| 1.1Background and Objective URG11 proved to exist in liver cancer, gastric cancer, thyroid cancer, bladder cancer,,it is also a proliferation factor, it plays a important role in the regulation of cell growth,a dhesion, migration, lymphocyte outcome, and signaling metastasis. The Studies have proved that the expression of URG11 in gastric cancer cell which is transfected si RNA is less and the proliferation and clone formation ability is weaken, it prevent the cell from Glperiod to enter Sphase,the ability of adhesion and invasion;metastasis is weaken, as well as the inhibition of tumor and metastasis ability in nude mouse transplantation tumor experiment is weaken, it had proved that the expression level of URG11 in prostate cancer tissues is higher than in benign prostate hyperplasia tissues, comparing with other prostate cancer cells,the expression of URG11 is the highest in LNCa P, This study wants t o prove the characteristics expression of URG11 and the inhibition of URG11 si RNA in URG11 by the method of MTS/CCK-8;FCM; scratch detection;Transwell;Real-time-PCR and Western blot in prostate cancer cells(LNCa P cell line), it further confirms that the characteristics of URG11 in prostate cancer cell and the effectiveness of gene targeting treatment, it is a new method for the treatment of prostate cancer gene therapy and theoretical basis.1.2Method1.Effects of silent URG11 gene on prostate cells biological characteristics1.Cell culture, the design of sequence fragment URG11 si RNA jamming and anti password chain(provided by the sigma company), screening the most appropriate URG11 si RNA by cell transfection technique and inhibition for concentration.URG11 si RNA interference fragment(3pairs) was designed, the carrier of URG11 si RNA plasmid was builded,it will virus vector plasmid mixed with LNCa P cell for cultivating, so that,the LNCa P cell was infected with plasmid, The method of Real-time-PCR and Western blot were used for carrier interference in suitable and effective concentration.2. Stable silent endogenous URG11 prostate cancer cells with URG11 siRNA interference fragment was builded.LNCa P cell was infected by the specificity interference fragment of URG11 si RNA,Comparison with empty plasmid, the method of Real-time-PCR and Western blot were used for to verify the expression of the silent LNCa P cell.3. The biological characteristics of the silent LNCa P cell.MTS/CCK 8 method was used to detect cell proliferation cells and growth cur ve; FCM was used to detect the growth cycle and apoptosis of the silent LNCa P cell; A fter the vitro invasion and athletic ability of the silent LNCa P cell was by migration experiment(scratch detection); Transwell was uaed in detection of the migration and invasi on ability of the silent LNCa P cell.1.4Result1. Screening of URG11 siRNA specificity interference fragment Providing with interference sequence fragment and password chain(SASI-Hs01-00182412,SASI-Hs01-00182413,SASI-Hs01-0018241) and blank UUCUCCGAACGUGUCACGUTT form sigma company, the LNCa P cell was transfected with it, Real- time PCR, Western blot and RNA Structure analysis software were used to the stable SASI-Hs01-00182413 fragments interference, the most stable; efficientt; effective concentration is 50 nm/ul.2.The stable and silent endogenous LNCa P prostate cancer cells was builded.The effective interference fragment SASI-Hs01-00182413 was used to construct infection LNCa P cells, FCMwas used to identify positive cells after separation interference; RT-qPCR and Western blot detection silence after prostate cancer cells LNCa P, RT-q PCR technology was developed for the determination of silence after the LNCa P prostate cancer cells compared with control group suppression inhibitory rate 78.72%, Western blot test group compared with control group(95.1285/1993.015), OD value through the above experiment confirmed the silence endogenous LNCa P successfully established prostate cancer cells.3. MTS/CCK 8 method was used to detect LNCa P cell proliferation after silent The MTS /CCK 8 method was used to detect LNCa P cell proliferation after silent,compared with control group, experimental group growth inhibition rate day1(7.15%); Day2(8.75%); Day3(10.75%)(P < 0.01), by comparison, confirmed by URG11 si RNA specificity interference fragment after silent,proliferation URG11 LNCa P cell proliferation is suppressed.4. FCM was used to analyse the the cell cycle and apoptosis of LNCa P cell after silent.FCM was used to analyse the the cell cycle and apoptosisof LNCa P cell after silent.,compared with control group, experimental group stay in S phase were 25.49 ± 0.463(48 h) and24.89 ±0.437(72 h), the difference was statistically significant(P < 0.00); apoptosis index of LNCa P cell was(8.48 + 0.436) %(24h), respectively(48 h) and(8.83 + 0.382) %(72 h), the difference was statistically significant(P < 0.05), by flow cytometry instrument analysis data,it can be confirmed that URG11 si RNA specificity interference fragment is useful,so that URG11 LNCa P cell growth is restrained.5. Transwell experiment and migration experiment By Transwell experiment of silent LNCa P cells, compared with control group, experi mental group LNCa P cell was 142.83 ± 10.91(mean + sd), and a control group of 234± 8.29(mean + sd.), silent LNCa P cell invasion ability decreased 2 times, the difference was statistically significant(t = 4.009, P < 0.05), migration experiment show that the experimental group the movement of LNCa P cell migration rate in 6 h(0.82%); 24 h(3.56%); 48 h(7.57%) and control group was 6 h(2.06%); 24 h(14.77%); After 48 h(25.75%), silent LNCa P cell invasion metastasis ability reduced three times, the difference was statistically significant(t = 4.437, P < 0.05).1.3Conclusions1. The growth;Invasive ability and transfer ability of silent LNCa P cells is weaken, the apoptosis of it obviously increases, it proves that URG11 si RNA interference fragment is useful.URG11 is the promoting genes of prostate cancer cell in proliferation, invasion and metastasis. |
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