2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38621Background and Purposes Human nonmetastatic clone 23 type 1 gene (nm23-H1) is a metastasis-associated gene in many types of cancer. The purposes of our study are to: 1. find the downstream genes of nm23-H1 using cervical cancer cells; 2. correlate cell invasion and metastatic potential of cervical cancer cells with nm23-H1 and its target downstream genes; 3. evaluate the expressions of nm23-H1 and its binding proteins (cofactors) such as h-prune, SET or Tiam1 and its downstream genes as well as nm23-H1 isoforms (such as nm23-LV or nm23-H1B) and some genes (such as c-myc) those it may regulate in cervical cancer tissues; and further evaluate the relationships among these factors and various clinicopathological variables, metastasis, cumulative recurrence hazard and 5-year survival rate of cancer patients. Materials and Methods 1. HeLa and SiHa cells, exhibiting higher levels of nm23-H1 protein, are knocked down for nm23-H1 gene with RNA interference (short hairpin of RNA; shRNA). Caski cells, exhibiting low levels of nm23-H1 protein, are transfected with exogenous nm23-H1 gene. Thereafter, the target downstream genes of nm23-H1 are found after these cancer cells are tested to have different expressions of nm23-H1 and some cancer genes using Oligo GEArray® Human Cancer Microarray. 2. The expression of nm23-H1 is correlated with cell invasive and metastatic potential of these cervical cancer cells using wound healing assay, soft agar colonization, modified Boyden chamber migration and invasion assay, extracellular matrix adhesion assay and gelatin zymography by independent Student t test. 3. After downstream genes of nm23-H1 are inhibited using RNA interference or overexpressed using genes transfection, these cervical cancer cells are also detected for cell invasion and metastatic potential to investigate the impacts of these genes. 2 4. The expressions of mRNA and protein of nm23-H1 and its cofactors and target downstream genes, as well as nm23-H1 isoformes and the genes it regulates (such as c-myc) are compared between cervical cancer tissues and their normal counterparts using real time reverse transcriptasepolymerase chain reaction andWestern blot. 5. The immunoreactivities of nm23-H1 and its binding proteins and downstream genes as well as nm23-H1 isoforms are evaluated using immunohistochemical method for tissue microarrays consisted of carcinomatous and normal cervices. The Image-Pro Plus is used for quantitative analysis of their immunoreactivities. The confocal microscopy is used for co-localization of nm23-H1 and its cofactors. The immunoprecipitation and immunoblot are used to define the expression of nm23-H1 and its binding proteins in cervical cancer tissues. 6. Expressions of mRNA or protein of these factors are correlated with the clinocopathological variables, metastasis, cumulative recurrence hazard and 5-year survival rates of cervical cancer patients. Predictive results 1. The cell invasion and metastatic potentials of cervical cancer cells are probably correlated with nm23-H1 and its some target downstream genes. 2. Nm23-H1 and its binding proteins and downstream genes or nm23-H1 isoforms may be correlated with some clinicopathological variables, 5-year survival rate or recurrence distribution for cervical cancer patients.一、背景 Human nonmetastatic clone 23 type 1 gene (nm23-H1 gene)是一metastasis-associated gene，在子宮 頸癌角色尚未清楚。我們初步發現，nm23-H1 蛋白於子宮頸侵入性癌和癌症轉移之病人，表現有 增強趨勢，但是，nm23-H1 和子宮頸癌臨床病理因子、轉移、復發和存活之關係仍需釐清。 二、目的 1. 對HeLa、SiHa 和Caski 子宮頸癌細胞，進行nm23-H1 基因重組，以癌症基因晶片檢測其 癌症或轉移相關基因(target downstream genes)。 2. 檢測nm23-H1 及其下游基因對癌細胞侵犯及轉移潛力之影響。 3. 分析nm23-H1 及其結合蛋白如h-prune、SET 和Tiam1 及其下游基因；暨nm23-H1 isoforms 和其所調節之基因如c-myc 等，於子宮頸癌組織表現情形；並和子宮頸臨床病理因子、轉 移、復發和存活作關聯性分析。 三、材料和方法 1. HeLa 癌細胞nm23-H1 蛋白表現量最高，SiHa 細胞次之，Caski 細胞很低。使用RNA interferenc 降低HeLa 及SiHa 細胞nm23-H1 基因表現；並將nm23-H1 基因轉染給Caski 細 胞。 2. 以cancer oligo DNA microarray 檢測以上六種癌細胞，癌症基因表現之差異，找出nm23-H1 之 下游基因。 3. 以wound healing assay、soft agar colonization、modified Boyden chamber migration and invasion assay、extracellular matrix adhesion assay 及gelatin zymography分析六種癌細胞之表現，並和 nm23-H1基因及其downstream genes作關聯性分析。 4. 蒐集子宮頸癌組織，作癌組織晶片，核質分離及免疫沉澱法及real time RT-PCR 等，測 nm23-H1 及其結合蛋白暨nm23-H1 isoforms 和其所調節之基因之mRNA 或蛋白表現，並和 子宮頸癌臨床病理特徵、復發或存活率作關聯性分析。 四、預期結果 1. 子宮頸癌細胞經nm23-H1 基因重組後，會改變侵犯或轉移潛力，且和某些癌基因有關。 2 2. 某些子宮頸癌臨床病理特徵、復發或存活和nm23-H1 及其結合蛋白及下游基因或nm23-H1 isoforms 有關。Human nonmetastatic clone 23 type 1子宮頸癌子宮頸癌細胞株下游基因結合蛋白nm23-H1 isoforms癌症基因晶片轉移潛力組織晶片臨床病理特徵五年存活率累計復發危險性Human nonmetastatic clone 23 type 1cancer of uterine cervixcervical cancer cellsdownstream genesbinding proteinsnm23-H1 isoformsgenechipinvasionmetastaticpotentialtissue microarraysProject