Identification of key genes and pathway related to chemoresistance of small cell lung cancer through an integrative bioinformatics analysis.

Background: Small cell lung cancer (SCLC), the most malignant of all the lung cancer subtypes, is characterized by drug resistance. This study sought to explore the key genes and pathways associated with the chemoresistance of SCLC. Methods: The drug sensitivity of chemosensitive and chemoresistance SCLC cell lines was measured by Cell Counting Kit-8 assays. The total RNA from chemosensitive cell line H69 and chemoresistance cell line H69AR cells was extracted and subjected to messenger RNA (mRNA) and long non-coding RNA (lncRNA) microarray analyses. The differentially expressed genes (DEGs) and the differentially expressed lncRNAs (DELs) were screened out with a threshold of a |log fold change | ≥1 and an adjusted P value <0.05. A protein-protein interaction network was constructed, and hub genes were screened out. A lncRNA-mRNA co-expression network was also constructed. Gene Ontology and Kyoto Encyclopedia of Genes, Genomes enrichment analyses and Cis-regulatory element analyses were conducted on the DEGs and the top 10 upregulated DEL-co-expressed DEGs. The expression of the key genes was further analyzed in the GSE149507 data set and validated in H69/H69AR and H446/H446DDP cells by quantitative polymerase chain reaction assays. Results: The microarray results showed that a total of 609 mRNAs and 394 lncRNAs were differentially expressed in the chemoresistant SCLC cells. The mammalian target of rapamycin (mTOR) signaling pathway was enriched among the DEGs, the top 10 upregulated DEL-co-expressed DEGs, and the NCRNA00173-co-expressed DEGs, which included IGF1, INS, WNT6, WNT11, WNT2B, and SESN2. IGF1, WNT2B, and SESN2 were downregulated, and WNT11 was upregulated in the SCLC tumor tissues in the GSE149507 data set. Further, IGF1, WNT6, WNT11, and WNT2B were lowlier expressed and SESN2 and NCRNA00173 were more highly expressed in the chemoresistant cells than sensitive cells. Conclusions: The top 10 upregulated DELs containing NCRNA00173 may be involved in the regulation of drug resistance in SCLC. These DELs may regulate the genes related to the mTOR signaling pathway. These genes may also be biomarkers and potential targets for the treatment of SCLC.

HuR affects chemoresistance of small cell lung cancer by regulating FGFRL1 expression.

Human antigen R (HuR), an RNA-binding protein, has been demonstrated to serve an oncogenic role in various types of cancer. Fibroblast growth factor receptor-like 1 (FGFRL1) has been shown to regulate small cell lung cancer (SCLC) chemoresistance. In the present study, the role of HuR in chemoresistance of SCLC, as well as its possible molecular mechanism involving FGFRL1, was explored by reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8 assay, flow cytometry and RNA immunoprecipitation. The results revealed that HuR expression levels were markedly upregulated in drug-resistant SCLC cell lines (H69AR and H446DDP) compared with in the parental cell lines (H69 and H446). Knockdown of HuR in drug-resistant SCLC cells enhanced drug sensitivity, cell apoptosis and cell cycle arrest. Furthermore, molecular mechanism studies indicated that HuR could bind and regulate FGFRL1 expression levels to increase FGFRL1 mRNA stability. Taken together, the present study suggested that HuR may mediate chemoresistance of SCLC by regulating FGFRL1 expression. HuR may represent a prognostic predictor and a potential target for overcoming chemoresistance in SCLC.

Decreased TMEM40 expression is associated with malignant behavior of cutaneous squamous cell carcinoma and inhibits tumor progression.

Cutaneous squamous cell carcinoma (CSCC) is one of the most common types of skin cancer in humans worldwide. The identification and characterization of cancer-associated transmembrane proteins are important for understanding the molecular biology of CSCC. The aim of the present study was to evaluate the expression pattern of transmembrane protein 40 (TMEM40) in CSCC and its clinical significance. The underlying mechanisms were also examined. Reverse transcription-quantitative PCR, western blot and immunohistochemistry analysis were used to determine the relative expression of TMEM40 in CSCC cell lines and clinical tissue samples. The effect of TMEM40 gene silencing on cell proliferation was also evaluated using Cell Counting Kit-8 assays. Wound healing assays, flow cytometry and Transwell assays were used to explore the migration, cell cycle distribution/apoptosis and invasion of CSCC cells following TMEM40 silencing, respectively. In the present study, increased TMEM40 expression was observed in CSCC tissue samples, compared with normal skin, and TMEM40 expression was associated with large tumor size in patients with CSCC. In vitro functional assays indicated that TMEM40 was involved in the regulation of A431 and SCL1 cell growth through its effects on the cell cycle and apoptosis. Silencing TMEM40 in A431 and SCL1 cells resulted in cell cycle arrest at the G0/G1 phase and promoted apoptosis. In addition, migration and invasion were significantly inhibited following silencing of TMEM40 expression in CSCC cells. Taken together, the results of the present study indicated that reduced TMEM40 expression could inhibit CSCC development and that TMEM40 may represent a therapeutic target in CSCC.

ATM mutations as an independent prognostic factor and potential biomarker for immune checkpoint therapy in endometrial cancer.

The morbidity and mortality of endometrial cancer has been increasing over years. Ataxia telangiectasia mutated (ATM) gene, encoding a protein kinase participated in the response to DNA damage, is frequently mutated in endometrial cancer patients. However, the potential relationship between ATM mutations and the progression of endometrial cancer remains unclear. We performed an integrative bioinformatics analysis to investigate the relationship between ATM mutational status with clinical outcomes and tumor microenvironment in endometrial cancer patients. The whole exome sequencing data, RNA sequencing data and clinical information were collected from The Cancer Genome Atlas (TCGA) dataset. We found that mutation in the ATM gene was an independent prognostic factor for endometrial cancer. Antitumor immune pathways were enriched in endometrial tumors with ATM mutations. The tumor-infiltrating T lymphocytes, especially cytotoxic lymphocytes, were generally more abundant in tumors with ATM mutations. Furthermore, patients with ATM mutations exhibited higher tumor mutational burden, higher neoantigen load and increased expression levels of some immune checkpoints. In conclusion, the present study indicated that ATM mutations were linked to longer overall survival of endometrial cancer. Our findings may add better understanding for potential immunotherapy of endometrial cancer.

CHILD Syndrome: Case Report of a Chinese Patient and Literature Review of the NAD[P]H Steroid Dehydrogenase-Like Protein Gene Mutation.

Congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome is an X-linked autosomal dominant disorder characterized by unilateral congenital hemidysplasia with ichthyosiform erythroderma and ipsilateral limb defects caused by a mutation in the gene encoding NAD[P]H steroid dehydrogenase-like protein (NSDHL) at Xq28. The histopathologic hallmark of skin lesions in CHILD syndrome is psoriasiform epidermis with hyperkeratosis and parakeratosis, and its most striking feature affecting the upper dermis is filling of the papillary dermis with foam cells. Here we present the case of a 9-year-old Chinese girl born with the typical clinical features of CHILD syndrome. Histologic and immunohistochemical evaluation of the skin lesions confirmed the diagnosis and led to identification of a heterozygous point mutation in exon 8 of the NSDHL gene. In addition, we provide a literature review of 26 unrelated CHILD syndrome patients from different countries, caused by 20 unique gene mutations occurring throughout the entire NSDHL gene, to promote understanding and provide a more comprehensive description of this unusual disorder.

[miR-126 modulates the expression of epidermal growth factor-like domain 7 in human umbilical vein endothelial cells in vitro].

OBJECTIVE: To investigate the regulatory effect of miR-126 on epidermal growth factor-like domain 7 (EGFL7) in ECV-304 cells. METHODS: The miR-126-expressing plasmid targeting EGFL7 (plegfp-N1-miR-126) was constructed and transiently transfected into ECV-304 cells via liposome. The changes in the mRNA and protein expressions of EGFL7 in the transfected cells were analyzed by fluorescence quantitative RT-PCR and Western blotting. RESULTS: Transfection with the recombinant plasmid plegfp-N1/miR-126 resulted in decreased EGFL7 expression with the passage of time, and the expression reached the lowest level at 48 h after the transfection. The expression of EGFL7 protein was reduced by 67% following the transfection in comparison with the control level, while the transfection with the empty vector resulted in a reduction only by 6.5% relative to the control level. CONCLUSIONS: miR-126 can downregulate EGFL7 expression at the protein level in ECV-304 cells.

Activation of transcription factors NF-kappaB and AP-1 and their relations with apoptosis associated-proteins in hepatocellular carcinoma.

AIM: To study the distribution pattern of transcription factors NF-kappaB and AP-1 and their relations with the expression of apoptosis associated-proteins Fas/FasL and ICH-1L/S in human hepatocellular carcinoma (HCC). METHODS: We performed in situ hybridization and immunohistochemical techniques for NF-kappaB, AP-1, Fas/FasL and ICH-1 in 40 cases of human HCC along with corresponding nontumoral tissues and 7 cases of normal liver tissues. RESULTS: Twenty-two (55%) and 25 (62.5%) of 40 cases for NF-kappaB and AP-1 were presented for nuclear or both nuclear and cytoplastic staining respectively, while less cases were presented for only cytoplastic staining for NF-kappaB (18%) and AP-1 (10%) in adjacent nontumoral tissues and negative staining in normal liver tissues. There was no statistically significant difference of NF-kappaB or AP-1 activation between well differentiated tumors and poorly differentiated tumors (P>0.05). NF-kappaB activity is positively corresponded to AP-1 activation. The expression of ICH-1L/S was associated with the activation of NF-kappaB and AP-1 (P<0.05), but no significant relationship was found between Fas/FasL and NF-kappaB or AP-1(P>0.05). CONCLUSION: Activation of both NF-kappaB and AP-1 may be required for ICH-1L/S-induced apoptosis in HCC, but not for Fas/FasL-mediated apoptosis. NF-kappaB and AP-1 may play important roles in the pathogenesis of human HCC.

Detection of bcl-2 and bax expression and bcl-2/JH fusion gene in intrahepatic cholangiocarcinoma.

AIM: To investigate the relationship between bcl-2 gene and its related protein bax and intrahepatic cholangiocellular carcinoma (CCC). METHODS: Semi-nested in situ PCR (SNISPCR) and immunohistochemistry were performed to detect bcl-2/JH fusion gene and bcl-2, bax protein expression in 29 cases of CCC. RESULTS: No bcl-2/JH fusion gene was found in all cases of CCC, 72.4% of 29 cases expressed bcl-2 protein. Bcl-2 protein expression was related to histopathological grades (P<0.05). There was no corresponding relationship between bcl-2/JH fusion gene formation and bcl-2 protein expression in CCC (P<0.05). Bax was expressed in 10.3% of 29 cases. The ratio of bcl-2 to bax in normal liver tissues (3.5 to 1) was different from that in tumor tissues (7.0 to 1). CONCLUSION: It is suggested that bcl-2/JH fusion gene formation is not a frequent event and may not play an important role in the pathogenesis of CCC. However, aberrant ratio of bcl-2 to bax protein expression may be involved in the course of tumorigenesis of CCC. Abnormal bcl-2 protein expression may not be solely resulted from bcl-2/JH fusion gene.

[A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation].

OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro. METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay. RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition. CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.

Identification of lung micrometastatic tumor foci in nude mice with implanted tumor using laser capture microdissection and PCR-single-strand conformation polymorphism.

OBJECTIVE: To assess the value of the laser capture microdissection (LCM) combined with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique for diagnosing micrometastatic cancer cells in the lung of nude mice with implanted tumor. METHODS: Isolation of the cells from the suspected tumor loci in the lung of nude mice with implanted tumors was performed using laser capture microdissection technique, and the genomic DNA extracted from the cells was amplified by 2 sequential PCRs. Non-radioisotopic single-strand conformation polymorphism (SSCP) was subsequently performed to analyze the point mutation of K-ras gene. RESULTS: K-ras gene (codon 12) mutation in AGT was identified in the suspected metastatic cancer cells but not in the benign nodular lesion, where wild type K-ras gene (GGT) was detected. CONCLUSION: The utilization of LCM combined with PCR-SSCP technique may serve as a crucial aid for molecular diagnosis of morphologically suspicious cancer cell populations.

[High-dose glucose induces human retinal endothelial cell apoptosis].

OBJECTIVE: To examine the direct effect of high glucose levels on primary cultured human retinal capillary endothelial cells (HRCEC). METHODS: HRCECs were isolated from donated eyes and cultured for 6 days in the media containing 5 or 25 mmol/L glucose. The cell viability was determined by trypan blue exclusion assay and cell cycle analyzed by flow cytometry, with the cell apoptosis assayed by TUNEL method. RESULTS: The cell viability was significantly decreased after exposure to 25 mmol/L glucose, and the number of apoptotic cells determined by flow cytometry and TUNEL was significantly increased in response to high-dose glucose treatment. CONCLUSION: High-dose glucose induces apoptosis in HRCEC, which may contribute to the development of diabetic retinopathy.

[Relationship between hepatitis C virus infection and expression of apoptosis-related gene bcl-2, bax and ICH-1 in hepatocellular carcinoma tissues].

OBJECTIVE: To study the association between hepatitis C virus (HCV) infection and expression of apoptosis-related gene bcl-2, bax and ICH-1, and explore the significance of HCV infection in the tumorigenesis of hepatocellular carcinoma (HCC). METHODS: The expression of HCV antigen (NS5) along with bcl-2, bax and ICH-1 proteins was investigated in 40 specimens of hepatocellular carcinomas by immunohistochemistry method, with 13 normal liver tissues serving as control. RESULTS: Eleven carcinoma specimens were positive for NS5 antigen, accounting for a positive rate of 27.5 %, and 5 tissue specimens adjacent to the carcinomas were identified as positive, with a rate of 12.5 %. Of the 11 carcinoma tissues positive for NS5, bcl-2 and bax were positive respectively in 1 case (showing a ratio of 1:1), and ICH-1L and ICH-1S were positive in 5 and 6 cases respectively (showing a ratio of 1:1.2). Among the 29 NS5-negative cases, bcl-2 and bax were positive in 6 and 7 cases respectively constituting a ratio of 1:1.4, while ICH-1L and ICH-1S were identified in 10 and 15 cases respectively, at the ratio of 1:1.5. No significant differences were found between NS5-positive and -negative groups (P>0.05). CONCLUSION: Aberrant expression of bcl-2, bax and ICH-1 may be related to HCC genesis, but HCV infection may not be the principal cause for their expression aberrance.