發(fā)布時(shí)間：2018-07-29 08:31

【摘要】：背景 宮頸癌是全世界范圍內(nèi)同時(shí)也是我國(guó)最常見的惡性腫瘤之一，其發(fā)生率位居女性生殖系統(tǒng)惡性腫瘤第一位，且有逐年年輕化的趨向。近年來，隨著手術(shù)切除、化學(xué)治療、放射治療、及其它綜合性治療措施的應(yīng)用，對(duì)宮頸癌的治療有了一定程度的進(jìn)展，但療效并不十分理想。宮頸癌的病因及發(fā)生機(jī)制目前尚不十分明了，許多研究證明其發(fā)生、發(fā)展和浸潤(rùn)、轉(zhuǎn)移是多因素、多階段、多基因共同參與的非常復(fù)雜的過程，因此，探討宮頸癌的發(fā)病機(jī)制，尋找更有效的治療措施是當(dāng)前研究的熱點(diǎn)課題之一。 LRIGs是最近發(fā)現(xiàn)的一組腫瘤相關(guān)基因，該家族共有三個(gè)成員：LRIG1、LRIG2、LRIG3。LRIGs編碼一組結(jié)構(gòu)上較為相似的膜整合蛋白，蛋白分為胞外段、跨膜區(qū)和一個(gè)胞漿內(nèi)尾巴，胞外段蛋白含有富含亮氨酸重復(fù)序列（LRRs）和3個(gè)免疫球蛋白樣結(jié)構(gòu)域。研究表明LRIG的表達(dá)能夠?qū)GFR信號(hào)通路進(jìn)行調(diào)控，從而起到抑制腫瘤生長(zhǎng)的作用，但是我們對(duì)其分子和信號(hào)機(jī)制尚不清楚。近年來許多研究表明，，LRIGs基因家族在垂體瘤、神經(jīng)膠質(zhì)瘤和皮膚鱗狀細(xì)胞癌等多種腫瘤中具有抑癌作用。 LRIG3定位于人類基因組12q13，在人類腫瘤中LRIG3是高表達(dá)還是低表達(dá)尚不十分清楚，對(duì)于其在宮頸癌中表達(dá)的相關(guān)研究較少，對(duì)于其在宮頸癌細(xì)胞株中發(fā)揮的生物學(xué)作用尚未見報(bào)道。LRIG3和宮頸癌的發(fā)生發(fā)展是否有相關(guān)性，抑制LRIG3表達(dá)對(duì)宮頸鱗癌的影響是本研究需要探討的核心。 目的 檢測(cè)宮頸鱗癌組織、宮頸上皮內(nèi)瘤變組織和正常宮頸粘膜組織中LRIG3、EGFR和Ki67的表達(dá)，并分析其與宮頸鱗癌浸潤(rùn)轉(zhuǎn)移的關(guān)系。觀察LRIG3反義核苷酸轉(zhuǎn)染到宮頸癌Hela229細(xì)胞中后對(duì)LRIG3和EGFR的表達(dá)以及對(duì)細(xì)胞增殖、轉(zhuǎn)移以及細(xì)胞周期的影響。觀察LRIG3反義核苷酸對(duì)宮頸癌裸鼠移植瘤的影響。 第一部分LRIG3在宮頸鱗癌組織中的表達(dá)及意義 方法 應(yīng)用免疫組織化學(xué)和原位雜交檢測(cè)45例宮頸鱗癌組織、20例CIN組織和30例正常宮頸黏膜組織中LRIG3、EGFR和Ki67表達(dá)，并結(jié)合臨床病理資料對(duì)結(jié)果進(jìn)行分析。 結(jié)果 1. LRIG3蛋白和mRNA在宮頸鱗狀細(xì)胞癌組織中的陽(yáng)性表達(dá)率分別為35.56%和48.89%，顯著低于CIN組織（分別為70%、80%）和正常宮頸粘膜組織（皆為100%），兩兩間比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。其蛋白和mRNA的表達(dá)強(qiáng)度與/和患者的年齡以及腫瘤大小無關(guān)（P均0.05），而與腫瘤的浸潤(rùn)程度、臨床分期以及淋巴結(jié)轉(zhuǎn)移成負(fù)相關(guān)（P均0.05）。 2. EGFR蛋白和mRNA在宮頸鱗狀細(xì)胞癌組織中的陽(yáng)性表達(dá)率分別為80%和84.44%，顯著高于CIN組織（分別為35%、45%）和正常宮頸粘膜組織（分別為6.67%、16.67%），兩兩間比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。在宮頸鱗狀細(xì)胞癌組織中EGFR蛋白和mRNA的表達(dá)強(qiáng)度與患者的年齡、腫瘤大小、臨床分期無關(guān)（P均0.05），而與腫瘤的浸潤(rùn)程度以及淋巴結(jié)轉(zhuǎn)移成正相關(guān)（P均0.05）。 3. Ki67蛋白和mRNA在宮頸鱗狀細(xì)胞癌組織中的陽(yáng)性表達(dá)率分別為86.67%和91.11%，顯著高于CIN組織（分別為30%和50%）和正常宮頸粘膜組織（分別為3.33%、23.33%），兩兩間比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。Ki-67的表達(dá)與宮頸鱗癌各臨床病理因素均無相關(guān)性((P0.05)。 4. LRIG3和EGFR在宮頸鱗狀細(xì)胞癌組織中的表達(dá)呈負(fù)向相關(guān), LRIG3和Ki67在宮頸鱗狀細(xì)胞癌組織中的表達(dá)亦呈負(fù)向相關(guān)。 第二部分LRIG3的表達(dá)調(diào)控對(duì)宮頸鱗癌Hela229細(xì)胞周期、凋亡、侵襲力的影響 方法 1.設(shè)計(jì)合成LRIG3反義寡核苷酸（LRIG3ASODN）、正義寡核苷酸序列（LRIG3SODN）以及錯(cuò)義寡核苷酸序列（LRIG3MSODN），用脂質(zhì)體Lipofectamine2000包裹轉(zhuǎn)染Hela229細(xì)胞，作為轉(zhuǎn)染組細(xì)胞。將脂質(zhì)體轉(zhuǎn)染的細(xì)胞作為空白對(duì)照組細(xì)胞。 2. Western blot檢測(cè)不同濃度(150μg/ml、200μg/ml、250μg/ml) LRIG3ASODN、LRIG3MSODN和LRIG3SODN轉(zhuǎn)染細(xì)胞24h、48h及72h后LRIG3和EGFR蛋白表達(dá)情況。 3. RT-PCR檢測(cè)不同濃度(150μg/ml、200μg/ml、250μg/ml) LRIG3ASODN、LRIG3MSODN和LRIG3SODN轉(zhuǎn)染細(xì)胞24h、48h及72h后LRIG3和EGFRmRNA表達(dá)情況。 4.應(yīng)用MTT檢測(cè)細(xì)胞增殖情況，細(xì)胞流式法測(cè)定細(xì)胞凋亡，細(xì)胞基質(zhì)粘附實(shí)驗(yàn)檢測(cè)細(xì)胞粘附能力，Transwell小室法檢測(cè)細(xì)胞的侵襲能力。 結(jié)果 1. ASODN組中LRIG3蛋白和mRNA表達(dá)較SODN組、MSODN組、空白對(duì)照組明顯降低（P0.05），且有濃度依賴性和時(shí)間依賴性，LRIG3的表達(dá)隨轉(zhuǎn)染濃度的增加以及時(shí)間的延長(zhǎng)而減少。 2. ASODN組中EGFR蛋白和mRNA表達(dá)較SODN組、MSODN組、空白對(duì)照組明顯增加（P0.05），隨轉(zhuǎn)染濃度的增加及時(shí)間的延長(zhǎng)EGFR的表達(dá)隨之升高。EGFR表達(dá)和LRIG3表達(dá)呈負(fù)相關(guān)。 3. MTT實(shí)驗(yàn)表明LRIG3ASODN組Hela229細(xì)胞增殖率較其它組別明顯升高（P0.05），隨著LRIG3ASODN轉(zhuǎn)染濃度增加，表達(dá)抑制時(shí)間延長(zhǎng)，細(xì)胞增殖率明顯升高，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 4.轉(zhuǎn)染LRIG3ASODN后Hela229細(xì)胞凋亡率明顯降低，隨著轉(zhuǎn)染濃度的增加，時(shí)間延長(zhǎng)，細(xì)胞的凋亡率隨之降低。 5.細(xì)胞基質(zhì)粘附實(shí)驗(yàn)表明轉(zhuǎn)染LRIG3ASODN后使Hela229細(xì)胞粘附率明顯增高，隨著轉(zhuǎn)染濃度的增加，時(shí)間延長(zhǎng)，細(xì)胞的粘附率隨之升高。 6. Transwell實(shí)驗(yàn)表明轉(zhuǎn)染LRIG3ASODN后使得Hela229細(xì)胞的遷徙能力明顯增高，ASODN組穿膜細(xì)胞數(shù)（246±10）明顯高于SODN組（168±10）、MSODN組（177±8）和空白對(duì)照組（176±6）。 第三部分LRIG3表達(dá)調(diào)控對(duì)人宮頸鱗癌裸鼠移植瘤生長(zhǎng)的影響 方法 將1×107個(gè)Hela229細(xì)胞注射到裸鼠皮下構(gòu)建宮頸鱗癌裸鼠移植瘤模型，將LRIG3ASODN皮下注射到瘤體內(nèi)后觀察裸鼠移植瘤的生長(zhǎng)狀態(tài)。測(cè)量各組種植瘤瘤體體積。Western blot和RT-PCR檢測(cè)裸鼠移植瘤中LRIG3和EGFR蛋白和mRNA表達(dá)。 結(jié)果 1. ASODN LRIG3組注射后18-30天裸鼠細(xì)胞瘤體積較其它兩組明顯增高（P0.05）。 2. Western blot和PT-PCR檢測(cè)裸鼠移植瘤中LRIG3蛋白和mRNA表達(dá)，顯示ASODN組LRIG3表達(dá)較MSODN和空白對(duì)照組明顯降低（P0.05）。 3. Western blot和PT-PCR檢測(cè)裸鼠移植瘤中EGFR蛋白和mRNA表達(dá)，顯示ASODN組EGFR表達(dá)較MSODN和空白對(duì)照組明顯升高（P0.05）。 結(jié)論 1.宮頸鱗癌組織和CIN組織中LRIG3呈低表達(dá)，EGFR高表達(dá)，其表達(dá)量與宮頸鱗癌的浸潤(rùn)和轉(zhuǎn)移密切相關(guān)。提示LRIG3通過對(duì)EGFR的負(fù)反饋調(diào)節(jié)起到抑制宮頸鱗癌發(fā)生發(fā)展的作用。 2. LRIG3ASODN能顯著下調(diào)宮頸鱗癌Hela229細(xì)胞中LRIG3蛋白和mRNA的表達(dá)，同時(shí)EGFR蛋白和mRNA表達(dá)上升，能抑制宮頸鱗癌細(xì)胞凋亡，增強(qiáng)細(xì)胞侵襲能力。提示LRIG3在宮頸鱗癌細(xì)胞增殖、凋亡及侵襲力中起著重要作用。 3. LRIG3ASODN可導(dǎo)致宮頸鱗癌裸鼠成瘤速度加快，體積增加，同時(shí)移植瘤組織中LRIG3蛋白和mRNA的表達(dá)顯著降低，EGFR蛋白和mRNA表達(dá)增高。
[Abstract]:background
Cervical cancer is one of the most common malignant tumors in the world and is one of the most common malignant tumors in China. Its incidence is the first malignant tumor in female reproductive system, and it has a tendency to be young year by year. In recent years, with the application of surgical resection, chemical therapy, radiation therapy, and other comprehensive treatment measures, the treatment of cervical cancer has a certain course. The etiology and mechanism of cervical cancer are still not very clear. Many studies have proved that the occurrence, development and infiltration of cervical cancer are a very complex process involving multiple factors, multi stages and multiple genes. Therefore, the study of the pathogenesis of cervical cancer and the search for more effective treatment measures are currently studied. One of the hot topics in the study.
LRIGs is a recently discovered group of tumor related genes. There are three members of the family: LRIG1, LRIG2, and LRIG3.LRIGs, which encode a group of relatively similar membrane integrins. The proteins are divided into extracellular, transmembrane and cytoplasmic tails, and exosin contains high leucine rich repeat (LRRs) and 3 immunoglobulin like domains. Studies have shown that the expression of LRIG can regulate the EGFR signaling pathway, thus inhibiting the growth of the tumor, but we are not clear about its molecular and signal mechanisms. In recent years, many studies have shown that the LRIGs gene family has a tumor suppressor effect in a variety of tumors, such as pituitary adenoma, glioma and skin squamous cell carcinoma.
LRIG3 is located in the human genome 12q13. The high expression or low expression of LRIG3 in human tumor is not very clear. There are few related studies on its expression in cervical cancer. There is no report on the correlation between the development of.LRIG3 and cervical cancer and the inhibition of the expression of LRIG3 in cervical cancer cell lines. The impact on cervical squamous cell carcinoma is the core of this study.
objective
To detect the expression of LRIG3, EGFR and Ki67 in cervical squamous cell carcinoma, cervical intraepithelial neoplasia and normal cervical mucosa, and to analyze the relationship with invasion and metastasis of squamous cell carcinoma of the cervix. The expression of LRIG3 antisense nucleotides transfected to Hela229 cells of cervical cancer and the expression of LRIG3 and EGFR and the shadow of cell proliferation, metastasis and cell cycle were observed. To observe the effect of LRIG3 antisense nucleotide on cervical cancer xenografts in nude mice.
Expression and significance of LRIG3 in cervical squamous cell carcinoma
Method
Immunohistochemistry and in situ hybridization were used to detect the expression of LRIG3, EGFR and Ki67 in 45 cases of cervical squamous cell carcinoma, 20 CIN tissues and 30 normal cervical mucosa tissues, and the results were analyzed with the clinicopathological data.
Result
The positive expression rates of 1. LRIG3 protein and mRNA in cervical squamous cell carcinoma were 35.56% and 48.89%, respectively, significantly lower than that of CIN tissues (70%, 80%, respectively) and normal cervical mucosa (100%). The 22 differences were statistically significant (P0.05). The expression intensity of protein and mRNA and the age of the patients and the size of the tumor None (P = 0.05), but negatively correlated with tumor infiltration, clinical stage and lymph node metastasis (P = 0.05).
The positive expression rates of 2. EGFR protein and mRNA in cervical squamous cell carcinoma were 80% and 84.44% respectively, which were significantly higher than those of CIN (35%, 45%) and normal cervical mucosa (6.67%, 16.67%, respectively, 6.67%, 16.67%, respectively). The expression of EGFR protein and mRNA in the squamous cell carcinoma of the cervix was significant. No correlation was found with age, tumor size and clinical stage (P = 0.05), but positively correlated with tumor infiltration and lymph node metastasis (P 0.05).
The positive expression rates of 3. Ki67 protein and mRNA in cervical squamous cell carcinoma were 86.67% and 91.11% respectively, which were significantly higher than those of CIN tissues (30% and 50%) and normal cervical mucosa (3.33%, 23.33%, respectively). There were significant differences in 22 differences (P0.05).Ki-67 expression was not different from the clinical and pathological factors of cervical squamous cell carcinoma. P0.05.
4. the expression of LRIG3 and EGFR in cervical squamous cell carcinoma was negatively correlated, and the expression of LRIG3 and Ki67 in cervical squamous cell carcinoma was also negatively correlated.
The second part is the effect of LRIG3 expression regulation on cell cycle, apoptosis and invasiveness of cervical squamous cell carcinoma Hela229.
Method
1. the LRIG3 antisense oligonucleotide (LRIG3ASODN), the just oligonucleotide sequence (LRIG3SODN) and the missense oligonucleotide sequence (LRIG3MSODN) were designed and synthesized. The transfected Hela229 cells were transfected with liposome Lipofectamine2000 as the transfected cells. The cells transfected with liposomes were used as blank control cells.
The expression of 24h, 48h and 72h after transfection of 24h, 48h and 72h was detected by 2. Western blot (150 g/ml, 200, g/ml, 250 mu g/ml) LRIG3ASODN, LRIG3MSODN and LRIG3SODN.
3. RT-PCR was used to detect the expression of 24h and 48h and 72h after transfection at different concentrations (150 g/ml, 200 g/ml, 250 g/ml) LRIG3ASODN, LRIG3MSODN and LRIG3SODN.
4. the cell proliferation was detected by MTT, cell apoptosis was measured by flow cytometry, cell adhesion test was used to detect cell adhesion ability, and cell invasion ability was detected by Transwell chamber method.
Result
The expression of LRIG3 protein and mRNA in group 1. ASODN was significantly lower than that in group SODN, in group MSODN and in blank control group (P0.05), and there was a concentration dependence and time dependence. The expression of LRIG3 decreased with the increase of transfection concentration and the prolongation of time.
The expression of EGFR protein and mRNA in group 2. ASODN was significantly higher than that in group SODN, and in MSODN group, the blank control group was significantly increased (P0.05). The expression of.EGFR and LRIG3 expressed a negative correlation with the increase of the transfection concentration and the prolongation of the expression of EGFR.
3. MTT test showed that the proliferation rate of Hela229 cells in group LRIG3ASODN was significantly higher than that of other groups (P0.05). With the increase of LRIG3ASODN transfection concentration, the expression inhibition time was prolonged and the cell proliferation rate was significantly increased, the difference was statistically significant (P0.05).
4. the apoptosis rate of Hela229 cells decreased significantly after transfection of LRIG3ASODN. With the increase of transfection concentration and the prolongation of time, the apoptosis rate of cells decreased.
The adhesion test of 5. cell matrix showed that the adhesion rate of Hela229 cells increased obviously after transfection of LRIG3ASODN. With the increase of transfection concentration, the time was prolonged and the adhesion rate of cells increased.
6. Transwell test showed that the migration ability of Hela229 cells increased significantly after transfection of LRIG3ASODN. The number of membrane cells in ASODN group (246 + 10) was significantly higher than that in group SODN (168 + 10), MSODN group (177 + 8) and blank control group (176 + 6).
The third part is the effect of LRIG3 expression regulation on the growth of human cervical squamous cell carcinoma xenografts in nude mice.
Method
1 x 107 Hela229 cells were injected into nude mice to construct nude mice model of nude mice, and the growth state of nude mice was observed after subcutaneous injection of LRIG3ASODN into the tumor. The expression of LRIG3 and EGFR protein and mRNA in the transplanted tumor of nude mice was measured by.Western blot and RT-PCR.
Result
In 1. ASODN LRIG3 group, the tumor volume in nude mice was significantly higher than that in the other two groups on the 18-30 day after injection (P0.05).
2. Western blot and PT-PCR detected the expression of LRIG3 protein and mRNA in nude mice xenograft. The expression of LRIG3 in ASODN group was significantly lower than that in MSODN and blank control group (P0.05).
3. Western blot and PT-PCR detected the expression of EGFR protein and mRNA in nude mice xenograft. The expression of EGFR in ASODN group was significantly higher than that in MSODN and blank control group (P0.05).
conclusion
1. the expression of LRIG3 in cervical squamous cell carcinoma tissues and CIN tissues is low, and the expression of EGFR is highly expressed. The expression of LRIG3 is closely related to the invasion and metastasis of cervical squamous cell carcinoma. It suggests that the negative feedback regulation of EGFR plays a role in inhibiting the development of cervical squamous cell carcinoma.
2. LRIG3ASODN can significantly reduce the expression of LRIG3 protein and mRNA in the Hela229 cells of cervical squamous cell carcinoma, and increase the expression of EGFR and mRNA, which can inhibit the apoptosis of cervical squamous cell carcinoma cells and enhance the invasive ability of the cells, suggesting that LRIG3 plays an important role in the proliferation, apoptosis and invasion of cervical squamous cell carcinoma cells.
3. LRIG3ASODN could lead to accelerated tumor formation and increased volume of cervical squamous cell carcinoma in nude mice. The expression of LRIG3 protein and mRNA in the transplanted tumor tissue was significantly decreased, and the expression of EGFR protein and mRNA was increased.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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