發(fā)布時(shí)間：2018-03-04 00:12

  本文選題：hsa-miR-6841-3p　切入點(diǎn)：三葉因子3　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本研究檢測(cè)了MI RNA-6841-3P在HPV陽性宮頸癌組織、HPV陰性宮頸癌組織和正常宮頸組織中的表達(dá),進(jìn)一步探討MIRNA-6841-3P對(duì)宮頸癌細(xì)胞生物學(xué)行為的影響,預(yù)測(cè)并驗(yàn)證MIRNA-6841-3P的靶基因,闡明MIRNA-6841-3P在宮頸癌發(fā)生發(fā)展中的作用,為深入理解宮頸癌發(fā)病的潛在分子機(jī)制和開發(fā)新的有效分子治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。方法:通過實(shí)時(shí)熒光定量PCR方法,檢測(cè)MIRNA-6841-3P在38例HPV陽性宮頸癌組織和10例HPV陰性宮頸癌組織及8例正常宮頸組織中的表達(dá);體外利用脂質(zhì)體轉(zhuǎn)染技術(shù)將MIRNA-6841-3P模擬物、MIRNA-6841-3P抑制物及其各自陰性對(duì)照轉(zhuǎn)染至宮頸癌細(xì)胞系CASKI和SIHA,將轉(zhuǎn)染后細(xì)胞系分為4組,實(shí)驗(yàn)分組分為MIR-6841-3P模擬物轉(zhuǎn)染組(MIR-6841-3P),即含MIR-6841-3P模擬物(MIMICS);MIR-6841-3P模擬物對(duì)照組(MIR-6841-3P-NEGATIVE CONTROL-MIMICS,NC);MIR-6841-3P抑制物轉(zhuǎn)染組(MIR-6841-3P-IN),即含MIR-6841-3P抑制物(INHIBITOR);MIR-6841-3P抑制物對(duì)照組(MIR-6841-3P-NEGATIVE CONTROL-INHIBITOR,NC-IN)。應(yīng)用實(shí)時(shí)熒光定量PCR檢測(cè)宮頸癌細(xì)胞系CASKI和SIHA轉(zhuǎn)染后MIRNA-6841-3P M RNA以及其預(yù)測(cè)靶基因TFF3的轉(zhuǎn)錄表達(dá),CCK-8法檢測(cè)細(xì)胞增殖能力,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力,TRANSWELL小室檢測(cè)細(xì)胞侵襲能力。運(yùn)用基因預(yù)測(cè)軟件TARGET SCAN、MIRANDA以及PICTAR共同預(yù)測(cè)并分析MIRNA-6841-3P可能的潛在靶基因。WESTERN BLOT檢驗(yàn)細(xì)胞轉(zhuǎn)染后TFF3的表達(dá)水平的變化。采用的是SPSS 22.0統(tǒng)計(jì)學(xué)軟件進(jìn)行處理。結(jié)果:1.應(yīng)用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)發(fā)現(xiàn)與正常組織相比MIRNA-6841-3P在宮頸癌組織表達(dá)明顯降低,MIRNA-6841-3P在HPV陽性組織中表達(dá)量低于HPV陰性組織(P0.05),差異有統(tǒng)計(jì)學(xué)意義。2.實(shí)時(shí)熒光定量PCR顯示,MIRNA-6841-3P模擬物轉(zhuǎn)染組(MIR-6841-3P)與MIRNA-6841-3P模擬物對(duì)照組相比(NC),M RNA轉(zhuǎn)錄表達(dá)明顯上調(diào)(P0.01),MIRNA-6841-3P抑制物轉(zhuǎn)染組(MIR-6841-3P-IN)M RNA的轉(zhuǎn)錄表達(dá)低于MIRNA-6841-3P抑制物轉(zhuǎn)染對(duì)照組(NC-IN)(P0.01),差異有統(tǒng)計(jì)學(xué)意義。3.與MI RNA-6841-3P抑制物轉(zhuǎn)染對(duì)照組(NC-IN)比較,MIRNA-6841-3P抑制物轉(zhuǎn)染組(MIR-6841-3P-IN)CASKI和SIHA細(xì)胞活性、遷移侵襲能力明顯增強(qiáng),與MIRNA-6841-3P模擬物轉(zhuǎn)染對(duì)照組(NC)比較,轉(zhuǎn)染MIRNA-6841-3P模擬物組(MI R-6841-3P),宮頸癌細(xì)胞系CASKI和SIHA細(xì)胞活性、遷移侵襲能力明顯減弱(P0.05)。4.其預(yù)測(cè)靶基因TFF3 MRNA的轉(zhuǎn)錄表達(dá)在MIRNA-6841-3P模擬物轉(zhuǎn)染組(MI R-6841-3P)明顯低于MIRNA-6841-3P模擬物轉(zhuǎn)染對(duì)照組(NC),MIRNA-6841-3P抑制物轉(zhuǎn)染組(MI R-6841-3P-IN)明顯高于MIRNA-6841-3P抑制物轉(zhuǎn)染對(duì)照組(P0.01)。5.WESTERN BLOT實(shí)驗(yàn)表明,抑制MIRNA-6841-3P導(dǎo)致CASKI和SIHA細(xì)胞中TFF3的蛋白水平增加。另外,過表達(dá)MIRNA-6841-3P模擬物降低了這兩種細(xì)胞中TFF3的表達(dá)。結(jié)論及意義:上調(diào)MIRNA-6841-3P表達(dá)可明顯抑制宮頸癌細(xì)胞系CASKI和SIHA增殖,抑制其遷移、侵襲能力。TFF3可能是MIRNA-6841-3P的靶基因,并有可能為宮頸癌的治療提供新的靶點(diǎn)。
[Abstract]:Objective: to detect the expression of MIRNA-6841-3P in HPV positive cervical carcinoma tissues and normal cervical tissues, to further investigate the effect of MIRNA-6841-3P on the biological behavior of cervical cancer cells, and to predict and verify the target gene of MIRNA-6841-3P. To elucidate the role of MIRNA-6841-3P in the carcinogenesis and development of cervical cancer, and to provide experimental basis for further understanding the underlying molecular mechanism of cervical cancer and to develop new effective molecular therapeutic targets. The expression of MIRNA-6841-3P was detected in 38 cases of HPV positive cervical carcinoma, 10 cases of HPV negative cervical carcinoma and 8 cases of normal cervix. MIRNA-6841-3P inhibitor (MIRNA-6841-3P) and its respective negative control were transfected into cervical cancer cell lines CASKI and SIHA by liposome transfection in vitro. The transfected cells were divided into 4 groups. The experiment was divided into two groups: MIR-6841-3PnP, MIR-6841-3P, MIR-6841-3P mimic, MIR-6841-3P-NEGATIVE ConNTROL-MISMICSMICMIR-6841-3P, MIR-6841-3P-INP, the MIR-6841-3P inhibitor, MIR-6841-3P inhibitor, MIR-6841-3P-NEGATIVE NTROL-INHIBITORNC-INNIN. The real-time quantitative PCR was used to detect the cervical cancer cell line CASKI and MIR-6841-3P, the MIR-6841-3P-NEGATE-CONTROL-INHIBITORNC-INP. The transcriptional expression of MIRNA-6841-3P M RNA and its predicted target gene TFF3 were detected by CCK-8 method after SIHA transfection. Scratch assay was used to detect cell migration ability and TRANSWELL chamber was used to detect cell invasion. Gene prediction software TARGET SCANN MIRANDA and PICTAR were used to predict and analyze MIRNA-6841-3P potential target gene. Western BLOT to detect the change of TFF3 expression level after transfection. SPSS 22.0 statistical software was used to process it. Results 1. The expression of MIRNA-6841-3P in cervical cancer tissues was significantly lower than that in normal tissues by real-time fluorescence quantitative PCR. The expression of MIRNA-6841-3P in HPV positive tissues was lower than that in HPV positive tissues. Real time fluorescence quantitative PCR showed that MIRNA-6841-3P mimic transfection group had significantly up-regulated MIR-6841-3P RNA expression compared with MIRNA-6841-3P mimic control group, and the expression of MIR-6841-3P RNA was significantly higher than that of MIRNA-6841-3P inhibition group compared with that of MIRNA-6841-3P mimetic control group (P < 0.05). The expression of MIR-6841-3P-INM RNA in MIR-6841-3P transfection group was significantly higher than that in MIRNA-6841-3P mimetic control group (P < 0.05). The expression of MIR-6841-3P-INM RNA was significantly higher in the transfected group than that in the MIRNA-6841-3P control group. The activity of MIRNA-6841-3P inhibitor was compared with that of MIRNA-6841-3P inhibitor transfection group (MIR-6841-3P-CASKI and SIHA cells). The ability of migration and invasion was significantly enhanced. Compared with the control group transfected with MIRNA-6841-3P mimics, the activity of CASKI and SIHA cells was increased in the MIRNA-6841-3P mimetic group. The transcriptional expression of the target gene TFF3 MRNA in the MIRNA-6841-3P mimic transfection group was significantly lower than that in the MIRNA-6841-3P mimic transfection group compared with the control group. The expression of MIR-6841-3P-INwas significantly higher than that of the MIRNA-6841-3P inhibitor transfection control group (P0.01N. 5. WESTERN BLOT). Inhibition of MIRNA-6841-3P induced an increase in TFF3 protein levels in CASKI and SIHA cells. In addition, overexpression of MIRNA-6841-3P mimics decreased the expression of TFF3 in these two cell lines. Conclusion: up-regulation of MIRNA-6841-3P expression can significantly inhibit the proliferation of CASKI and SIHA cells. Inhibiting its migration, invasiveness. TFF3 may be the target gene of MIRNA-6841-3P, and may provide a new target for the treatment of cervical cancer.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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