[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene].

OBJECTIVE: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene. METHODS: Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively. RESULTS: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05). CONCLUSION: The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.

[Clinical diagnosis and treatment of familial medullary thyroid carcinoma caused by a p.C618Y RET proto-oncogene mutation in a Chinese pedigree].

OBJECTIVE: To explore the clinical characteristics, therapeutic and clinical significance for RET proto-oncogene screening in a pedigree with familial medullary thyroid carcinoma. METHODS: Comprehensive medical history was obtained from 19 members in a 4-generate southern Chinese family. Systemic clinical investigations including biochemical testing, imaging examinations and germline RET screening. RESULTS: RET screening showed heterozygous missense mutations of TGC to TAC at codon 618 on exon 10 in 8 cases (p.C618Y) completely consistent with the clinical manifestations. The clinical data of 7 patients with medullary thyroid carcinoma (MTC) and 2 carriers of asymptomatic RET mutation from were analyzed. Single/bilateral multi-centric MTC with lymph node metastases was confirmed in 6 cases by histopathology and 1 case by clinical examination. There were 1 male and 6 females with an initial mean diagnostic age was 49.6 years (range: 24 - 78). All had palpable neck masses. And the mean maximum diameter of MTC was 2.6 cm (range 1.4 - 4.4). Seven patients underwent thyroidectomy except a 78-year-old female patient: right total and left subtotal thyroidectomy (n = 1), right total thyroidectomy (previous left total thyroidectomy for benign mass) (n = 1) and total thyroidectomy (n = 4) were performed. All procedures were accompanied by at least bilateral level VI lymph node dissection and/or with modified single/bilateral neck dissection. After the first operation, 6 patients still presented a high value of calcitonin: 1 patient died of metastasis 64 months postoperatively; 3 patients underwent reoperation at 6 months after initial operation, the calcitonin levels dropped to normal in 2/3 cases and stayed higher in 1 case; another two cases presented bilateral thyroid gland residua, local lymph node enlargement on imaging examination and elevated levels of calcitonin at 214 and 60 months postoperation respectively. However, 1/2 asymptomatic with elevated pre-operative calcitonin subjects underwent total thyroidectomy and histopathological examination showed bilateral C cell hyperplasia. The other carriers, without surgery, with normal neck images, close monitoring and a 10-month follow-up, still presented undetectable calcitonin. CONCLUSIONS: Based on family survey, integrated RET screening and serum levels of calcitonin facilitate an early diagnosis and normalize surgery to improve the prognosis. For asymptomatic RET mutation carriers, their levels of calcitonin shall guide the individualized regimen of prophylactic thyroidectomy or strict monitoring and follow-ups.

[The clinical patterns and RET proto-oncogene identification of pheochromocytoma in 13 multiple endocrine neoplasia type 2A pedigrees].

OBJECTIVE: To explore the clinical patterns and clinical significance for RET screening in adrenal pheochromocytoma (PHEO) associated with multiple endocrine neoplasia type 2A (MEN2A). METHODS: The clinical data of 32 PHEO patients with MEN2A from 13 unrelated MEN2A pedigrees from August 1989 to January 2013 were analyzed. The comprehensive medical data included systemic examinations and germline RET gene screening. RESULTS: Among 68 patients belonging to 13 MEN2A families, 32 (47.1%) presented with PHEO. There were 19 males and 13 females with a mean age of (41 ± 12) years. And the mean maximum diameter of PHEO was (4.6 ± 2.2) cm. The diagnosis of PHEO was made after medullary thyroid carcinoma (n = 12, 37.5%), simultaneously (n = 12, 37.5%), initially (n = 7, 21.9%) and death during appendectomy for PHEO-induced hypertensive crisis (n = 1, 3.1%). The diagnosis of PHEO was made before (n = 22) or after (n = 10) clinical screening. The former had 12 symptomatic cases while the latter only 1 case (12/22 vs 1/10, P = 0.024).Except for 5 asymtomatic fatal cases during non-PHEO operations, bilateral PHEO was found in 17 cases including 3 unilaterally treated cases developing another PHEO in contralateral adrenal with a lag period of 5, 10 and 17 years. There were 7 symptomatic patients in bilateral cases versus 6 in unilateral cases (7/17 vs 6/10, P = 0.440). Twenty-five patients underwent PHEO surgery: laparascopic approach in 14 cases (8 with bilateral simultaneous adrenalectomy) and open approach in 11 (2 with bilateral simultaneous adrenalectomy). And 10 patients undergoing bilateral adrenal-sparing operations or adrenalectomy required hormonal replacement therapy. During a mean observation period of 72 (1-282) months, no local recurrence, distant metastasis or Addisonian crisis were noted in 25 cases (contralateral relapse in 3 cases). Among them, 2 cases developed adrenocortical insufficiency unresponsive to an adjustment of hormonal doses.RET screening showed 4 recurrent missense substitutions in 32 MEN2A-PHEO patients: p. C634Y exon 11 (n = 27, 84.4%), p. C634R exon 11 (n = 3, 9.4%), p. C634F exon 11 (n = 1, 3.1%) and p. C618R exon 10 (n = 1, 3.1%). CONCLUSIONS: The mutations of RET proto-oncognene of PHEO in MEN2A are frequently located at codon 634. A combination of pedigree examination and RET gene screening may facilitate an early diagnosis and early treatment of asymptomatic PHEO patients in MEN2A.Laparoscopic cortical-sparing adrenalectomy for preserving adrenocortical function is a preferred surgical approach.

Medullary and papillary thyroid carcinomas in a patient with a C634Y mutation in the RET proto-oncogene: A case report.

A 41 year old man presented with a familial history of multiple endocrine neoplasia type 2A (MEN2A) and severe hypertension. Rearranged during transfection (RET) gene sequencing confirmed a Cys634Tyr mutation of TGC to TAC. Total thyroidectomy and bilateral neck dissection were performed and the pathological assessment revealed a medullary thyroid carcinoma (MTC), 0.6 cm in size on the right side (number of lymph nodes: 0/2, 0/15, 0/12, and 0/8 in areas VI, II, III, and IV, respectively) and a papillary thyroid carcinoma (PTC), 0.2 cm in size on the left side (numbers of lymph nodes: 2/6, 0/3, 0/10, and 0/6 in areas VI, II, III, and IV, respectively). There were no pathological changes in the MTC observed in the thyroid tissues on the left side. We believe that the follow-up of patients with both MTC and PTC should utilize a combination of the respective principles for rational disease reassessment.

High-dose vitamin C suppresses the invasion and metastasis of breast cancer cells via inhibiting epithelial-mesenchymal transition.

PURPOSE: Vitamin C (VC) is a kind of essential nutrient in the body regarded as a canonical antioxidant during the past hundred years. However, the anti-cancer effect of VC is controversial. Our study is trying to clarify the relationship between VC dosage and breast cancer metastasis. METHODS: Human breast cancer cell lines Bcap37 and MDA-MB-453 were treated with VC at three different concentrations (low-dose, 0.01 mM; medium-dose, 0.1 mM; high-dose, 2 mM). Wound healing assays were conducted for migration assay; transwell tests were performed to detect the ability of cell invasion. The protein levels were evaluated by Western blot analysis or immunohistochemistry. Tumor xenografts in nude mice were built to test the effects of VC on breast cancer cell proliferation and metastasis. RESULTS: 0.01 and 0.1 mM VC promoted cell migration and invasion when compared with the control group, but 2 mM VC significantly suppressed cell migration and invasion of breast cancer cell lines. High-dose VC increased E-cadherin and reduced Vimentin, indicating that high-dose VC suppressed epithelial-mesenchymal transition (EMT) in breast cancer cells. Besides, high-dose VC inhibited cell invasion promoted by TGF-ß1 in breast cancer cells. Meanwhile, high-dose VC reversed the suppression of E-cadherin and enhancement of Vimentin induced by TGF-ß1 in breast cancer cells. Furthermore, high-dose VC significantly inhibited breast cancer metastasis in vivo. CONCLUSION: High-dose VC inhibits cell migration and invasion of breast cancer cell lines through suppressing EMT. Thus, it may be considered as an anticancer drug candidate for breast cancer patients.

The novel EZH2 inhibitor, GSK126, suppresses cell migration and angiogenesis via down-regulating VEGF-A.

PURPOSE: To explore the effects and mechanisms of GSK126, a novel inhibitor of histone methyltransferase enhancer of zeste homologue 2, on cancer cell migration. METHODS: Gastric cancer cell line MGC803 and human lung adenocarcinoma cell line A549 were treated with GSK126 at three doses. Transwell and wound healing assays were conducted to detect cell migration. Human umbilical vein endothelial cells tube formation assay and chick embryo chorioallantoic membrane assay were performed to assess the effects of GSK126 on angiogenesis in vitro and in vivo, respectively. The mRNA level of VEGF-A was detected by quantitative PCR, and the protein levels of VEGF-A were detected both by western blot analysis and immunohistochemistry. Epi-fluorescent intensity was obtained by in vivo imaging. RESULTS: GSK126 inhibited cell migration in both MGC803 and A549 in a dose-dependent manner, as revealed by transwell and wound healing assays. The effects of GSK 126 were similar to those of gefitinib at the same doses. Moreover, GSK126 at doses of 20 and 50 µM inhibited angiogenesis both in vitro and in vivo. GSK126 reduced both the mRNA and protein expression of VEGF-A in a dose-dependent manner. Finally, in vivo imaging assay revealed that GSK126 at 200 mg/kg significantly inhibited cancer cell migration. CONCLUSIONS: GSK126 inhibits cell migration and angiogenesis in solid tumor cell lines through down-regulation of VEGF-A expression. Thus, it may be considered as a novel anticancer drug candidate for solid tumor.

RET proto-oncogene genetic screening of families with multiple endocrine neoplasia type 2 optimizes diagnostic and clinical management in China.

BACKGROUND: Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families. METHODS: Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed. RESULTS: The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period. CONCLUSIONS: Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.

RET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family.

BACKGROUND: Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years). CONCLUSIONS/SIGNIFICANCE: The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.