| [Objective] Using mi RNA gene chip technology, Real-time PCR technology and mi RNA function experiments in order to explore the relationship between the mi RNAs and the occurrence and development of laryngeal carcinoma.[Methods] 1.Extracted the total RNA of laryngeal carcinoma tissue samples and then identified RNA quality by spectrophotometer and agarose gel electrophoresis.The RNA was dealed with fluorescent labeling, hybridization and so on.Screened out the differentially expression mi RNAs in four pairs of laryngeal carcinoma tissue samples and adjacent non-neoplastic tissue samples.2.Using real-time PCR technology tested the expression of mi R-99a-5p, mi R-195-5p,mi R-3195,mi R-101-3p,mi R-126-3p in four pairs of laryngeal carcinoma tissue samples and adjacent non?neoplastic tissues amples and verified the result reliability of microarray chip.And then, tested the expression of mi R-99a-5p,mi R-195-5p, mi R-3195,mi R-101-3p, mi R-126-3p in twenty-eight pairs of laryngeal carcinoma tissue samples and adjacent non?neoplastic tissues amples.The ABI 7500 PCR as detection system,data analysis used CT analysis, Sn RNA U6 as Internal reference. quantitative: Cycle threshold method Relative quantitative. A formula to calculate:the ratio=2-??ct=2-[?Ct( samples)-?Ct(standard)], ?ct=ct(target gene)-Ct(Internal reference).3.The mi R-3195 mimic and mi R-3195 inhibitor were transfected into laryngeal cancer Hep2 cell lines. The experiment was divided into three groups:mi R-3195 mimic group or mi R-3195-inhibitor group,negative control group(NC), blank group.we observed the effect of overexpression or inhibition of mi R-3195 on Hep2 cell proliferation by the MTT assay and colony formation experiments.4.The mi R-3195 mimic was transfected into laryngeal cancer Hep2 cell lines. The experiment was divided into three groups:mi R-3195 mimic group,negative control group(NC), blank group. we observed the effect of overexpression of mi R-3195 on Hep2 cell apoptosis and cell cycle by flow cytometry assay.[Results]The identification result of total RNA quality showed that the samples were high-quality and suitable for microarray experiments. Screened out fourty-two laryngeal carcinoma related differentially expression mi RNAs in the result of microarray chip by relevant Softwares analysis.mi R-21-5p,Mi R-3651,mi R-222-3P,mi R-1246,mi R-181 d,mi R-193b-3P 22 mi RNAs were up-regulated in four Laryngeal carcinoma tissue samples(Foldchange>2,P<0.05).Mi R-99a-5P,mi R-195-5P,mi R-3195,mi R-101-3P,mi R-126-3P 20 mi RNAs were down-regulated in four Laryngeal carcinoma tissue samples(Foldchange<0.5,P<0.05).2.The result of Real- time PCR confirmed that mi R-99-a-5p, mi R-195-5p, mi R-3195,mi R-101-3p,mi R-1 26-3p were down-regulated in four laryngeal carcinoma tissue samples.It was consistent with the result of microarray chip. It revealed that the result of microarray chip was credible and reliable.mi R-3195 was significantly down-regulated in twenty-eight Laryngeal carcinoma tissue samples and it was statistically significant(P<0.05).3.The result of MTT showed that:the living cells number was significantly less in mi R-3195 mimic group than the control group(P<0.05),and the proliferation ability of Hep2 cell was significantly inhibited; On the contrary,the living cells numer was significantly more in mi R-3195 inhibitor group than the control group(P<0.05),and the proliferation ability of Hep2 cell was significantly incresed.4.Colony formation assay revealed that: the colony formation number was significantly less in mi R-3195 mimic group than the control group(P<0.05),and the proliferation ability of Hep2 cell was significantly inhibited;On the contrary,the colony formation number was significantly more in mi R-3195 inhibitor group than control group(P<0.05),and the proliferation ability of Hep2 cell was significantly increased.5.Flow cytometry showed that: the apoptosis rate of Hep2 cell in mi R-3195 mimic group was significantly higher than the control group(P<0.05). the G1 phase cells percentage in mi R-3195 mimic group were signficantly hiher than the control group(P<0.05),The laryngeal carcinoma Hep2 cell arrested in G1 phase, the G1 phase cells increased,G2 phase and S phase cells decresed,lead to the proliferation of Hep2 cells was restrained.The overexpression of mi R-3195 promoted Hep2 cell apoptosis and suppressed its proliferation.[Conclusions]1.We firstly discover mi R-3195 was significantly down-regulated in laryngeal carcinoma tissue.2.Mi R-3195 take part in the regulation of the laryngeal carcinoma by suppress laryngeal carcinoma Hep2 cell proliferation and cell cycle progression,promot Hep2 cell apoptosis. |
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