本文選題：miR-193a-3p + 食管癌��； 參考：《安徽醫(yī)科大學》2016年碩士論文

【摘要】：目的：探討miR-193a-3p在食管鱗癌放射抵抗及化療耐受機制中的作用。方法：通過6MV-X射線對四株食管癌細胞進行照射,采用克隆形成法檢測出相對敏感株及耐受株細胞；利用MTT方法檢測四種化療藥物對四株食管鱗癌細胞的生存率影響；莖環(huán)引物實時定量PCR法檢測miR-193a-3p在敏感株及耐受株細胞中的表達,分別合成并轉染miR-193a-3p的mimic(3PM)或antagomiR(3PA)序列或小干擾RNA(si-PSEN1)以提高或抑制其在細胞中的表達水平,克隆形成法和流式細胞分析術檢測miR-193a-3p及其下游基因PSEN1對于放射耐受性及化療耐受性的影響,Western blot檢測r-H2AX、 PSEN1蛋白的表達；細胞增殖實驗檢測miR-193a-3p與PSEN1對細胞生長的影響。結果：篩選出相對放射敏感細胞株(KYSE150)及耐受細胞株(KYSE410);同時對四種藥物最敏感的細胞株為KYSE150,最耐受的細胞株為KYSE410,故將這兩株細胞作為后續(xù)研究的標本。miR-193a-3p在兩株細胞中的表達水平差異顯著(1.00：192)；KYSE410細胞中轉染miR-193a-3pantagomiR,抑制其在細胞中的表達水平,與對照組相比,其放射敏感性及化療敏感性均增加,細胞凋亡比例顯著增加3.63%,差異有統(tǒng)計學意義(P0.05),同時r-H2AX蛋白表達增加(1.00：1.66)。KYSE510細胞中轉染miR-193a-3pmimic,提高其表達水平,與對照組相比,其放射敏感性及化療敏感性均減小,細胞凋亡比例顯著下降11.10%,差異有統(tǒng)計學意義(P0.05),同時r-H2AX蛋白表達減少(1.00：0.85)；在KYSE150中沉默PSEN1表達,與對照組相比,其放射敏感性降低,細胞凋亡比例下降7.07%,差異有統(tǒng)計學意義(P0.05)；在KYSE150中沉默PSEN1及上調(diào)miR-193a-3p表達后,與對照組相比,細胞增殖力增加,差異有統(tǒng)計學意義(P0.05)。結論：miR-193a-3p通過調(diào)控基因PSEN1促進食管癌細胞放射抵抗性及化療耐受性,miR-193a-3p和PSEN1可能成為食管癌放化療耐受的一個潛在標志物。
[Abstract]:Objective: to investigate the role of miR-193a-3p in the mechanism of radiation resistance and chemotherapy tolerance in esophageal squamous cell carcinoma. Methods: four esophageal cancer cell lines were irradiated by 6MV-X rays, the relative sensitive and tolerant cell lines were detected by clone forming method, and the survival rates of four chemotherapeutic drugs on four esophageal squamous carcinoma cells were detected by MTT method. Stem loop primer real-time quantitative PCR assay was used to detect the expression of miR-193a-3p in sensitive and tolerant cell lines, and to synthesize and transfect miR-193a-3p mimico-3PM3 or antagomiRN3PAA sequences or small interfering RNA-PSEN1, respectively, in order to increase or inhibit the expression level of miR-193a-3p in the cells. The effects of miR-193a-3p and its downstream gene PSEN1 on radiation tolerance and chemotherapeutic tolerance were detected by clone formation and flow cytometry. The expression of r-H2AX, PSEN1 protein was detected by Western blot, and the effects of miR-193a-3p and PSEN1 on cell growth were detected by cell proliferation assay. Results: the relative radiosensitive cell line (KYSE150) and the resistant cell line (KYSE410P) were screened, and the most sensitive cell line to the four drugs was KYSE150, and the most tolerant cell strain was KYSE410. therefore, these two cell lines were used as the specimen for further study. MiR-193a-3p was used in the two cell lines. The expression level of miR-19a-3pantagomiR was inhibited by transfection of miR-193a-3pantagomiR in KYSE410 cells. Compared with the control group, the radiosensitivity and chemosensitivity of the cells were increased, and the percentage of apoptosis was increased significantly (P 0.05). Meanwhile, the expression of r-H2AX protein was increased by 1.00: 1.66N. KYSE510 cells transfected with miR-193a-3 pmimicl, and the expression level of miR-193a-3 pmimiccells was increased compared with the control group. Its radiosensitivity and chemosensitivity decreased, the percentage of apoptosis decreased significantly (P 0.05), and the expression of r-H2AX protein decreased by 1.00: 0.85. In KYSE150, the expression of PSEN1 was silenced, compared with the control group, the radiosensitivity was decreased. After silencing PSEN1 and upregulating the expression of miR-193a-3p in KYSE150, the cell proliferation was increased and the difference was statistically significant compared with the control group (P 0.05). ConclusionMiR-193a-3p may be a potential marker of radiotherapy and chemotherapeutic tolerance of esophageal cancer cells by promoting radioresistance and chemotherapeutic tolerance of esophageal cancer cells by regulating gene PSEN1.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.1

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本文編號：1959777