The Expression And Clinical Significance Of Musashi-1 And α-SMA In Gastric Carcinogenesis

Backgrounds and aims:Carcinoma tissue was composed of epithelial and stromal cells. Carcinoma stem cells are carcinoma cells found within cancers that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. Carcinoma stem cells may generate tumors through the stem cell self-renewal and differentiation into multiple cell types. Researches indicate that gastric carcinoma occurs where gastric stem cells settle down. Stem cells may play an important role in formative process of gastric carcinoma. Myofibroblast is an atypical fibroblast combining the ultrastructural features of a fibroblast and a smooth muscle cell. According Lauren Classification, gastric cancers are categorized as diffuse and intestinal type gastric cancer. The clinical difference between diffuse and intestinal type gastric cancer may due to the number of stem cells and genetic differences. The relationship among gastric stem cells, tumor stem cells and stromal cells remain unclear. The epithelial stem cell and stromal myofibroblasts may play critical roles in gastric carcinogenesis and cancer progression. Therefore, the aims of this study are as follows:1. To investigate the expression changes of gastric epithelial stem cell marker Musashi-1 and stromal myofibroblast marker a-SMA in normal gastric mucosa, hyperplastic polyps, intestinal metaplasia, intraepithelial neoplasia, gastric cancer. By analyzing the relationship between Musashi-1,α-SMA and PCNA, we discuss the change of epithelial stem cells and stromal myofibroblasts in the gastric carcinogenesis and cell proliferation.2. To evaluate the expression difference of Musashi-1, PCNA andα-SMA in intestinal type gastric cancer and diffuse type gastric cancer. To observe whether there is any difference between intestinal type and diffuse type gastric cancer cells on the expression of MMP-7, which can degrade extracellular matrix, and IGF-2, which can promote the proliferation. Also, we discuss the distinction between intestinal type and diffuse type gastric cancer on the epithelial stem cells and stromal myofibroblasts, and the relationship between this distinction with the invasion and metastasis of gastric cancer.Methods:1. Endoscopic biopsy antral specimens and surgical antral specimens were obtained from September 2008 to August 2010 in Qilu Hospital of Shandong University. There are 182 cases of endoscopic biopsy specimens, including 21 cases of normal gastric mucosa (mild inflammation),32 cases of hyperplastic polyp,31 cases of intestinal metaplasia,57 cases of low grade intraepithelial neoplasia and 41 cases of the high grade intraepithelial neoplasia; The other 50 cases are gastric cancer surgical specimens which were confirmed to be adenocarcinoma by three pathologists. Specimens were fixed by 10% polyphosphate formaldehyde, embedded in paraffin and were sliced in a 4μm serial sections. Microwave EDTA antigen retrieval method and two-step immunohistochemical method were used to detect the Musashi-1 which is epithelial stem cell marker, andα-SMA which is myofibroblast marker. Correlation analysis was made between the two markers and PCNA expression.2. Clinical and pathological data of the 50 cases of gastric surgical cancer specimens were collected, including age, gender, Lauren classification, tumor size, TNM staging and so on. Musashi-1,α-SMA, PCNA, MMP -7, IGF-2 expression of gastric cancer tissue were detected by immunohistochemistry to compared those in intestinal type with those in diffuse type gastric carcinoma and make correlation analysis with clinical pathological data. Results:1. PCNA and Musashi-1 positive cells were restricted to the proliferative zone of the neck of gastric glands in normal gastric mucosa. PCNA expression was higher in hyperplastic polyps than in the normal gastric mucosa (U=164.00, P<0.01), but Musashi-1 positive cells are rarely found. Musashi-1 in intestinal metaplasia mucosa expressed higher than in normal mucosa (U=227.50,P<0.05), and the distribution of Musashi-1 positive cell was more diffuse. Compared with hyperplastic polyps, PCNA expression in intestinal metaplasia mucosa also increased (U=108.00,P<0.01). The expression of PCNA and Musashi-1 showed no significant difference among intestinal metaplasia, low and high grade intraepithelial neoplasia. Musashi-1 in gastric cancer was higher than that in high grade intraepithelial neoplasia (U=789.00,P<0.05), and PCNA in gastric cancer was also higher than that in high grade intraepithelial neoplasia (U=650.00,P<0.01). Musashi-1 and PCNA expression were positively correlated (rs=0.34, P<0.01)2.α-SMA positive cells scattered irregularly among the glands in normal mucosa and hyperplastic polyps, and slightly increased in hyperplastic polyps (U=276.00,P>0.05). Compared with hyperplastic polyps,α-SMA positive cells were significantly higher in the mucosa of intestinal metaplasia (U=256.00,P<0.01), and behaved in parcel gland-like distribution around the intestinal metaplasia glands; In intraepithelial neoplasia the expression of a-SMA also showed pericryptal distribution, but was obviously not as significant and regular as intestinal metaplasia mucosa. Compared with intestinal metaplasia mucosa, the number of a-SMA positive cells decreased in low and high grade intraepithelial neoplasia (χ2=6.42,P<0.05); In gastric cancer, a-SMA-positive cells were not distributed in pericryptal pattern, and distributed irregularly among cancer cells nests in small pieces, mass or cord-like pattern; a-SMA expression was significantly higher in gastric cancer than that in intraepithelial neoplasia (U=600.00,P<0.01), but slightly higher than that in intestinal metaplasia mucosa (U=624.50,P>0.05).α-SMA and PCNA were positively correlated (rs=0.39,P<0.01) 3. Musashi-1 was expressed higher in the diffuse type gastric cancer than in the intestinal type gastric cancer (χ2=16.96,P<0.01). Musashi-1 expression was higher in gastric cancer with lymph node metastasis than that in gastric cancer without lymph node metastasis (χ2=13.45, P<0.01). Musashi-1 expression was higher in the group of stageⅡ-Ⅳthan that in stageⅠgroup (χ2=11.88, P<0.01). There was no significant difference between intestinal type gastric cancer and diffuse type gastric cancer on a-SMA expression (χ2=2.98,P>0.05).α-SMA expression was higher in the depth of tumor invasion T2-4 group than in the≤T group (χ2=11.38,P<0.05), and higher in gastric cancer with lymph node metastasis than without lymph node metastasis (χ2=9.67,P<0.05),and higher in gastric cancer> 5cm group than in the≤5cm group (χ2=9.79,P<0.05).4. MMP-7, PCNA expression was higher in diffuse type gastric cancer than those in the intestinal type gastric cancer (χ2=6.52,P<0.05;χ2=19.54,P<0.01). In the diffuse type gastric cancer, most of IGF-2 was expressed in the cancer cell nucleus (21/23) while most of IGF-2 was expressed in the cytoplasm of intestinal type gastric cancer cells(24/27). There was significant differences(χ2=32.00, P<0.01). PCNA expression was correlative with lymph node metastasis and tumor size; MMP-7 expression was correlative with gastric cancer depth of invasion, lymph node metastasis, distant metastasis and staging; IGF-2 nuclear expression was correlative with depth of tumor invasion, lymph node metastasis and staging.Conclusions:1. In the hyperplastic polyps, during the sustained injury and repair process of the gastric mucosa, the epithelial stem cells proliferate limitedly and differentiate into a large number of epithelial cells. The increase of PCNA expression prompts DNA replication activity, but the Musashi-1 expression decreased rapidly with the stem cells differentiation, indicating that the self-replication of epithelial stem cell in benign lesions is controlled in a limited range.2. Musashi-1 and PCNA expression increased in the intestinal metaplasia mucosa, with no significant differences compared with low and high grade intraepithelial neoplasia, suggesting that there is a similar epithelial stem cells proliferation activity in the gastric precancerous lesions. Musashi-1 positive cells in the intestinal metaplasia mucosa may be the early events of initiating the way "intestinal metaplasia-intraepithelial neoplasia-adenocarcinoma". Stem cells amplification caused by the disorder during the process of epithelial stem cells replicating may play a key role in the early events of gastric carcinogenesis.3. The expression of Musashi-1 and PCNA in gastric cancer is higher than that in gastric precancerous lesions, suggesting that gastric cancer stem cells have greater ability of proliferation than stem cells in gastric precancerous lesions.4. During the process of gastric mucosa malignant transformation, not only quantity of myofibroblasts cells, but also their distribution pattern and position have changed. Myofibroblasts cells increase gradually from normal mucosa to intestinal metaplasia mucosa, and the pericryptal distribution of myofibroblasts was observed in intestinal metaplasia. But from the mucosa of intestinal metaplasia to high grade gastric intraepithelial neoplasia, myofibroblasts reduce gradually and the pericryptal distribution pattern is weakened. Pericryptal myofibroblasts disappear in gastric cancer, and are replaced by tumor associated myofibroblasts with disorganized distribution, suggesting that myofibroblasts have functional heterogeneity during the different development stage of gastric cancer. Pericryptal myofibroblasts in the precancerous lesions may have an early defense function, while tumor associated myofibroblasts can promote the invasion and metastasis of gastric cancer. There is no significant difference between intestinal type gastric cancer and diffuse type gastric cancer on stromal myofibroblasts. The process of malignant transformation of gastric cancer is the joint action of epithelial cells and stromal myofibroblasts.5. The overexpression of Musashi-1 in gastric cancer may promote self-replicating of stem cells. Musashi-1 and PCNA expression increases in the diffuse type gastric cancer than intestinal type gastric cancer. Diffuse type gastric cancer stem cells may have higher proliferative capacity than intestinal type gastric cancer and may be relevant with the poor differentiation of diffuse type gastric cancer. 6. The overexpressed expression of MMP-7 in gastric cancer cells and IGF-2 nucleus expression are related with gastric cancer invasion and metastasis. Diffuse type gastric cancer has higher expression of Musashi-1 and MMP-7 than intestinal type gastric cancer, and IGF-2 expression patterns are significant different between two gastric cancer subtypes. Several parts of gene of diffuse and intestinal type gastric cancer stem cells may have many differences, such as MMP-7, IGF-2 gene, or the activity of signal transduction which controls cancer stem cells is different. These mechanisms work together, through the complex interaction mechanism between the whole cancer cells and body to promote invasion and metastasis of diffuse type gastric cancer.