time (43 vs. 56 min) and shorter duration to go through the LF (1 vs. 13 min, p 0.001). No dural sac or nerve root injury resulting from blunt perforation of the LF was observed. CONCLUSION: Compared to cutting, blunt perforation of the LF could reduce surgical time and injury to LF and surrounding tissues. Thus, it could be a safe and efficient surgical technique for patients undergoing intralaminar lumbar discectomy.

Systematic review and meta-analysis of robotic versus open pancreaticoduodenectomy.

BACKGROUND: Although robotic pancreaticoduodenectomy (RPD) has been successfully performed since 2003, its advantages over open pancreaticoduodenectomy (OPD) are still uncertain. The aim of this systematic review and meta-analysis was to compare the clinical outcomes of RPD to those of OPD. METHODS: A systematic literature review was performed to identify RPD versus OPD comparative studies published between January 2003 and January 2016. Intraoperative outcomes, post-operative outcomes and oncologic safety were evaluated. Pooled odds ratios (ORs) and weighted mean differences (WMDs) with a 95% confidence interval (95% CI) were calculated using fixed-effect or random-effect models. RESULTS: Nine non-randomized observational clinical studies involving 680 patients met the inclusion criteria and involved 245 RPDs and 435 OPDs. The overall complication rate was significantly lower in RPD (OR 0.65, 95% CI 0.47-0.91, P = 0.012), as well as the margin positivity rate (OR 0.40, 95% CI 0.20-0.77, P = 0.006), the wound infection rate (OR 0.18, 95% CI 0.06-0.53, P = 0.002) and the length of hospital stay (WMD = -6.00, 95% CI -9.80 to -2.21, P = 0.002). There was no significant difference in the following: the number of lymph nodes harvested; the operation time; the reoperation rate; the incidence of delayed gastric emptying, bile leakage, pancreatic fistula and clinically significant pancreatic fistula; and mortality. The mean conversion rate was 7.3% (range 0-14%). CONCLUSIONS: According to the results of this meta-analysis, RPD is as safe and efficient as OPD and is even favourable in terms of margin-negative resection, overall complication and wound infection rates and length of hospital stay. Given that there have not yet been any high-quality randomized controlled trials (RCTs), the evidence is still limited. Additional prospective, multi-centre RCTs are needed to further define the role of the robotic technique in PD.

Primary pancreatic paraganglioma: a case report and literature review.

BACKGROUD: Primary pancreatic paraganglioma is an extremely rare extra-adrenal paraganglioma. CASE PRESENTATION: We report a case of primary pancreatic paraganglioma undergoing middle segment pancreatectomy in a 42-year-old woman. Histological examination showed that the tumor was composed of well-defined nests of cuboidal cells separated by vascular fibrous septa, forming the classic Zellballen pattern. The chief cells showed positive staining to neuron-specific enolase, chromogranin A, synaptophysin, and the chief cells were surrounded by S-100 protein-positive sustentacular cells. The patient has remained tumor free for 12 months after surgery. A brief discussion about the histopathological features, clinical behavior, and treatment of primary pancreatic paraganglioma, and review of the relevant literature is presented. CONCLUSIONS: Primary pancreatic paraganglioma is a rare clinical entity, its diagnosis mainly depends on histopathological and immunohistochemical examinations. Complete surgical resection is the first choice of treatment and close postoperative follow-up is necessnary.

The severity of operative invasion to the posterior muscular-ligament complex influences cervical sagittal balance after open-door laminoplasty.

PURPOSE: The aim of this study was to investigate how the severity of operative invasion to the posterior muscular-ligament complex impacts postoperative cervical sagittal balance. MATERIALS AND METHODS: Ninety cases of open-door expansive laminoplasty due to cervical spondylotic myelopathy were reviewed. Fifty-three patients underwent laminoplasty with unilateral preservation of the muscular-ligament complex (unilateral elevation group). Thirty-seven patients underwent traditional open-door laminoplasty (bilateral elevation group). Preoperative and postoperative cervical sagittal parameters, including C2-C7 sagittal vertical axis (SVA), C0-2 Cobb angle and T1 slope, were compared. The cervical curvature, range of motion (ROM) and JOA score were also compared. RESULTS: The average follow-up time was 16.7 months (range 3-40 months). C2-C7 SVA significantly increased in the bilateral elevation group (+4.9 mm, P = 0.005) but remained unchanged in the unilateral elevation group (-0.2 mm, P = 0.414). The C0-2 Cobb angle increased in both groups (+4.1°, P < 0.001; +2.5°, P = 0.002). The T1 slope also increased in both groups (+1.1°, P = 0.015; +0.7°, P = 0.042). The postoperative C3-C7 curvature significantly decreased in the bilateral elevation group (-4.1°, P < 0.001). The C3-C7 ROM decreased in both groups (-17.9°, P < 0.001; -15.1°, P < 0.001). C2-C7 SVA was positively correlated with the T1 slope (Pearson = 0.468, P < 0.001) and negatively correlated with the C3-C7 curvature (Pearson = -0.322, P = 0.001). The C0-2 Cobb angle was positively correlated with C2-C7 SVA (Pearson = 0.303, P = 0.004) and negatively correlated with the C3-C7 curvature (Pearson = -0.362, P < 0.001). There was no significant between-group difference in the JOA improvement rate. CONCLUSIONS: Open-door laminoplasty significantly affected postoperative cervical sagittal balance, with the cervical vertebra appearing to tilt forward. As the severity of surgical invasion to the posterior muscular-ligament complex increased, the loss of cervical sagittal balance also increased.

[Changes of sagittal balance of cervical spine after open-door expansive laminoplasty].

OBJECTIVE: To explore the changes of sagittal balance of cervical spine after open-door expansive laminoplasty. METHODS: The clinical and radiological data were analyzed retrospectively for 90 patients undergoing open-door expansive laminoplasty due to cervical spondylotic myelopathy. The Japanese Orthopedic Association (JOA) score and 3 cervical sagittal parameters including C2-C7 SVA, C0-2 Cobb angle and T1-Slope on lateral view radiographs were recorded before operation and at the final follow-up. RESULTS: The average follow-up period was 16.7 (3-40) months. The post-operative JOA score rose to 14.6 ± 0.2 from pre-operative 12.2 ± 0.3 with 43.5% ± 4.2% recovery rate. The post-operative values of C2-C7 SVA, C0-2 Cobb angle and T1-Slope were significantly different from pre-operative ones (P = 0.022, P < 0.001, P = 0.002) . C2-C7 SVA increased to (23.0 ± 1.2) mm from pre-operative (20.7 ± 1.1) mm. C0-2 Cobb angle increased (23.1 ± 0.8) ° from pre-operative (19.9 ± 0.8)°; T1-Slope increased to (26.2 ± 0.7)° from pre-operative (25.1 ± 0.7)°. The changes of C0-2 Cobb angle and T1-Slope were correlated with that of C2-C7 SVA respectively (Pearson = 0.469, P < 0.001) (Pearson = 0.303, P = 0.004) . Patients with higher preoperative T1-Slope had less JOA improvement (31.5% vs 53.7%, P = 0.019) than those with lower preoperative T1-Slope after laminoplasty. CONCLUSION: The sagittal balance of cervical spine significantly changes after open-door expansive laminoplasty with forward tilting of cervical vertebra. And compensation occurs by excessive high-strength contraction of posterior muscles to maintain lordosis in upper cervical spine. A higher pre-OP T1-Slope affects the outcomes of open-door expansive laminoplasty.

Methylation-associated silencing of MicroRNA-335 contributes tumor cell invasion and migration by interacting with RASA1 in gastric cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes, previous studies have shown that miR-335 play an important role in suppressing metastasis and migration in human cancer including gastric cancer (GC). However, the mechanisms which result in aberrant expression of miR-335 in GC are still unknown. Recent studies have shown that the silencing of some miRNAs is associated with DNA hypermethylation. In this study, we find the promoter of miR-335 we embedded in CpG island by accessing to bioinformatics data and the low expression of miR-335 in 5 gastric cell lines can be restored by 5-aza-2'-deoxycytidine (5-Aza-dC) treatment. So we postulated that the miR-335 genes undergo epigenetic inactivation in GC. Subsequently, in GC cells and tissues, we performed quantitative real-time PCR (RTQ-PCR) to assess the expression of miR-335, and methylation-specific PCR (MSP) and bisulfite sequence-PCR (BSP) to evaluate the DNA methylation status in the CpG islands upstream of MiR-335. The result showed that the expression of miR-335 was significantly reduce in gastric cancer cell lines and tumor tissues compared to matched normal gastric tissues, and cell lines, and which is inverse correlation with DNA hypermethylation of miR-335 both in GC cells lines and tissues, but not in normal tissues. In addition, we found that the lower miR-335 expression induced by abnormal methylation may be mainly involved in gastric cell invasion and metastasis in GC tissues. No statistical significance was found about miR-335 expression and methylation level between healthy individuals with and without H. pylori (HP) infection. Finally, we carry out miRNA transfection, RTQ-PCR and western blot assay to find the RAS p21 protein activator (GTPase activating protein) 1 (RASA1) may be the possible target genes which lead to the gastric cell invasion and metastasis, furthermore, the re-expression of endogenous miR-335 by 5-Aza-dC treatment can exert effects similar to exogenous miRNAs transfection. Taken together, our results suggest that miR-335 may be silenced by promoter hypermethylation and play important roles in gastric cell invasion and metastasis through its target genes, such as RASA1. Its methylation level might be a predictive epigenetic marker of GC and remodeling on the expression by demethylation can provided a potential therapeutic strategy.

Expression of c-Kit and PDGFRα in epithelial ovarian tumors and tumor stroma.

The purpose of this study was to investigate the expression of c-Kit and platelet-derived growth factor receptor α (PDGFRα) in epithelial ovarian tumor cells and tumor stroma. The expression of c-Kit and PDGFRα in 71 malignant or benign epithelial ovarian tumor tissues and 20 normal ovarian tissues was evaluated by immunohistochemical staining. The expression of c-Kit and PDGFRα in 71 malignant epithelial ovarian tumors and tumor stroma tissue samples was analyzed. A significant increase (P<0.01) of c-Kit expression was observed in malignant ovarian tumors (50.7%) when compared to normal ovarian tissues (10.0%) or benign ovarian tumors (20.0%). The PDGFRα expression rate in malignant ovarian tumors (63.4%) was also significantly higher (P<0.01) than that in normal ovarian tissues (15.0%) or benign ovarian tumors (25.0%). c-Kit was expressed in only 4.2% of the tumor stroma samples, which was significantly lower than the expression of malignant ovarian tumors (P<0.01), whereas the PDGFRα expression in tumor stroma (87.3%) was significantly higher than that of the malignant ovarian tumors (P<0.01). The expression levels of c-Kit and PDGFRα are higher in the malignant ovarian tumors than in the benign ovarian tumors or normal tissues. In the malignant ovarian tumor stroma, c-Kit expression is low and PDGFRα expression is high, and the differential changes of c-kit and PDGFRα suggest distinct roles in ovarian cancer.

Identification of rare DNA variants in mitochondrial disorders with improved array-based sequencing.

A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation. The confidence of each base-call was ranked using two quality measures. In comparison to Sanger capillary sequencing, we achieved a false discovery rate of 2% (false positive rate 1.2 × 10⁻5, false negative rate 5%), which is similar to automated second-generation sequencing technologies. Applied to the analysis of 39 nuclear candidate genes in disorders of mitochondrial DNA (mtDNA) maintenance, we confirmed mutations in the DNA polymerase gamma POLG in positive control cases, and identified novel rare variants in previously undiagnosed cases in the mitochondrial topoisomerase TOP1MT, the mismatch repair enzyme MUTYH, and the apurinic-apyrimidinic endonuclease APEX2. Some patients carried rare heterozygous variants in several functionally interacting genes, which could indicate synergistic genetic effects in these clinically similar disorders.