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Objective To analyze the changes of Survivin,MSH6 and MSH2 expression in colorectal cancer and explore their relationship with clinical and pathological parameters.Methods 197 cases of colorectal adenocarcinoma and 20 cases of inflammatory intestinal mucosa were detected by immunohistochemistry with Survivin,MSH6 and MSH2,and the correlation between Survivin,MSH6 and MSH2 expression was analyzed.Results In the control group of 20 cases with inflammatory non-tumor intestinal mucosa,the positive expression rate of MSH2,MSH6,Survivin were 95％,95％,10％,respectively.While the positive expression rate of MSH2,MSH6,Survivin were 88.3％,74.1％ and 84.3％ in 197 cases of colorectal cancer.Survivin positive expression rate in colorectal cancer group was significantly higher than that in inflammatory control group (P ＜ 0.05).Survivin expression was correlated with invasion depth and lymph node metastasis in colorectal cancer tissue (P ＜ 0.05),but not with gender,age,tumor size,gross type or degree of differentiation (P ＞ 0.05).MSH2 expression was correlated with tumor size and lymph node metastasis (P ＜ 0.05),but not with gender,age,gross type,depth of infiltration and degree of differentiation (P ＞ 0.05).MSH6 expression was related to gender and lymph node metastasis (P ＜ 0.05),but not to age,tumor size,gross type,infiltration depth and differentiation (P ＞ 0.05).Colorectal cancer tissues showed positive correlation between MSH6 and MSH2 (r =0.326,P ＜ 0.01),positive correlation between MSH2 and Survivin positive expression (r =0.277,P ＜ 0.01),and positive correlation between MSH6 and Survivin positive expression (r =0.435,P ＜ 0.01).Conclusions The positive expression rate of Survivin in colorectal cancer is high.MSH2,MSH6 and Survivin in colorectal cancer play an important role in the development and progression of colorectal cancer,and can provide evidence for the detection of these three factors,including metastasis risk,prognosis assessment and clinical treatment.In particular,Survivin gene may provide evidence for gene therapy.
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Objective@#To investigate the expression of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2) in colorectal cancers and to explore the relationship between MMR expression and clinicopathologic features.@*Methods@#Six hundred and fifty-eight colon cancers were collected from January 2016 to January 2017 at Shengjing Hospital of China Medical University. Of the 658 patients there were 409 male and 249 female. The patients were 20 to 92 years old, with average age of (63±5) years old. Expression of MLH1, MSH2, MSH6 and PMS2 protein was detected by immunohistochemical method. Immunohistochemistry for BRAF V600E was performed in colorectal cancers with loss of MLH1 protein expression. Relationship between MMR protein expression and clinicopathologic features was analyzed statistically. @*Results@#Forty-four cases of 658 cases (6.7%) lost at least one MMR protein expression. Expression deficiency rates of MLH1, MSH2, MSH6 and PMS2 were 4.1%(27/658), 2.3%(15/658), 2.4% (16/658), and 4.3% (28/658), respectively. MMR expression deficiency mainly consisted of combined loss of MLH1/PMS2 (61.4%, 27/44) and MSH2/MSH6 (34.1%, 15/44). Two unique mutations were identified including one MSH6-deficient(2.3%, 1/44) and PMS2-deficient(2.3%, 1/44). Seven cases (25.9%, 7/27) had positive BRAF V600E expression, suggesting BRAF gene mutation related sporadic colorectal cancers. No correlation was observed between the expression of MMR and depth of tumor infiltration, lymph node metastasis, vascular tumor emboli, clinical stage or hematogenous metastasis (P>0.05). MMR status was associated with tumor cell differentiation, histological type and tumor location (P<0.01). Tumors with combined MLH1 and PMS2 loss were associated with mucinous differentiation (P=0.049, P=0.013) and located in the right hemi-colon (P=0.006, P=0.002). Combined MSH2 and PMS2 loss was related to gender, while loss of MSH2 protein was observed more frequently in female patients (P=0.048) and loss of PMS2 protein was seen more frequently in male patients (P=0.031). @*Conclusions@#Patients with MMR protein deficiency have a younger onset age and poorly differentiated tumors. Most tumors are located in the right hemi-colon and have mucinous differentiation.
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Objective To explore the expression and clinical significance of mismatch repair (MMR)protein and MLH1 promoter methylation testing in endometrial cancer(EC). Methods A total of 420 cases with EC diagnosed by the surgical pathology examination from the Department of Pathology of PLA General Hospital, MLH1,MSH2,MSH6 and PMS2 protein in EC were detected by immunohistochemistry and methylation-specific multiplex ligation-dependent probe amplification(MS-MLPA) testing. Results (1)Of the 420 tumor cases, the total expression loss rate of MMR protein was 34.5%(145/420), the expression loss rates of MLH1,MSH2,MSH6 and PMS2 protein were respectively 17.1%(72/420), 8.1% (34/420), 7.4%(31/420), 26.2%(110/420)and loss rates of MLH1 and PMS2,MSH2 and MSH6 were 16.7%(70/420), 6.2%(26/420). When there was a loss of MMR protein expression, any one or more protein expression deletions in MLH1, PMS2, MSH2 and MSH6, it could be Lynch syndrome related endometrial carcinoma(LS-EC). The expression loss rate of MMR protein in the poorly differentiated endometrioid adenocarcinoma was higher than that in the well differentiated endometrioid adenocarcinoma(P<0.05).(2) The expression loss rate of MMR and PMS2 protein had statistically significant between the endometrioid adenocarcinoma and non-endometrioid adenocarcinoma(P<0.01). The expression loss rate of MSH2 protein had statistically significant in the stage Ⅲ(P<0.01). Moreover, there were also significant differences in depth of myometrial invasion and lymph node metastasis between the expression loss rate of MMR protein (P<0.05).(3)The expression loss rate of MLH1 protein was 72 cases and 57 cases had MLH1 promoter methylation testing(excluding those who were not qualified for DNA testing). The positive rate was 47.4% (27/57). Therefore, these patients were sporadic endometrial cancer, not non-LS-EC. Conclusions MMR protein may be play an important role in the development of endometrial cancer and be indicated poor prognosis. Immunohistochemical staining and MLH1 promoter methylation detection may be play an important role in the screening of the LS-EC.
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Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells.Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination.The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule.The expression of hMSH2 molecule in NCI-H520 was detected by Western blot.The expression of hMSH2 on the cell membrane was measured by flow cytometry.Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro.Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size.Exogenous hMSH2-EGFP fusion protein was expressed in NCIH520 cells.The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P＜0.001).Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P＜0.05).Conclusions Lung cancer cell model that overexpresses hMSH2 molecule is successfully constructed,hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.
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Objective To analyze the expression of excision repair cross-complementation group 1 (ERCC1),MutS protein homolog 2 (MSH2) and poly ADP-ribose polymerase 1 (PARP1) in non-small cell lung cancer (NSCLC) and their prognostic value for patients receiving platinum-based postoperative adjuvant chemotherapy.Methods Immunohistochemistry was applied to detect the expression of ERCC1,MSH2 and PARP1 in 111 cases of NSCLC paraffin embedded surgical specimens.Through OG-Rank survival analysis,the prognostic value of the ERCC1,MSH2,PARP1 and the related clinic pathological factors were evaluated.Cox regression analysis was used to determine whether ERCC1,MSH2 and PARP1 were independent prognostic factors or not.Results Among the 111 NSCLC patients,the positive expression rates of ERCC1,MSH2 and RARP1 were 33.3 ％(37/111),36.9 ％(41/111) and 55.9 ％(62/111),respectively.Besides,a total of 72 patients who received platinum-based chemotherapy had complete follow-up data.ERCC1 (P＜ 0.001) and PARP1 (P=0.033) were found to be correlated with the survival time while there was no correlation for MSH2 (P =0.298).Patients with both ERCC1 and PARP1 negative had significant longer survival time than those with ERCC1 (P=0.042) or PARP1 (P=0.027) positive alone.Similarly,the survival time of patients with both ERCC1 and PARP1 positive was shorter than those with ERCC1 (P=0.048) or PARP1 (P=0.010) positive alone.Conclusions The NSCLC patients with ERCC1 and (or) PARP1 negative may benefit from platinumbased postoperative adjuvant chemotherapy.The detection of ERCC1 and PARP1 can be used as an important method to assess the prognosis of patients with NSCLC.
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Objective To investigate the reversal effect of hMSH2 small interference RNA(siRNA) on chemo-resistance of ovarian carcinoma cell line OC3/TAX300,explore the clinical significance.Methods The specific hMSH2 siRNA (experimental group) and non-specific hMSH2 siRNA (negative control group) was designed,synthesized and transfected into ovarian carcinoma cell line OC3/TAX300.The expression mRNA and protein levels of hMSH2 were detected by real-time reverse transcription (RT)-PCR and western blot.The cell proliferation was detected by methyl thiazolyl tetrazolium(MTT) method after 12,24,48,72 hours of 2 μg/ml taxol,the apoptosis rate after 24,48 hours of 2 μg/ml taxol was analyzed by flow cytometry.Morphological changes and ultramicrostructure of cells after 48 hours of 2 μg/ml taxol were observed with transmission electron microscope.Results (1) The mRNA levels of hMSH2 were 0.004 ± 0.000,0.053 ±0.006 and 0.057 ± 0.012 in experimental group,negative control group and non-infected group,respectively.The protein levels of hMSH2 were 0.19 ± 0.04,1.00 ± 0.07 and 0.95 ± 0.03 in experimental group,negative control group and non-infected group,respectively.(2) Compared with the noninfected group and the negative control group.The cell proliferation was effectively inhibited after 12,24,48,72 hours of 2 μg/ml taxol(P ＜0.05).The cell cycle was arrested at G2/M phase,the apoptotic rate was significantly increased after 24,48 hours of 2 μg/ml taxol (P ＜ 0.05).The experimental group after 48 hours of 2 μg/ml taxol was found to have more visible cell shrinkage,more serious chromatin margination,nucleus condensation,fragmentation and apoptotic body formation,nucleolus disappeared,markedly swollen mitochondria,mitochondrial cristae disappeared and other signs of apoptosis.While the nucleus was located in the cells of the central and nucleolus is clear,only mild chromatin pyknosis and marginalized,mild swelling of mitochondria in the control group and blank group.Conclusion siRNA targeting hMSH2 may reverse the chemo-resistance of ovarian carcinoma cell line OC3/TAX300 and may become a treatment or a new direction in the adjuvant therapy of ovarian cancer.
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Objective To study the role of human MutS homolog 2 (hMSH2), a newly identified protein ligand that was recognized by Vγ9δ2 T cells , in innate anti-gastric carcinoma immunity .Methods Flow cytometry and laser confocal microscopy were used to identify hMSH 2 that ectopically expressed on gas-tric carcinoma cell line 803.An anti-hMSH2 antibody was used to block hMSH2 to evaluate its effects on the cytotoxicity of Vγ9δ2 T cells and their cytokines secretion .Subcellular distribution of hMSH 2 in gastric car-cinoma tissues was examined by tissue microarray immunohistochemistry analysis .Results Ectopic mem-brane expression of hMSH 2 was observed on 803 cells at a relatively high level .Vγ9δ2 T cells blocked with specific anti-hMSH2 antibody showed a decreased cytotoxicity and a reduced IFN-γbut an increased TNF-αsecretion.Ectopic expression of hMSH2 was found in various types of gastric carcinoma tissues at different stages.Enhanced expression of hMSH2 was detected in specimens collected from patients with chronic super-ficial gastritis.Conclusion Ectopically expressed hMSH2 served as a stress-induced endogenous ligand which could promote the cytotoxicity of Vγ9δ2 T cells against gastric carcinoma cells and enhance their IFN-γsecretion.hMSH2 played an essential role in innate anti-gastric carcinoma immunity .
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OBJECTIVE: This study aimed to investigate the expression of the MSH2 DNA repair protein in head and neck squamous cell carcinoma (HNSCC) in order to analyze its association with clinicopathologic factors and overall survival of patients. MATERIAL AND METHODS: Clinical data and primary lesions of HNSSC were collected from 55 patients who underwent surgical resection with postoperative radiotherapy in Montes Claros, state of Minas Gerais, Brazil, between 2000 and 2008. Immunohistochemical reactions were performed to analyze MSH2 protein expression. RESULTS: Bivariate analysis showed no significant correlation or association between MSH2 expression and clinicopathologic parameters by Mann-Whitney and Kruskal-Wallis tests. Patients with locoregional metastatic disease (OR=4.949, p<0.001) and lower MSH2 immunohistochemical expressions (OR=2.943, p=0.032) presented poorer survival for HNSCC by Cox regression models. CONCLUSIONS: Our data demonstrated that lower MSH2 expression might contribute to a higher clinic aggressiveness of HNSCC by promoting an unfavorable outcome. .
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Humans , Male , Female , Adult , Middle Aged , Aged , Carcinoma, Squamous Cell/metabolism , DNA Repair , Head and Neck Neoplasms/metabolism , /metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Kaplan-Meier Estimate , Neoplasm Staging , Prognosis , Retrospective Studies , Statistics, Nonparametric , Time FactorsABSTRACT
Objective To explore the role of hMSH2, a novel endogenous tumor-associated pro-tein ligand recognized by Vγ9δ2 T cells, in innate anti-cervix cancer immunity .Methods hMSH2 that ex-pressed on the surface of cervical cancer cell line HeLa cells was blocked by specific antibody .Then the differences in their effects on Vγ9δ2 T cells before and after antibody blockage were evaluated by cytotoxicity of Vγ9δ2 T cells and cytokines secretion .The serum levels of hMSH2 in patients with cervical cancer were detected by ELISA .Distribution of hMSH2 in cervical cancer tissues was analyzed by TMA immunohisto-chemistry .Results Decreased cytotoxicity and IFN-γsecretion were observed in Vγ9δ2 T cells against He-La cells blocked with specific anti-hMSH2 antibody .Serum concentration of hMSH 2 in patients with cervical cancer was slightly higher than that of healthy control .Altered distribution pattern of hMSH 2 was observed in cervical cancer tissues .Conclusion hMSH2 is involved in Vγ9δ2 T cells-mediated innate anti-cervical cancer immunity by enhancing their cytotoxicity and IFN-γsecretion.
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Objective To explore the role of methylation of Human MutS homolog 2 (MSH2) and Human runt-related transcription factor 3 genes (RUNX3) in DNA prepared from nasopharyngeal swabs in early diagnosis and prognosis prediction of nasopharyngeal carcinoma. Methods The methylation-specific PCR was used to detect hypermethylation of MSH2 and RUNX3 genes in DNA prepared from nasopharyngeal swabs from 54 nasopharyngeal carcinoma patients, 18 chronic nasopharyngitis patients and 20 healthy volunteers. The specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma by detecting MSH2 and/or RUNX3 gene methylation were evaluated. The relationship between methylation of MSH2 or RUNX3 gene and the biological behavior of nasopharyngeal carcinoma was analyzed. Results Hypermethylation of MSH2 and RUNX3 gene was respectively detected in 38 out of 54 (70.37%) and in 28 out of 54 (51.85%) nasopharyngeal swabs obtained from nasopharyngeal carcinoma patients, while there was no methylation in nasopharyngeal swabs from 18 chronic nasopharyngitis patients and 20 healthy volunteers. The differences were statistically significant (P0.05). Conclusions Parallel combined testing of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs determined by methylation-specific PCR could increase the specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma, and is of important clinical significance. However it may not serve as an index in evaluating the clinical prognosis of nasopharyngeal carcinoma at present.
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Objective To explore the role of methylation of Human MutS homolog 2 (MSH2) and Human runt-related transcription factor 3 genes (RUNX3) in DNA prepared from nasopharyngeal swabs in early diagnosis and prognosis prediction of nasopharyngeal carcinoma. Methods The methylation-specific PCR was used to detect hypermethylation of MSH2 and RUNX3 genes in DNA prepared from nasopharyngeal swabs from 54 nasopharyngeal carcinoma patients, 18 chronic nasopharyngitis patients and 20 healthy volunteers. The specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma by detecting MSH2 and/or RUNX3 gene methylation were evaluated. The relationship between methylation of MSH2 or RUNX3 gene and the biological behavior of nasopharyngeal carcinoma was analyzed. Results Hypermethylation of MSH2 and RUNX3 gene was respectively detected in 38 out of 54 (70.37%) and in 28 out of 54 (51.85%) nasopharyngeal swabs obtained from nasopharyngeal carcinoma patients, while there was no methylation in nasopharyngeal swabs from 18 chronic nasopharyngitis patients and 20 healthy volunteers. The differences were statistically significant (P0.05). Conclusions Parallel combined testing of MSH2 and RUNX3 gene methylation in DNA prepared from nasopharyngeal swabs determined by methylation-specific PCR could increase the specificity and sensitivity in early diagnosis of nasopharyngeal carcinoma, and is of important clinical significance. However it may not serve as an index in evaluating the clinical prognosis of nasopharyngeal carcinoma at present.
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ObjectiveTo explore the relationship between the expression of human proliferating cell nuclear antigen (PCNA) and human MutS honolog2 gene (hMSH2) in breast cancer and its significance. Methods PCNA and hMSH2 expression were detected in 68 cases of breast cancer tissues by immunohistochemistry.ResultsAmong the 68 cases of breast cancer, the expression rate of PCNA and hMSH2 was 44.1％ (30/68) and 54.4％ (37/68) respectively.The expression of PCNA and hMSH2 was positively correlated with lymph nodes metastasis(P ＜0.05).PCNA expression was positively correlated to hMSH2 expression in breast cancer (P ＜0.05).ConclusionInteraction between PCNA and hMSH2 may be related to the carcinogenesis of breast cancer.Effective detection of PCNA and hMSH2 proteins may contribute to the evaluation of malignancy and biological behavior of breast cancer.
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To detect the presence of point mutations in a small section of the mutS homolog2 (MSH2) gene in both healthy and affected persons treated at the General Hospital of the State of Sonora, a 353 base pair section of the MSH2 gene was amplified and sequenced from six persons affected by hereditary nonpolyposis colorectal cancer and from 19 healthy persons. The affected persons did not show the mutations reported in the scientific literature; however, six healthy persons were heterozygote and mutant-allele carriers. The heterozygote condition implies that carriers are candidates for the development of colorectal cancer. However, it is important to know the family medical history when investigating hereditary mutations.
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MutL and MutS or their homologues are two crucial proteins of DNA mismatch repair (MMR) system. A new method was described for observation of the interaction between MutS and MutL which is based on the fusion gene/fusion protein technique. Three fusion proteins, MutL-GFP fusion (Trx-His6-GFP-(Ser-Gly)6-MutL), MutL-Strep tag Ⅱ fusion (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) and MutS fusion (Trx-His6-(Ser-Gly)6-MutS), were constructed and expressed in E. coli AD494 (DE3). Interaction assay between MutS and MutL was performed in a 96-well microtiter plate.MutS fusion protein was immobilized on the wells and provided a surface for the interaction between MutS and MutL.Results showed that only after binding of MutS to the mismatched DNA, there was an interaction between MutS and MutL.The binding events could be indicated by GFP signal or the signal generated from alkaline phosphatase and its substrate. In addition, the method based on fusion molecular system also serve as a model for studies on the interactions among other proteins or biomolecules.