Antitumor effect of RNA interference on non-small- cell lung cancer in vivo

Objective： Lung cancer has emerged as a leading cause of cancer death in the world. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. RNA interference （RNAi） has shown promise in gene silencing in vitro, the potential of which in developing new methods for the therapy of non-small-cell lung cancer （NSCLC） needs to be further tested in vivo. In this study, chemically synthesized double-stranded RNA （dsRNA） targeting epidermal growth factor receptor （EGFR） was transfected into NSCLC cell line SPC-A1 cells and established the tumor burdened athymic nude mice model to investigate whether dsRNA could induce gene silencing in NSCLC cells in vivo. Methods： SPC-A1 was transfected with EGFR sequence-specific dsRNA formulated with Lipofectamine 2000. SPC-A1 cells （1 × 107/ mL） in 200 pL were injected s.c. into the left flank area of the mice to establish the tumor burdened athymic nude mice model. Calculate the tumor growth inhibition rate by measuring the diameter and the weight of the tumor. Immunohistochemistry and Westem blot were used to monitor the reduction in the production of the EGFR protein. Realtime RT-PCR was used to detect the silencing of the EGFR mRNA level. Results： It displayed that EGFR sequence specific dsRNA （dsRNA-EGFR） significantly inhibited the tumor growth in vivo. The tumor growth inhibition rate was 75.03%. The dsRNA-EGFR sequence specifically silenced EGFR with 53.6% of down-regulation of EGFR protein production and 32.3% of silencing of EGFR mRNA level. Conclusion： DsRNA-EGFR showed a blockbuster effect in downregulation of EGFR mRNA level and protein production, and inhibition of tumor growth in vivo.     

Effects Of the anti-tumor composition of the acetoacetate extract of vitex negundo seed on the growth of human cervical carcinoma xenografts in nude mice

Objective： The aim of our study was to investigate the effects of the anti-tumor composition of the acetoacetate extract of Vitex Negundo Seed （EVn-50） on the growth of human cervical carcinoma HeLa cells xenografts in nude mica and its possible molecular mechanism. Methods： Models of human cervical cancer HeLa cells xenografts transplanted subcuta- neously in nude mice were established and randomly divided into 7 groups （each group including 5 nude mice）： saline group, Taxol group, EVn-50 group, comp-6 group, comp-7 group, comp-8 group and comp-10 group. The volume and weight of Xe- nograts were observed and compared. The alteration of the weight of nude mice, and the change of serum levels ofLDH, ALT, Cr and WBC counts were examined and compared. The apoptotic rate of human cervical carcinoma HeLa cells xenografts was analyzed by FCM. The expressions of P53 and Bcl-2 proteins of HeLa cells xenografts were determined by Western blot- ting. Results： EVn-50 and its fractionated extracts could significantly suppress the increasing volume and weight of human cervical carcinoma HeLa cells xenografts in nude mice models in time-dependent manner, yet had no significant effect on the weight of nude mice, the serum levels of LDH, ALT, Cr and WBC were counted. When the xenografts were treated with EVn-50 and its fractionated extracts for 16 days, the apoptotic rate of xenografts cells were significantly increased, and the expression of P53 protein was up-regulated and protein level of Bcl-2 was decreased. Conclusion： EVn-50 and its fractionated extracts could suppress the growth of human cervical carcinoma HeLa cells xenografts in nude mice, which may be related to its pro- motion on xenografts cells apoptosis through down-regulation of Bcl-2 expressionPand activation of P53 expression.     

Relationship between prognosis and angiogenesis,lymphangiogenesis, proliferative cell distribution in gastric cancer

Objective： We investigated the relationship between lymphangiogenesis, angiogenesis and cell proliferation in gastric cancer. Methods： We observed the central cancer tissues and the peritumoral tissues of 75 patients with gastric carcinoma by immunohistochemistry. Using D2-40, VEGFR-C and VEGFR-3 detected the microlymphatic density （MLD）, CD34 and CD31 detected the microvessel density （MVD）. The proliferation of the cells was labeled by Ki-67. Results： There were a few atresic streak lymphatic vessels in the central cancer tissues, but in the peritumoral tissues lymphatic increased and dilated, it appears adenoid structure. LMVD in the gastric central area （33.7 ± 14.7） decreased significantly than in the peripheral zone （61.8 ± 22.6; P 〈 0.01）. The differences in the distribution and amount of microvessels were similar to lymphatic vessels. The central area of gastric cancer has a small amount of the distribution of focal Ki-67 positive tumor cells, while around the central area there were a large number of Ki-67 positive tumor cells, especially in one low power field （× 10）. Gastric central area of the Ki-67 positive cells in gastric central area （49.5%） were significant decreasing than in the cancer peripheral zone （73.2%; P 〈 0.05）. Conclusion： The mainly distribution of the neonatal lymphatic vessels, blood vessels and Ki-67 positive carcinoma in gastric cancer is anterior border of carcinoma tissues, especially in one low power field （x 10）. The MLD, MVD and Ki-67 positive carcinoma might be an important index for the prognosis of gastric carcinoma. The more lymphatic microvessel, microvessel and Ki-67 positive carcinoma in the peritumoral tissues, the poorer prognosis, vice versa.     

Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells

Objective： The aim of this study was to construct THY1 eukaryotic expression plasmid ,and study its effects on ovarian cancer SKOV3 cells. Methods： The gene fragment coding for THY1 was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） to construct the recombinant plas- mid pcDNA3.1（＋）-THY1, which was transfected into SKOV3 cells. The experimental cells were classified into three groups： SKOV3-THY1, SKOV3-Null and SKOV3. The expression of gene was measured using RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Both SKOV3-THY1 and SKOV3-null cells were inoculated subcutaneously into nude mice to determine in vivo tumorigenicity. Results： The gene fragment of THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） and veri- fied by PCR, restriction endonucleases digestion and DNA sequencing and the plasmid of pcDNA3.1（＋）-THY1 （THY1 gene overexpression） has been stably transfected into SKOV3 cells. The analysis of flow cytometry indicated that the pcDNA3.1（＋）- THY1 transfected ceils in G1 phase were significantly elevated, but in S phase were decreased. The growth of transfected cells was suppressed, and more apoptosis cells were identified in pcDNA3.1（＋）-THY1 transfectants compared with vector vehicle transfectants. The tumor suppressing activity of THY1 in SKOV3 cells was associated with inhibition of SKOV3 cellular proliferation, in vivo tumorigenesis in nude mice. Conclusion： THY1 transfection can inhibit the growth of SKOV-3 cells in vitro and in vivo. THY1 gene may play an important role in generation and development of ovarian cancers.     

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

Objective： To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. Methods： T24-3 ceil subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 10^7 hu-PBL and subcutaneously injected with 3 × 10^6 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3^＋, CD4^＋, CD8^＋ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhlL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. Results： All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group （P 〈 0.01）. Conclusion： DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.     

The expression and clinical significance of β-catenin and colorectal cancer stem cells marker EpCAMhigh/CD44＋ in colorectal cancer

Objective： The aim of the study was to explore the role of Wnt βcatenin signalling pathway in the maintenance, invasion and metastasis of colorectal cancer stem cells. Methods： Double immunohistochemical staining was used to detect the expression of EpCAMhigh/CD44~ which is regarded as the marker of colorectal cancer stem cells in 80 cases of colorectal cancer and their corresponding liver metastases. The SP method of immunohistochemistry was used to detect the expression of the key protein βcatenin in the Wnt pathway in these tissue. The expression and correlation of ~-catenin and EpCAMh~gh/ CD44＋ in colorectal cancer were analyzed and their role on the biological behavior of colorectal cancer was explored. Results： The abnormal expression of βcatenin was significantly higher in colorectal cancer than in the paraneoplastic normal intes- tinal mucosa [55% （44/80） vs 10% （2/20）, P 〈 0.05]. The positive expression of EpCAMhigh/CD44＋ was significantly higher in colorectal cancer than in the paraneoplastic normal intestinal mucosa [66.25% （53/80） vs 0% （0/20）, P 〈 0.05]. In the 80 cases of colorectal cancer, the abnormal expression of ~-catenin has no correlation with gender （P = 0.079）, age （P = 0.416） and the magnitude （P = 0.816） of the tumor （P 〉 0.05）, but it was significantly correlated with degree of differentiation （P = 0.001）, depth of invasion （P = 0.001）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the colorectal cancer, the expression of EpCAMhi~h/CD44~ cells has no correlation with gender （P = 0.934） and the magnitude （P = 0.160） of the tumor （P 〉 0.05）, but was significantly correlated with age （P = 0.021）, degree of differentiation （P = 0.013）, depth of invasion （P = 0.000）, clinical stage （P = 0.000） and metastasis （P = 0.000）. In the corresponding liver metastases, we could also detecte EpCAMhih/CD44＋ cells. In cases with abnormal expression of βcatenin, the positive expression rate of EpCAMhigh/CD44＋ was significantly higher than those with normal expression of β-catenin （84.1% vs 44.4%）, and the difference was statistically significant （P 〈 0.05）. Conclusion： The abnormal activation of Wnt β-catenin signalling pathway may prompt the abnormal proliferation of the colorectal cancer stem cells, which leads to the recurrence and metastasis of the cancer.     

S100A6 gene have a positive influence on the growth and proliferation of gastric cancer cell MKN45

Objective： S100A6 （a.k.a., calcyclin） is over-expressed in several human tumors, including gastric carcinoma, human melanoma, pancreatic carcinoma, squamous cell carcinoma, malignant fibrous histiocytoma （MFH）, and carcinomas of the thyroid, breast, and colon. However, little is known about the role S100A6 plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of S100A6 on the growth, proliferation, apoptosis, invasion and cell cycle of the gastric cancer cell MKN45. Methods： As an important member of S100 family, S100A6 cDNAwas subcloned into a constitutive vector pcDNA3.1 followed by transfection in gastric cancer cell line MKN45 by using liposome. Then stable transfectants were selected and appraised. The apoptosis and cell cycles of these clones were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay respectively. The S100A6 stable expression clones （MKN-S100A6） were detected and compared with their control groups respectively. Results： MKN- S100A6 grew faster than MKN45 and MKN-PC （MKN45 transfected with pcDNA3.1 vector）. The cell counts of MKN-SI00A6 in the fifth, sixth and seventh days were significantly more than those of control groups （P 〈 0.05）. Cell cycle analysis showed that proportions of MKN-S100A6 in G0-G1 and G2-M were different significantly with those of its control groups respectively （P 〈 0.05）. The apoptosis rate of MKN-S100A6 was significantly lower than those of control groups （P 〈 0.05）. Results of colony-forming assay showed that the colon formation rate of MKN-S100A6 was higher than those of control groups （P 〈 0.05）. Conclusion： S100A6 can promote the growth and proliferation of gastric cancer cells. It can help tumor cell maintain malignant phenotype. In gastric cancer, S100A6 could be thought as a tumor-enhancing gene in some distance, but its role could be complicated.     

Effect of grape proanthocyanidins on tumor growth and angiogenesis in H22 liver cancer xenograft mode

Objective： The aim of this study was to investigate the effect of grape proanthocyanidins （GPC） on the growth and angiogenesis of hepatocellular carcinoma H22 cells xenograft in mice. Methods： The xenograft model was established using injected subcutaneously H22 cells into the right axilla of the mice. Each group was treated with different doses of GPC and Endostar. All these treatments were maintained for 10 days, and mice were sacrificed. The xenograft tumors in mice were measured. The proliferation activity level of H22 cells was determined by MTT assay, and the levels of vascular endothelial growth factor （VEGF） protein were examined by immunohistochemistry. Results： When treated with 50, 100 and 200 mg/kg of GPC and Endostar, the tumor inhibition rates were 13.17%, 23.37%, 36.15% and 14.71%, respectively. The tumor weight of xenograft was significantly lighter in high GPC group than the control group （P 〈 0.05）. The ODs in GPC groups were 0.835, 0.666 and 0.519, respectively. The absorbances in middle and high GPC groups were statistically significant, compared with control group （P 〈 0.01）. Immunohistochemical technique showed the expression of VEGF of the GPC groups was down- regulated significantly compared with the control group （P 〈 0.01）. Conclusion： GPC can inhibit the growth of hepatocellular carcinoma H22 cell xenograft in mice. The inhibition of angiogenesis by the down-regulation of VEGF expression may play a key role in the anti-neoplastic effect of GPC.     

Application of non-small cell lung cancer pleural effusion cell blocks in molecular pathological detection

Objective： The tumor tissues used in molecular pathological detection were usually obtain,ed by surgery, which would cause trauma and may not be suitable for the terminal cancer patients. This paper evaluated the value of the non-small cell lung cancer （NSCLC） pleural effusion cell blocks as tumor tissues replacement materials in the application of molecular pathological detection. Methods： Tumor cells were made into cell blocks through stratified centrifugal from 30 NSCLC pa- tients with the pleural effusion. The immunohistochemistry, fluorescence in situ hybridization （FISH） and gene sequencing methods were employed in our experiments. Results： The tumor cells of cell block section were rich and could keep part of histological structure. Immunohistochemistry staining could assist diagnosis and tumor parting. Epidermal growth factor receptor （EGFR） FISH-positive was found in 33.33% of the group, high polysomy in 6 cases, amplification in 4 cases. EGFR gene mutations were found in 8 cases of 30 samples, with an incidence of 26.67%, 6 cases were detected in the exon 19, and 2 cases were detected in the exon 21. Conclusion： The NSCLC pleural effusion cell blocks are useful for the diagnosis and determining the primary source of tumor, instructed targeted therapy.     

Up-regulation of Raf kinase inhibitor protein enhances chemosensitivity of cervical cancer cell

Objective： The purpose of the study is to investigate the effects of up-regulation of Raf kinase inhibitor protein （RKIP） on the chemosensitivity of cervical cancer Hela cells. Methods： Eukaryotic expression plasmid pcDNA3.1（±）-ssRKIP containing human overall length RKIPcDNA was transfected into cervical cancer Hela cell by lipofectin assay, establishing a stable cell line containing a target gene by G418. Expression of RKIP in Hela cells was measured by Western blot analysis. After treatment with cisplatin of different concentrations and intervals of time, the effect of RKIP on the proliferation of Hela cells was evaluated by MTT method. The flow cytometry was used to investigate whether the RKIP could inhibit apoptosis in Hela cells induced by cisplatin. Results： The expression of RKIP in Hela cells transfected with pcDNA3.1-ssRKIP was increased obviously. After different concentrations of cisplatin treatment cells for 24, 48 and 72 h, the growth inhibition rate in Hela cells transfected with pcDNA3.1-ssRKIP was significantly higher than in control cells （P 〈 0.05）. With 5 pg/mL cisplatin treatment for 24 h, pcDNA3.1-ssRKIP-transfected Hela cells had an obviously higher percentage of apoptosis （23.2 ± 0.24）% than non-transfected cells （12.4 ± 0.31）% and empty vector-transfected cells （13.4 ± 0.47）%. Without treatment of cisplatin, the percentage of apoptosis for Hela cells transfected with pcDNA3.1-ssRKIP was （5.7 ± 0.12）%, which was still higher than those of the non-transfected cells （2.9 ± 0.21）% and empty vector-transfected cells （3 ± 0.08）%. Conclusion： Higher expres- sion of RKIP gene can improve chemosensitivitv of cervical cancer Hela cells to cisplatin.     

Diagnostic value of transbronchial lung biopsy in peripheral lung cancer

Objective： The purpose of this study was to evaluate the diagnostic value of transbronchial lung biopsy （TBLB） in peripheral lung cancer. Methods： 78 cases of peripheral lung cancer which could not be observed by bronchoscope were selected from the Second Affiliated Hospital of Sun Yat-sen University （China）, of which 42 cases were diagnosed by TBLB. Among the 36 cases of peripheral lung cancer that could not be able to be diagnosed by TBLB, 22 cases were diagnosed by percutaneous lung biopsy （PNLB） and 14 cases being left were diagnosed by surgical specimens biopsy, lymphadenopathy biopsy, pleural biopsy or pleural effusion cytology. Results： The positive rates produced by TBLB and transbronchial brush biopsy were 53.8% and 8.9%, respectively, and the combined positive rate was 57.7%. The positive rate produced by TBLB was higher than that of transbronchial brush biopsy （P 〈 0.01）. As the tumor＇s diameter increased, the positive rate of the biopsy was higher （P 〈 0.05）. The positive rate of biopsy of the right lung was not significantly higher than that of the left lung （P 〉 0.05）. The positive rate of biopsy of the inferior lobes was not significantly higher than that of the upper lobes of the lung （P 〉 0.05）. The lesions of the tumors which were nearer to the infield and hilar of the lung got a higher positive rate （P 〈 0.01）. The incidence of complications in PNLB was much higher than that in TBLB （P 〈 0.05）. Conclusion： TBLB is an important method in the diagnosis of peripheral lung cancer. Combination of TBLB and other methods can increase the positive rate in the diagnosis of peripheral lung cancer.     

Analysis of clinicopathological and immunohistochemical features of 15 cases with olfactory neuroblastoma

Objective： We studied the clinicopathological and immunohistochemical features of olfactory neuroblastoma （ONB）. Methods： The clinic pathological data and immunohistochemical features of 15 cases with ONB were analyzed retrospectively, and the related literatures were reviewed. Results： The tumors were all located in the nasal cavity in 15 cases. According to Kadish＇s staging system, 7 cases were in stage A, 5 cases in stage B, and 3 cases in stage C. The morphological features showed small round or ovoid tumor cells divided into pieces or trabeculae by connective tissues which were rich in capillaries. The tumor cells had round or oval nuclei and fine chromatins and lack of cytoplasma. Flexner rosette and Homer- Wright rosette were found in some cases. Acidophilic fibrins were composed of cytoplasmic projection among tumor cells. All cases were positive for NSE, Syn, CgA, 1 case was positive for Vimentin, 2 cases were positive for S-100, while CKpan, LCA and HMB45 were all negative. Conclusion： ONB is a type of very rare malignant tumors, which could be diagnosed by pathology, and immunohistochemistry is helpful in the diagnosis and differential diagnosis.     

Changes in the clinicopathological characteristics of gastric cancer in Hehuang Valley of China -2379 cases of gastric cancer in recent 10 years

Objective： The aim of our studywas to discuss changes in the clinicopathological characteristics of gastric cancer in Hehuang Valley of China in recent 10 years. Methods： A retrospective case-control study was performed on 2379 newly-diagnosed gastric cancer patients. All of them came from Hehuang Valley. The patients were divided into two groups [recent 5 years （R5Y） and late 5 years （L5Y）] from February 2003 to February 2013, and the clinicopathological data were surveyed retrospectively. Results： The constituent ratio of upper 1/3 gastric cancer in R5Y and L5Y was 33.5% and 20.7%, respectively, and it showed a significant difference between the two groups （X2 = 21.28, P = 0.00）, The constituent ratio of squamous carcinoma/adenosquamous carcinoma was 2.7% and 1.6%, respectively, and it also showed a significant differ- ence between two groups （X2 = 50.91, P = 0.00）. The constituent ratio of moderately-poorly differentiated/poorly differentiated gastric carcinoma was 84.2% and 50.2%, respectively, and it showed statistically significant difference （X2 = 30.28, P = 0.00）. The detection rate of early gastric cancer （EGC） was 1.47% （35/2379）. The constituent ratio of the types of Borrmann II and Borrmann IV of advanced gastric cancer （AGC） among 2379 cases was 47.6% and 40.8%, respectively, and that still showed statistically significant difference （X2 = 18,80, P = 0.00）. The constituent ratio of diffuse-type of gastric cancer in R5Y and L5Y was 36.2% and 30.8%, respectively, even there was a significant difference （X2 = 7.49, P = 0.01）. Furthermore, there were also significant differences in regional lymph nodes metastasis, perineural infiltration and vascular invasion （P 〈 0.05）. The positive detection rate of HER2, ER and PR was 14.88%, 17.23% and 15.93%, respectively. The constituent ratio of HP in two groups was 43.8% and 36.2% respectively, and it also showed a significant difference （~2 = 13.51, P = 0.00）. Conclusion： The pathogenic sites in gastric cancer change to the upper stomach in Hehuang Valley in recent 10 years, and the detection rate of squamous carcinoma/adenosquamous carcinoma reveals a sharp rise. Borrmann III is still one of the main types of advanced gastric cancer, but the detection rates of Borrmann II and IV are increasing. The main type of gastric cancer is the intestinal- type, but the ratio of diffuse-type is also increasing in recent 10 years. The HP detection rate is 40.65% （967/2379）, and it has a slight rise in recent 10 years. The detection rate of poorly differentiated adenocarcinoma is increasing despite the fact that the moderately/moderately-poorly differentiated adenocarcinoma is the main histologic types. High detection rates of lymph nodes metastasis, perineural infiltration and vascular invasion indicate poor prognosis in patients with gastric cancer. There is no change in HER2 positive rate, on the contrary, there are a little increase in ER and PR expression in Hehuang Valley.     

Expression of BI-1 protein and its significance in breast cancer

Objective： To investigate the correlations of expression of Bax inhibitor-1 （BI-1） gene and the receptors of estrogen and progestogen in breast cancer and its significance. Methods： Immunohistochemical methods had been used to detect the expressions of BI-1 gene and receptors of estrogen and progestogen in breast cancer. Results： The positive rates of expressions of BI-1 gene, estrogen receptor （ER） and progestogen receptor （PR） in breast cancer were 77.08%, 60.42% and 54.17%, respectively. The positive rate of expression of BI-1 gene was higher in the group with negative expression of ER than the positive group, their positive rates were 76.92% and 52.27%, respectively; but there was no statistical difference between the two groups with positive and negative expressions of PR. The positive rate of expression of BI-1 gene was also higher in the group with positive lymph node metastasis than the non-lymph node metastasis group, and their positive rates were 64.58% and 36.36%, respectively. The difference was statistically significant （P 〈 0.05）. Conclusion： BI-1 gene, in combination with ER, has guiding significance for patients with breast cancer to choose individual chemotherapy and radiotherapy after operation and can become an important indicator for judging the prognosis of breast cancer.     

Peripheral primitive neuroectodermal tumors： a rare case report

We aimed to explore the diagnosis and treatment of peripheral primitive neuroectodermal tumors （pPNETs）. We retrospectively analyzed the diagnosis and treatment process of a patient who was diagnosed with pPNETs by pathology. This case was a man with soft masses arising from the left chest wall near the armpit and left supraclavicular of a 47-year-old man. The patient mainly presented with the masses which increasing gradually with obvious pain. Needle biopsy showed that they were both metastatic adenocaroinoma. Ultrasonography B revealed blood flow of these two low density placeholders can be seen in the signal, not oppression axillary and vein. Radical resection of the masses were performed. Histopathologic study and immunohistochemistry （IHC） confirmed the masses to be peripheral primitive neuroectodermal tumors, pPNETs is a rare malignant small round cell tumor. CT and MRI examination can estimate the resectability of the tumor; Ultrasound B can make sure its inside blood supply and the positional relationship between the mass and the surrounding vasculature. The diagnosis of pPNETs is based primarily on histopathologic study and IHC, especially those with the characteristics of the Homer-Wright and neuroendocrine markers. Radical resection of the tumor is the most effective therapeutic method. The effect of adjuvant chemo-radiation is worth affirmation. Autologous stem cell rescue besides adjuvant chemotherapy has been associated with prolonged survival.     

The 5-year incidence of male breast cancer in Southwest of China from 2007 to 2011

Objective： Male breast cancer is a rare disease with an incidence of about 1% of breast cancers in USA, but relatively lack of the information of male breast cancer in China, especially in Southwest of China, led us to study its incidence trends. Methods： Chongqing is one of the biggest and the most important areas that is located in Southwest of China. There are around 31.4 million people who live in approximate 82 402.95 km2 area of Chongqing. Data about breast cancer patients registered in the Center for Disease Prevention and Control of Chongqing （China） were statistically collected from 187 hospi- tals, about 58 hospitals in city and 129 hospitals in country, and over 6.2 million people were studied every year. It was tried to represent all the people in villages and cities in Chongqing, China. Results： The incidence of male breast cancer in Southwest of China ranged from 0.34/100 000 to 1,45/100 000 between 2007 and 2011, while the incidence of female breast cancer ranged from 15.40/100000 to 21.66/100000 at the same time. The rate of male breast cancer to female breast cancer ranged from 0.02：1 to 0.07：1, male breast cancer accounted for 1.96% to 6.5% （with the mean value of 2.9%） of breast cancers in Southwest of China from 2007 to 2010. Conclusion： In Southwest of China male breast cancer accounts for about 2.9% of breast cancers which is higher than that in United States. It is important for policy makers and health manager to seriously consider breast cancer in future plan in Southwest of China.     

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.     

Expression profiles of miRNAs in human pancreatic cancer cell lines

Objective： To analyze initially the differences of miRNAs expression profiles in human pancreatic cancer cell lines by microarray technique. Methods： A total of 743 probes were designed according to the known miRNAs sequences of human, mice, and rats. miRNAs microarray was manufactured and its credibility was verified. Total RNAs were extracted and miRNAs were separated from human pancreatic cancer cell lines （SW1990, Capan-2, BxPC-3, Aspc-1, and Pancl） and immortal human pancreatic duct epithelial cell line H6C7. They were labeled with T4 RNA ligase, then were hybridized with microarray. Through array scan and analysis, miRNAs expression profiles in pancreatic cancer were obtained. The results were verified by Northern blotting and RT-PCR. Results： A total of 63 rniRNAs related to pancreatic cancer were found to be differentially expressed in 5 pancreatic cancer cell lines, including 25 down-regulated and 38 up-regulated miRNAs. Expressions of mir-21 and let-7 were also confirmed： Conclusion： The results suggested that miRNAs expression profiles could be found in pancreatic cancer cells.     

Construction of eukaryotic expression plasmid pEGFP-N1-WWOX and its transient expression in SMMC-7721 cells

Objective： To construct eukaryotic expression plasmid pEGFP-NI-WWOX and transiently express it in SMMC-7721 cells. Methods： Total mRNA was extracted from normal human liver tissue. RT-PCR was used to amplify the aimed segments WWOX cDNA which was then digested with Hindlll and BamHI and inserted into a eukaryotic expression plasmid pEGFP-N 1 to construct pEGFP-N 1-WMVOX. The constructed plasmid was transfected into SMMC-7721 cells by lipofectamine 2000 - mediated transfer method. The expression of WWOX in transfected SMMC-7721 cells was detected 24, 36 and 48 h post-transfection with fluorescence microscope and the expression level of WWOX mRNA in transfected SMMC-7721 cells was assay by using RT-PCR. The change of MMWOX expression and cell proliferation rates were detected by immunocyto- chemistry and MTT methods respectively. Results： The results showed pEGFP-N1-WWOX was successfully constructed and expressed transiently in SMMC-7721 cells. At 48th hour post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected, while no green fluorescence was detected in the control group. In SMMC-7721 cells transfected with pEGFP-NI-WWOX a high level of porcine WWOX was detected. WWOX ex- pressed by transfected cells could significantly inhibit the proliferation of SMMC-7721 cells. Conclusion： pEGFP-N1-WWOX was expressed successfully in SMMC-7721 cells, which suggested that might be used as a new therapeutic method for liver cancer.     

Influence of human mutant p27 gene on the biological behaviors of colon cancer cells

Objective： To observe the influence of human mutant p27 gene （p27mt） on the growth and apoptosis of colon cancer cells so as to investigate the function mechanism of p27mt in gene therapy for colon cancer. Methods： Colon cancer cell line SW480 was infected with recombinant replication defective adenovirus Ad-p27mt, and expression of p27mt protein was detected by Western blot; the inhibition effect of p27mt on SW480 cells was detected with cytometry. Cell cycle was decided with flow cytometer, and DNA fragment analytic process identified the occurrence of apoptosis. Results： After transfected SW480 cells with Ad-p27mt, high expression of p27 protein was identified with immunoblotting assay. PI staining and flow cytometer assay showed 77.96% colon cancer cells was blocked in phase G0/G1, while in Ad-LacZ group and blank control group, 27.57% and 25.29% cells were blocked in the same phase, respectively. Growth curve showed Ad-p27mt has an obvious inhibition effect on the growth of SW480 cells, DNA fragment assay demonstrated that p27mt was able to induce the apoptosis of colon cancer cells. Conclusion： p27mt has an obvious blocking effect on colon cancer cell cycle, and most cells were blocked in phase G0/G1. This blockage is related with the growth inhibition and apoptosis induction effect of p27mt.