摘要
研究目的
1.检测microRNA let-7a-1、let-7b、let-7c在新鲜宫颈鳞癌组织和癌旁组织中的表达量是否存在差异。
2.检测microRNA miR-127-5p、miR-218在宫颈鳞癌和癌旁石蜡切片中的表达量是否存在差异,并探讨miR-127-5p、miR-218与先期化疗、临床分期、癌灶大小、病理类型、分化程度、深肌层浸润、淋巴结转移、血管浸润及复发的相关性。
材料和方法
1.收集9例未经放化疗的宫颈鳞癌及其配对的癌旁组织新鲜标本。
2.提取组织中的总RNA,用实时荧光定量PCR方法检测样本中let-7a-1、let-7b、let-7c的相对表达量。
3.查找8年内北京协和医院行宫颈癌手术治疗的42例患者资料及石蜡包埋的病理标本。
4.手工显微切割获取目标细胞,提取总RNA。应用实时荧光定量PCR方法检测microRNA miR-127-5p和miR-218。并分析表达量与先期化疗、临床分期、癌灶大小、病理类型、分化程度、淋巴结转移等因素之间的相关性。
实验结果
1.let-7a-1在宫颈鳞癌和癌旁组织的平均相对表达量分别是0.8828±0.02075和0.9195±0.02104,统计学分析两组无显著性差异(P>0.05);let-7b在两组的平均相对表达量分别是0.8422±0.03928和0.8891±0.01052;let-7c在两组的平均相对表达量分别是0.9467±0.05266和0.9810±0.07344。统计学分析三基因表达两组中无显著性差异(P>0.05)。
2.miR-127-5p在宫颈鳞癌和癌旁组织中的平均相对表达量分别是1.4931±0.03744和1.3662±0.05681,统计学分析两组无显著性差异(P>0.05)。miR-218在两组的平均相对表达量分别是1.3403±0.01891和1.3114±0.05458,统计学分析两组无显著性差异(P>0.05)。
3.miR-127-5p、miR-218在宫颈鳞癌先期化疗组和未行先期化疗组、FIGOⅠ期和FIGOⅡ期、大小病灶组、低分化组和高分化组、非角化鳞癌组和角化鳞癌组、深肌层浸润组组和非深肌层浸润组、复发组与未复发组的表达量在各组内的统计学分析无显著性差异(P>0.05)
4.miR-127-5p在初治宫颈鳞癌无淋巴结转移组与淋巴结转移组的相对表达量均值分别为1.3070±0.05912和1.1988±0.12589,统计学分析两组无显著性差异(P>0.05)。miR-218在两组的相对表达量均值分别为1.4641±0.07449和1.2988±0.27536,统计学分析两组无显著性差异(P>0.05)。
5.miR-127-5p在宫颈鳞癌血管未浸润组与浸润组的表达量统计学分析无显著性差异(P>0.05)。miR-218在两组的相对表达量均值分别为1.3583±0.01619和1.2431±0.0708,统计学分析两组有显著性差异(P=0.039)。
结论
1.手工显微切割是从宫颈组织标本中纯化目标细胞的有效手段。
2.石蜡标本也是研究microRNA的丰富资源。
3.let-7a-1、let-7b、let-7c在宫颈鳞癌和正常宫颈组织中的表达无显著性差异。
4.miR-127-5p和miR-218在宫颈鳞癌和正常宫颈组织中的表达无显著性差异。
5.宫颈鳞癌中miR-127-5p的表达与先期治疗、FIGO分期、病灶大小、病理类型、分化程度、深肌层浸润、血管浸润及复发无相关性。
6.miR-218的高表达与宫颈鳞癌血管浸润显著相关,miR-218的表达与先期治疗、FIGO分期、病灶大小、病理类型、分化程度、深肌层浸润及复发无相关性。
7.宫颈鳞癌中miR-127-5p、miR-218的表达与淋巴结转移的关系尚不确定,需扩大样本量进一步研究明确。
OBJECTIVES
1. The aim of this study is to compare the expression level of microRNA let-7a-1、let-7b、let-7c in fresh cervical invasive squamous cell carcimoma tissues(CISCC) to the matched normal cervical tissues(MNCT).
2. To compare the expression level of microRNA miR-127-5p、miR-218 in paraffin embedded cervical invasive squamous cell carcimoma tissues to the matched normal cervical tissues.
3. To elucidate corelation of miR-127-5p and miR-218 with neoadjuvant chemotherapy, FIGO stage, tumor size, pathological type, differentiation, deep muscular invasion, lymph node metastasis, vascular invasion, and recurrence in cervical caner.
METHODS
In this study, microRNA let-7a-1、let-7b and let-7c were quantified in 9 fresh frozen specimens of cervical squamous carcinoma by real-time quantitative PCR array methods. 42 paraffin-embedded cervical squamous carcinoma specimens and their clinical data were collected. microRNA miR-127-5p and miR-218 were quantified in those specimens by real-time quantitative PCR array methods. Correlation of relative expression level of those two microRNAs and several factors were analyzed by SPSS statistical software.
RESULTS
1. The mean expression of let-7a-1 in CISCC and MNCT was 0. 8828±0. 02075 and 0. 9195±0. 02104; the mean expression of let-7b in these two groups was 0. 8422±0. 03928 and 0. 8891±0. 01052; the mean expression of let-7c in these two groups was0.9467±0.05266 and 0. 9810±0. 07344. Expression of the 3 genes was no significant difference between two groups (P>0.05).
2. The mean expression of miR-127-5p in ISCC and MNCT was 1.4931±0.03744 and 1.3662±0.05681; the mean expression of miR-218 in these two group was 1.3403±0.01891 and 1.3114±0.05458. Expression of these two genes was no significant difference between two groups (P>0.05).
3. Expression of miR-127-5p and miR-218 in neoadjuvant chemotherapy group and non-neoadjuvant chemotherapy group, FIGO stage I group and stage II group, large tumor size group and small tumor size group, poor differentiated group and well differentiated group, keratinized group and non keratinized group, deep muscular invasion group and non-deep muscular invasion group, recurrent group and non-recurrent group was no significant difference within those groups.
4. The mean expression of miR-127-5p in lymph node metastasis group negative and positive group of primary CISCC was 1. 3070±0.05912 and 1. 1988±0. 12589; the mean expression of miR-218 in these two groups was 1. 4641±0. 07449 and 1.2988±0.27536. Expression of the two genes was no significant difference between two groups (P>0.05).
5. Expression of miR-127-5p in non-vascular invasion group and vascular invasion group was no significant difference between two groups (P>0.05). The mean expression of miR-218 in these two groups was 1. 3583±0. 01619 and 1. 2431±0. 0708, decreased miR-218 was significantly associated with vascular invasion (P=0.039).
CONLUSION
1. Manual microdissection was an effective method for collecting target cells from cervical tissue.
2. Paraffin-embedded tissues were abundant resources for microRNA research.
3. The expression of let-7a-1、let-7b and let-7c in cervical squamous cell carcinoma tissues was no significant difference compared to matched normal cervical tissues.
4. The expression of miR-127-5p and miR-218 in cervical squamous cell carcinoma was no significant difference compared to matched normal cervical tissues.
5. The expression of miR-127-5p was not significantly associated with neoadjuvant chemotherapy, FIGO stage, tumor size, pathological type, differentiation, deep muscular invasion, vascular invasion and recurrence in cervical caner.
6. Increased miR-218 expression was significantly associated with vascular invasion (P=0.039). miR-218 expression was not significantly associated with neoadjuvant chemotherapy, FIGO stage, tumor size, pathological type, differentiation, deep muscular invasion and recurrence in cervical caner.
7. Correlation between miR-127-5p、miR-218 and lymph node metastasis was undetermined, requiring larger sample size.
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