| BackgroundThe incidence of laryngeal squamous cell carcinoma (LSCC) accounts for otorhinolaryngological carcinoma is11-22%,1-5%for general systemic cancer. Laryngeal squamous cell carcinoma is not sensitive to radiotherapy and chemotherapy. Until now the major therapeutic method is still the operation.In the past5decades.the therapy has been improved constantly, the disease-free survival rate and the quanlity of life have not been increased.Patients always need undergo the tracheotomy or permanent tracheostomy fistula,hoarseness even aphonia,which will lead bad living quanlity. Therefore, finding a meaningful method to diagnose the laryngeal carcinoma is great significance. Epidermal growth factor receptor, P53, mitogen-activated protein kinases(MAPKs) have been reported that these biomolecules may be related with the growth,therapy and prognostic of tumor. Recently, exploring the molecular biological mechanism of the malignancy is a new trend, aiming to provide new evidents for cancerous diagnosis, treatments and prognosis.Transient receptor potential (TRP) cation channel was firstly reported in Drosophila melanogaster photoreceptors for maintained membrane depoisoforms.TRP channels contribute to change intracellular Ca2+concentrations, either by as Ca2+entry pathways in the plasma membrane or changing the membrane polarization, modulating the driving force for Ca2+entry mediated by alternative pathways.TRPM, a subfamily of the transient receptor potential superfamily,named of the first member, contains eight members:TRPM1-TRPM8. TRPM7is ubiquitously expressed in various tissues,participates in many physiological activities like cell proliferation, differentiation, adhesion and neurotransmitter release[1].TRPM7also takes part in several systerm pathological functions, for instance cardiovascular, digestive, urinary systerm diseases. Nelson et al silencing the expression of TRPM7in the pancreatic cancer which induced eplicative senescence in stead of apoptosis by up regulating expression of the senescence-associated genes,meanwhile with gemcitabine enhanced cytotoxicity in pancreatic cancer cells[2].Guilbert et al have proved that the expression of TRPM7in oestrogen receptor-negative metastatic breast cancer cells is higher than the normal tissue, which may provide a new theraputic target[3].The relationship between the TRPM7channel and head and neck malignancies has been reported in recent years. Chen et al found that TRPM7channel may play an important role in the metastasis of nasopharyngeal carcinoma,down-regulated the expression inhibite the proliferation of the cancer cell[4].Hanano et al found TRPM7channel could be found in retinoblastoma cells, and induced Ispont and non voltage-gated current spontaneously;silencing TRPM7channel could reduce the Ca2+influx and slow down the cell growth[5].Jiang et al demonstrated SCC25and FaDu cell,two kinds of head and neck carcinoma cell lines,contained TRPM7channel, suppressed the expression of TRPM7by siRNA inhibited the cell growth and proliferation[6].Nowadays,the experts focus on the function of TRPM7in maligant tumor gradually,but there is rarely report abou the expression level on laryngeal squamous cell carcinoma.ObjectiveIn this study,we examine the expression of the TRPM7channel in laryngeal squamous cell carcinoma and the normal laryngael tissue.investigate the expressive difference between cancerous tissue and adjacent mucosa tissue of larynx and its relationships with cell differentiation and lymph node metastasis in LSCC.TRPM7channel may exert its unique influence on early diagnosis, targeted therapy, and prognostic evaluation of human laryngeal squamous cell carcinoma. Method1.Clinical samples collecting and groupingThirty-one patients with LSCC were recruited in this study from October2013to December2014.We collect the cancerous tissue and adjacent mucosa tissue.Eighteen of them were made into formalin fixed and paraffin embedded samples,and the others were collected in non-RNase tube and stored in-80℃ultra low temperature refrigerator without any process after the surgery. All the samples should match the requirements as follows:None of patients has distant metastasis;larynx cancer is the primary malignancy;they didn’t undergo the radiothrepy or chemotherapy;the cancerous tissues were squamous cell carcinoma by pathologist. All the samples were confirmed there is no cancer cell invasing the mormal tissue by H-E staining method.2. Immunohistochemical staining (IHC)Paraffin sections of tissues prepared for detection were stained by S-P method and the results of IHC were observed by light microscope and judged by the modified formula used by Bresalier:sections were observed in10X objective lens and the staining intensities in5fields of random vision were counted, then scores were calculated according to intensity stained on tissue(four grades):no colour on cell,negative=0score; pale yellow stained on cell, weakly positive=1score; palm yellow stained on cell, moderate positive=2scores; dark brown stained on cell, strong positive=3scores, finally the average staining intensities of every section tissue were counted according to the following formula:the average staining intensities=Σ{(0xF0)+(1xF1)+(2xF2)+(3xF3)}, Fi=%10X fields of vision(i=0,1,2,3).3.Quantitative real-time pcr(Q-RT-PCR)Total RNA was extracted according to the protocol,then reversely transcripted into cDNA and Q-PCR were amplified according to the sequences of primers.Applying2-△△Ct method to evaluate the relative expression level between the cancer tissue and normal tissue by Microsoft Excle. The formula of this experiment is△△Ct=(Ct target-Ctinternal) tumor-(Ct target-Ctnternal)normal.4. Statistical analysis All data was statistically analyzed by SPSS13.0software.Firstly,examing whether the variable is normal distribution and homogeneity of variance. The data obayed normal distribution and homogeneity of variance. The results were represented Mean±standard deviation(x±s). Paired t-test was applied to identify the difference between tumor and normal tissue;the relationship between the TRPM7and pathological differentiation,lymph node metastasis of the laryngeal carcinoma were tested by using independent samples T test.The data did not obey normal distribution, the results were represented by M(P25～P75),Wilcoxon rank test was used. The difference was significant if the P value was less than0.05.Result1. All patients were male, the age were48-75years old.2case was low differentiation,9cases were moderate differentiation and7cases were high differentiation; five cases of them were identified lymph node metastasis.The fresh tissue2case low differentiation,10cases moderate differentiation and1cases high differentiation.2. Immunohistochemical staining(1HC staining) had proved TRPM7protein can be expressed in both laryngeal carcinoma and laryngeal normal mucosa tissue, and the antigen binding site is cytoplasm. Bresalier semi-quantitative method was used to get the section staining score:the value obay normal distribution, tumor tissue is7.830±2.572and normal tissue is4.560±2.148. TRPM7expression of cancer cells was significantly higher than the normal one by using paired sample t test (t=3.979, p=0.001).The expression level of low differiation,moderate differiationand high differiation were10.000±2.828,8.444±2.128and6.571±2.820,respectively. There was no significant experesion neither differiation (F=1.945,0.177) nor lymph node metastasis (t=1.591,p=0.131).3. The sample were relative quantitative analyzed by quantitative real-time PCR.△Ct did not obay nomal distribution.The mean rank of△Cttumor7.140(6.515～10.190)and△Ctnormal7.430(6.470～8.240).According2-△△Ct method, we found2patients were2-fold over expressed than the normal, using Wilcoxon rank sum test to analysis the difference between cancer cell and mucosa squamous cell because of the△Ct value did not comply with normal distribution,there was no significant difference between cancer tissue and the normal tissue (z=-0.664, p=0.507)ConclusionWe examined the expression of TRPM7channel in human laryngeal cancinoma tissue and adjacent mucosa tissue firstly.Aiming to find out the significance and relationship between TRPM7channel and the tissue.We found both laryngeal squamous cell carcinoma and adjacent mucosa tissue could express TRPM7channel protein by immunohistochemical staining,and the expression level has significant difference,but we didn’t prove that there were any relationship with the pathological differentiation and the lymph node metastasis. We use the frozen tissue to test the TRPM7-RNA by Quantitative real-time PCR. Meanwhile there was no significant difference. The process of RNA synthetize protein may be affected by some other molecular mechanism and RNA degradation may affect these results. In conclusion,we couldn’t deny the difference between carcinoma and adjacent mucosa tissue completely. |
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