TM4SF1, A Potential Predictor For Poor Prognosis, Overexpressed In Human Glioma

Glioma is one of the most common primary malignant brain tumors in humans with poor prognsis, accounting for 40 to 60 percent of primary brain tumors. Treatment for glioma is often a combined approach including microscopic surgery, radiation therapy and chemotherapy. Although there have been numerous advances in neuroimaging, neurosurgery, radiotherapy and chemotherapy, the recurrence and mortality rates of glioma remain high, with almost a 100% postoperative recurrence rate, and a median overall survival of less than 1 year. It was reported that the mean survival time of patients with low grade gliomas was 3-5 years, while the mean survival time of patients with low high gliomas was 1-2 years. Up to now, World Health Organization(WHO) grades for glioma patients are the gold standard for determining prognosis, but these are of limited use in assessing the survival time of individual patients. It was found that some molecular factors, e.g. phosphatase and tensin homolog on chromosome 10(PTEN), determine individual glioma patients’ prognosis, but only a few studies showed that a single molecule might be the prognosis indicator. It is therefore extremely important that further molecular markers of prognosis in human glioma are identified.TM4SF1, a tetraspanin-like four-transmembrane domain surface protein, is highly expressed by cultured endothelial cells(EC) and EC lining angiogenic tumor blood vessels. TM4SF1 belonged to a distinct family(L6 family) of the four-transmembrane-domain proteins, which also included TM4SF4, TM4SF5, TM4SF18, TM4SF19, TM4SF20. The TM4SF1 gene has been reported to be over-expressed in breast, ovarian, lung, pancreatic, prostate and colon carcinomas. In vitro and in vivo studies revealed that TM4SF1 is involved in similar cellular processes to tetraspanins, such as migration and invasion.To our knowledge, the association between TM4SF1 expression and survival in patients with glioma has not been described in the literature to date. In the present study we therefore measured TM4SF1 gene and protein expression in human glioma cell lines using q RT-PCR and western blot. We also measured TM4SF1 gene and protein expression in human glioma tissues using q RT-PCR, western blot and immunohistochemistry and sought to determine their association with patient outcome using Kaplan-Meier analysis and multivariate survival analysis using Cox’s regression. Part one The expression of TM4SF1 in human glioma tissues and therelationship between the expression of TM4SF1 and the WHO gradeObjective: To explore the expression of TM4SF1 in glioma tissues of different grades and find the relationship between the expression of TM4SF1 and the pathological grade.Menthods:1 Tumor material was collected from 72 individuals diagnosed with glioma and 8 with brain trauma hospitalized in the Department of Neurosurgery of the Second Hospital of Hebei Medical University in recent years. All glioma samples were identified by pathological investigation. Following surgical resection, both glioma and control brain tissues were immediately divided into two parts. One part was fixed in formalin for the immunohistochemistry assay, and the other part was stored in 2ml sterile vials and immediately frozen in liquid nitrogen.2 Immunohistochemistry was performed to detect the expression of TM4SF1 protein in 8 control brain tissue samples and 72 cases of glioma tissue and the expression difference was compared. The relationship between the expression of TM4SF1 and the pathological grade was also analyzed.3 The q RT-PCR was performed to examin TM4SF1 m RNA expression levels in 8 control brain tissue samples and 72 cases of glioma tissue and the expression difference was compared.4 Western blot was performed to measure TM4SF1 protein expression in 8 control brain tissue samples and 72 cases of glioma tissue and the expression difference was compared.Results:1 Immunohistochemical results: Immunohistochemical staining showed TM4SF1 expression was increased in glioma. Of the 72 gliomas, 38 gliomas were in high TM4SF1 expression(52.78%, 38/72). TM4SF1 expression was negative in 8 cases of control brain tissues. Significant differences in TM4SF1 expression were observed between control brain and glioma tissues(P < 0.01), as well as between high-grade(III-IV) and low-grade(I-II) tumor tissues(P < 0.01). Further analysis revealed that the expression levels of TM4SF1 were associated with the WHO grades(rs = 0.950, P < 0.05). Group difference in AOD of TM4SF1 was shown among control brain, low-grade and high-grade tumor tissues(P < 0.01). AOD of TM4SF1 was significantly higher in high-grade tumor tissues than in low-grade tumor tissues(P < 0.01). Group difference in cell densityof tumor tissues was not found between low-grade and high-grade tumor tissues(P>0.05).2 The q RT-PCR results: The q RT-PCR analysis of TM4SF1 m RNA showed that the expression of TM4SF1 was significantly higher in high-grade(III-IV) than that in low-grade(I-II) tumor tissues(P < 0.01). The mean of TM4SF1 expression in high-grade tumor tissues(2.117 ± 0.618) was 238.4% more than that in low-grade tumor tissues(0.626 ± 0.452).3 Western blot results: To investigate whether TM4SF1 was also elevated at the protein level, western blot analysis was performed. TM4SF1 protein expression in glioma tissues was significantly higher than that in control brain tissues. Moreover, TM4SF1 protein expression levels in the low-grade(I-II) gliomaswere lower than those in the high-grade gliomas(III-IV; P < 0.05).Conclusion: Immunohistochemistry, q RT-PCR and Western blot analysis concluded that TM4SF1 protein and m RNA was highly expressed in human glioma tissue, and the expression levels increased with glioma grade. TM4SF1 protein expression level in high grade gliomas(III-IV) was higher than that in low grade gliomas(I-II), with significant statistical significance. Part two The expression of TM4SF1 in human glioma cell lines U87 and U251Objective: To detect the expression of TM4SF1 in human glioma cell lines U87 and U251Menthods:1 The q RT-PCR was conducted to examin TM4SF1 m RNA expression in U87 and U251 cells.2 Western blot was performed to examin TM4SF1 protein expression in U87 and U251 cells.Results:1 The q RT-PCR results: q RT-PCR analysis concluded that TM4SF1 m RNA was expressed in human glioma cell lines, but there were no significant differences between U87 and U251(P>0.05).2 Western blot results: Western blot analysis concluded that TM4SF1 protein was expressed in human glioma cell lines, but there were no significant differences between U87 and U251(P>0.05).Conclusion: The q RT-PCR and Western blot analysis concluded that TM4SF1 m RNA and protein was expressed in human glioma cell lines, but there were no significant differences between U87 and U251. Part three Multivariate analysis of prognostic factors including TM4SF1 in 72 patients with gliomaObjective: To study the prognostic factors of the patients with glioma as the theoretical reference to the clinical treatment, and to make an accuracy judgment of prognosis of the patients with glioma, therefore we can improve the survival status of the patients with glioma.Methods: The medical records of the 72 patients were reviewed for age, gender, the pre-operative neurological statusas defined by the Karnofsky Performance Status(KPS), having epilepsy or not before operation, size of tumor, extent of resection, pathological grade, and the expression of TM4SF1 as possible prognostic factors. The survival data were first analyzed using the Kaplan-Meier method. Differences in survival between each group were tested for statistical significance by the log-rank test. After that, multivariate test method(Multiple Cox Regression) was performed to identify independent prognostic factors with P<0.05 in the results of univariate analysis.Results:1 Following a univariate regression analysis, lower age, good preoperative neurological status(higher KPS), macroscopically total resection, lower pathological grade, and lower expression of TM4SF1 were asssciated with better overall survival.2 Multivariate cox regression analysis found that pathological grade and the expression of TM4SF1 were independent prognositic factors influencing surival. Conclusion: Glioma patients with higher pathological grade and higher expression of TM4SF1 often haver worse prognosis. Pathological grade and the expression of TM4SF1 were better independent prognostic factors of glioma patients.