Premiminary Functional Studies Of Metastasis-associated Gene MYH-9in Gastric Carcinoma

BACKGROUND&OBJECTIVEGastric cancer (GC) is one of the top ten most common malignant tumors in the world,and also the most common malignant tumors of digestive system. According to the statistical results of the world health organization(WTO), In East Asian countries (such as China, Japan and South Korea), the numbers of gastric cancer patient are relatively big,but in the countries(such as The United States, New Zealand and Australia),the numbers of gastric cancer patient are relatively small.In the worldwide, the incidence rate of GC is the fourth and the mortality rate of GC is the second in the malignant tumor.In china,GC is also the most common malignant tumors.according the investigation of the cause of death in the nationwide,the second cause of death in male and the third cause of deathe in female are the cancer deathe. On average,160000people die from GC each year, accounting for23.03%of the total death of malignant tumor. Through persevering efforts for several decades,some progress has been made in the basic and clinical research of gastric cancer,while there still remain some problems. Five year overall survival in patients with gastric cancer at less than24%. Most patients with gastric cancer are diagnosed in advanced stages, when a curative resection is impossible, which leads to an overall poor prognosis. In a very long time,the prevention and control of invasion and metastasis of gastric cancer is a important content during Man’s triumph over the stomach cancer. It has been confirmed that the curative effect of only aim to the treatment of primary lesions of GC was not very ideal.We need focus on primary lesions of GC, and also make a great effort to explore the mechanism of invasion and metastasis of gastric carcinoma. To find new diagnostic markers for gastric cancer early diagnosis; To find new prognostic molecular markers as a predictor of prognosis of patients with gastric cancer;To find new therapeutic targets and provide the basis for individualized treatment in patients with gastric cancer. This will lay a solid foundation for the progress of diagnosis and treatment of GC.Nonmuscle myosin Ⅱ(NMⅡ),one of the myosin superfamily,which includes NMIIA(MYH-9). NMIIB(MYH-10)and NMIIC(MYH-14). NMⅡ molecules are hexamers composed of MHC dimers and two pairs of myosin light chains.The functions of MYH-9include roles in hereditary disease、thrombotic disease、hearing impairment、Inflammation and tumor metastasis. Many studies suggest that MYH9/NMIIA has a key role in tumorigenesis and tumor development.For example, Myosin IIA was expressed in all esophageal squamous cancer tissues.In cancer tissues, elevated myosin IIA expression level was significantly correlated with increasing metastatic lymph nodes, poorer cancer differentiation, and advanced tumor stage. Further univariate analysis suggested that strong myosin IIA expression was associated with a significantly shorter overall survival. In addition, MYH9SiRNA was transfected into esophageal squamous cancer cell line (KYSE-510) to study the role of myosin IIA in cell migration. SiRNA-mediated depletion of myosin IIA in KYSE-510cells significantly increased cell-matrix adhesion and attenuated cell migration ability; S100A4plays an important role in the process of invasion and metastasis of malignant tumor; Overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9.The investigation about MYH-9gene is new which has never been reported in gastric cancer both domestic and overseas.The aim of this research was to explore the function of MYH-9gene in the carcigenesis and development of GC.The frist,we investigated the expression of NMIIA in fresh, paired GC tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis;The second, we performed immunohistochemistry(IHC)on paraffin embedded specimens, including GC specimens, matched normal specimens and paired metastatic lymph node samples,and we evaluated the relationship between NMIIA expression level and the clinicopathological parameters and prognosis of gastric carcinoma patients;The third, cell models of human GC with MYH-9gene knock-down were established and study the effects of MYH-9gene on the biological behaviors of human GC.With our study, MYH9/NMIIA may serve as a useful biomarker for the prediction of metastasis and outcome of gastric cancer as well as a possible target to intercept the metastasis in this kind of carcinoma.METHODS1.Expression of MYH-9gene(NM ⅡA protein) in gastric carcinomaThirty-six fresh frozen GC and corresponding normal gastric mucosa tissue samples(more than10cm away from the edge of the GC) were taken from patients with GC.The expression of NMIIA was examined in fresh, paired GC tissues by reverse transcriptase polymerase chain reaction (RT-PCR; n=14) and Western blot analysis (n=36).2.The relationship between NMIIA expression and clinicopathologic parameters and prognosis of GC patientsThe expression of NMIIA was examined on paraffin embedded specimens (including96GC specimens,30matched normal specimens and30paired metastatic lymph node samples) by immunohistochemistry(IHC).To investigate the relationship between NMIIA expression and clinicopathologic parameters(age, gender, tumor location, tumor size, tumor differentiation, mean no. of tLNs, mean no.of mLNs, depth of wall invasion, mean no. of mLNs, lymph node metastasis, distant metastasis and TNM stage) and prognosis(including the GC patients, the curative resection GC patients,the TNM stage Ⅰ+Ⅱ subgroup GC patients,the TNM stage Ⅲ+Ⅳ subgroup GC patients, the intestinal type GC patients and the diffuse type GC patients)of GC patients.3. Chemosynthesis and screening of MYH-9SiRNAThe MYH-9SiRNA were synthesized by Qiagen.including one positive control (Hs-MAPK1-3);one negative control(Ctrl-AllStars-1);two MYH-9SiRNA (Hs-MYH-9-land Hs-MYH-9-2).According the MYH-9siRNA to HiPerFect Transfection Reagent ratio provided by Qiagen, we designed five different kinds of MYH-9siRNA to HiPerFect Transfection Reagent ratio.Five experimental groups:The frist goup:SiRNA (75ng)/HiPerFect Transfection Reagent (1.5μl); The second gourp:SiRNA (75ng)/HiPerFect Transfection Reagent(3μl); The third gourp:SiRNA(37.5ng)/HiPerFect Transfection Reagent (1.5μl); The fourth gourp:SiRNA (37.5ng)/HiPerFect Transfection Reagent (3μl); The fifth gourp:cancer cell group.The positive contro SiRNA (Hs-MAPK1-3) was specified as the interest SiRNA. To monitor MAPK1gene silencing by qRT-PCR and Western blot analysis after an appropriate time point(48h and72h) after transfection. To understand the effect of SiRNA (Hs-MAPK1-3) on the expression of MAPK1gene of GC cell and determine the optimization of the siRNA to HiPerFect Transfection Reagent ratio.To desigened five experimental groups:the blank group(cancer cell group);the negatie control group::Ctrl-AllStars-1; the positive control group:Hs-MAPK1-3; the frist experimental group:Hs-MYH-9-1and the second experimental group: Hs-MYH-9-2。According the optimization of the siRNA to HiPerFect Transfection Reagent ratio.To monitor MYH-9gene silencing by qRT-PCR and Western blot analysis after72h after transfection. To understand the effect of SiRNA on the expression of MYH-9gene of GC cell and determine the MYH-9SiRNA which have the best effects of knock-down.4.Effect of MYH-9down-expression on the biological behaviors of human GCTo analysize the MYH-9expression in five GC cell lines with different metastasis potential by RT-PCR and Western blot,and select the GC cell lines which have the overexpression of MYH-9gene and some carcigenesis and metastasis potential. Cell adhesion assay was used to analyze the effect of decreased NMIIA on cell adhesion ability. Transwell assay was used to assess the effect of decreased NMIIA on cell migration and invasion ability.RESULTS1.Expression of MYH-9gene(NM ⅡA protein) in gastric carcinomaRT-PCR analysis of NMIIA expression in matched normal and tumor tissues showed that NMIIA was upregulated in the majority of GC tissues compared with their normal counterparts (71.50%,10/14). The Mann-Whitney U test was used to compare the differences. Compared with benign gastric tissue,MYH-9mRNA expression in GC is significant (9.79vs.19.21, U=32.00,p=0.002); Western blot analysis of NMIIA expression in matched normal and tumor tissues showed that NMIIA was upregulated in the majority of GC tissues compared with their normal counterparts (3.34%,26/36). The Mann-Whitney U test was used to compare the differences.Compared with benign gastric tissue, NMIIA expression in GC is significant(22.88vs.50.13, U=157.50,p=0.000). 2.The relationship between NMIIA expression and clinicopathologic parameters and prognosis of GC patientsSamples from the entire group of96patients that contained both cancerous and noncancerous tissues were evaluated for NMIIA protein expression by immunohistochemistry. Absent (6/30) or weak (24/30) NMIIA protein expression was detected in the mucosa of normal gastric tissues.In53intestinal type gastric carcinomas,28showed strong NMIIA expression,18showed moderate NMIIA expression and5showed weak NMIIA expression. In43diffuse type gastric carcinomas,25showed strong NMIIA expression,12showed moderate NMIIA expression and8showed weak NMIIA expression. Low staining (SI<10) was noted in46cases, and high staining(SI≥10) was shown in50cases.The mean Sis of normal gastric tissue and GC tissue were3.20and9.53, respectively. Compared with benign gastric tissue,NMIIA protein expression in GC is significant (18.98vs.77.41,U=104..50,p<0.001).Furthermore,both in intestinal type GC(18.92vs.55.07,U=102.50, p<0.001), and in diffuse type GC (15.57vs.51.95, U=2.00,p<0.001). All cancer cells in metastatic lymph nodes of30cases GC showed strong expression of NMIIA, whether their primary tumors were NMIIA high expression or low expression.Expression of NMIIA in tumor tissue was not significantly associated with age (P=0.909), gender (P=0.884), tumor location (P=0.325), tumor size (P=0.534), lauren classification(P=0.871)tumor differentiation (P=0.078), mean no. of tLNs (P=0.180). However, elevated NMIIA expression was strongly correlated with the depth of wall invasion(p=0.026), mean no.of mLNs(p<0.001),lymph node metastasis (p=0.015), distant metastasis(p=0.021)and TNM stage (p=0.004).The mean overall survival time for the high NMIIA expression group was48.1months,and for the low NMIIA expression group was67.0months, These data suggest up-regulated NMIIA expression correlates with poor prognosis(p<0.001).In RO (radical operation) gastric cancer, the mean overall survival time for the high NMIIA expression group was49.5months and for the low NMIIA expression group was68.6months, These data suggest up-regulated NMIIA expression correlates with poor prognosis (p=0.005). Statistical significance of the difference between curves of NMIIA high-expressing and low-expressing patients was compared within subgroups of TNM stage Ⅰ+Ⅱ (p=0.01) and Ⅲ+Ⅳ(p=0.027). No statistical significance of the difference between curves of NMIIA high-expressing and low-expressing patients was compared within subgroups of intestinal type gastric carcinoma (p=0.071) and diffuse type gastric carcinoma (p=0.112).The following significant parameters were entered into a multivariate analysis:depth of wall invasion, lymph node metastasis, distant metastasis, TNM stage and NMIIA. The NMIIA expression(p=0.000)-. lymph node metastasis(p=0.000) and TNM stage (p=0.014)were the independent predictor, which suggests NMIIA expression served as an independent prognostic factor.3. Chemosynthesis and screening of MYH-9SiRNAThe MYH-9SiRNA(dry powder state) were synthesized by Qiagen.Including one positive control (Hs-MAPK1-3);one negative control(Ctrl-AllStars-1);two MYH-9SiRNA(Hs-MYH-9-1and Hs-MYH-9-2).According the MYH-9siRNA to HiPerFect Transfection Reagent ratio provided by Qiagen, we designed five different kinds of MYH-9siRNA to HiPerFect Transfection Reagent ratio.To monitor MAPK1gene silencing by qRT-PCR and Western blot analysis after an appropriate time point(48h and72h) after transfection.At the time point of48h after transfection,the expression quantity of MAPK1gene in five GC cancer cells are not different; At the time point of72h after transfection,the expression quantity of MAPK1gene in the frist group((SiRNA: 75ng/HiPerfect:1.5μl) are the least.To desigened five experimental groups:the blank group(cancer cell group);the negatie control group::Ctrl-AllStars-1; the positive control group:Hs-MAPKl-3; the frist experimental group:Hs-MYH-9-1and the second experimental group: Hs-MYH-9-2.According the optimization of the siRNA to HiPerFect Transfection Reagent ratio.To monitor MYH-9gene silencing by qRT-PCR and Western blot analysis after72h after transfection. the expression quantity of MYH-9gene in the GC cell line of experimental group1are the least.lt is indicated that the MYH-9SiRNA (Hs-MYH-9-1) have the best effects of knock-down. The silence efficacy was highest when the ratio of SiRNA/tansfection reagent was75ng/1.5ul at72h post-transfection with western blot analysis. MYH9mRNA was silenced up to90.0%demonstrated by qRT-PCR.4. Effect of MYH-9down-expression on the biological behaviors of human GCThe expression quantity of MYH-9gene(NMⅡA protein) in the SGC-7901GC cell line are the highest.lt is suitable to do the RNAi experiment.The result of in vitro migration assay showed that SGC-7901/Hs-MYH-9-1cells had significantly reduced invasiveness as compared with SGC-7901/Ctrl-AllStars-1cells.The result of in vitro invasion assay showed that SGC-7901/Hs-MYH-9-1cells had significantly reduced invasiveness as compared with SGC-7901/Ctrl-AllStars-1cells.The result of in vitro cell adhesion assay showed that SGC-7901/Hs-MYH-9-1cells had significantly improved adhesion potency as compared with SGC-7901/Ctrl-AllStars-1cells.CONCLUSION1. MYH-9gene play an important role in invasion and metastasis of GC. 2. Elevated NMII A expression is an independent prognostic factor for GC patients.