| BackgroundPrimary liver cancer is a malignant tumor derived from epithelial tissue of liver, which is the world’s fifth most common cancer and the third most common cause of cancer death in the world each year, and there are about 630,000 new cases of liver cancer and more than half of new cases in China [1], at present, surgical resection and liver transplantation are still the most effective treatments of the early stage of liver cancer patients, in spite of this, the surgical site recurrence rate is as high as 70% within five years[2], and tumor invasion and metastasisis is the main cause of poor prognosis. Strict surgical indications, liver donor restrictions and high costs caused by liver transplantation is only applicable to a small number of patients. In recent years, with the development of imaging and high-throughput techniques, high resolution, long non coding RNA(lnc RNA) is closely related to the occurrence and development mechanism of the disease,especially the regulating effect on tumor biological behavior such as cell proliferation,metastasis,apoptosis and so on [3-5].lnc RNAs widely exist in the nucleus and cytoplasm of eukaryotes, the general length is 200bp-100 kb. The lack of an open reading frame, not coding protein, at first even considered as "transcriptional noise" [6-8]. In recent years, research on lnc RNAgradual progress, there is increasing evidence that is closely related to the occurrence and development of cancer.In the previous experiment, we used gene chip technology to screen from hepatocellular carcinoma a gene named UC00 lkfo, whose length is 2043 bp. The coding region of UC00 lkfo and gene encoding part named ACTA2 of alpha smooth muscle actin overlap, and there is a complementary sequence, using genome browser and RNAplex predicted α-SMA as the target protein of UC001 kfo. Further studies showed that the expression of UC001 kfo and α-SMA in hepatocellular carcinoma tissues increased significantly, and the expression in different invasion of hepatocellular carcinoma cells in different quantity increased significantly:The expression of UC00 lkfo was significantly higher in low differentiation group and hepatocellular carcinoma with portal vein metastasis. On the basis of human hepatocellular carcinoma cell model, using RT-PCR, in situ hybridization, immunohistochemistry and cell invasion experiments to further demonstrated UC001 kfo has a regulatory function, it is possible to promote the invasion and metastasis of liver cancer cells. In conclusion, previous results showed that the target gene of UC001 kfo is ACTA2, the target protein is α-SMA, UC001 kfo related to invasion and metastasis of HCC. In order to further analyze the interaction mechanism between UC001 kfo and α-SMA expression, we have used si RNA interference technology to reduce UC001 kfo, RT-PCR method to detect the expression of α-SMA m RNA, using Western method to detect the α-SMA protein expression, both significantly correlated. Conclution, after down-regulation of UC001 kfo expression, α-SMA reduced, was verified from the m RNA and protein levels separately [9]. α-SMA is an actin, which exists in both monomeric and multimeric forms. Theα-SMA is an actin, which exists in both monomeric and multimeric forms. The actinmonomer is globular actin. Actin polymerization of actin filaments that form fibrous actin. Actin can express at least 6 isomeric forms, according to the different isoelectric point, which can be divided into alpha, beta, gamma is divided into three categories, α distributs in a variety of muscle cells, and α-SMA can maintain cell morphology, participate in the forming of microfilament structure of eukaryotic cells, which is an important part of the cytoskeleton and the shrinkage component.Polymerization and depolymerization of actin can remodeling involved in cytoskeleton, cell movement. Therefore, α-SMA is the base of regulating cell movement[10].In addition, a-SMA is an important cellular components of carcinomaassociated fibroblasts(CAF), with the hepatocellular carcinoma cells paracrine mechanism, paired peritumoral tissue fibroblasts(PTF) begin to express a-SMA and turn to CAF, as a result, PTF started the migration and invasion[11]. The research shows that the expression of a-SMA, E-cadherin and vimentin can also promote epithelial-mesenchymal transition(EMT) [12], and through specific procedures epithelial cells will be transformed into mesenchymal cells, such as the cells lose their polarity, to obtain a higher ability to degrade extracellular matrix, etc, EMT mainly involved in the development of growth, wound healing, cancer metastasis, and a variety of fibrotic disease process [13], Decreased expression of cell adhesion molecules, increased expression of Vimentin and a-SMA, With the morphological characteristics of mesenchymal cells[14-15], is an important biological process for migration of malignant tumor cells derived from the epithelial cells[16-17]. In short, a-SMA bring to cytoskeletal remodeling, composition of microfilament and the formation of EMT, which affect cell migration, a-SMA is the key link of strengthening the cell movement capability, play an important role in the process of metastasis of hepatocellular carcinoma. In recent years, the study researchers made great progress in nuclear skeleton, which has become a new growth point in cytology.The nuclear cytoskeleton is relative to the cytoplasmic cytoskeleton, both of which constitute the cytoskeleton assembly. RNA replication, nuclear matrix and DNA transcription and processing of chromosome, is closely related to the. In the nucleus has been shown that the presence of actin, probably one of the reasons is the cytoplasmic actin translocated to the nucleus, while actin play the function of dynamic behavior of the cytoskeleton in the emerging as everyone knows, but the role of research in the new emphasis on actin. Some studies have found thatactivity in the function of actin in the nucleus. As one of the components of nuclear actin, it has its own rules of structure and function, such as something relative to nuclear matrix, chromatin remodeling, transcription and processing of m RNA[18].This study adopts the over expression and siRNA interference to change theexpression of UC001 kfo to remodel the cytoskeleton, promote the metastasis ofhepatocellular carcinoma cells by regulating a-SMA; study the regulation effects ofUC001 kfo on proliferation and apoptosis of hepatocellular carcinoma cells.Purpose1.Using lncRNA overexpression and siRNA interference technique to change the expression of UC001 kfo, then UC001 kfo remodel the cytoskeletoncytoskeleton morphology by regulating a-SMA, finally to investigate the effect of the gene on liver cancer cell invasion and metastasis.2.TO discusse the regulation effect of UC001 kfo on proliferation of liver cancer cell.3.To investigate the regulation effect of UC001 kfo on apoptosis of liver cancer cel.MethodHepatocellular carcinoma cell line Hep G2 cells were used as a model, in culture box 37 5% CO2 sterilization culture, timely passage; up℃-regulated experiment was divided into three groups: p CDNA3.1-UC001 kfo group(experimental group) and pc DNA3.1 group(negative control group) and Hep G2 group(blank group); downregulated experiment was divided into three groups: UC001kfo-si RNA group(experimental group), si RNA group(negative control group) and Hep G2 group(control group), three holes for each group; p CDNA3.1-UC001 kfo carrier in previous experiments had been constructed, using liposome transfection technique(refer to Lipofectamine 2000 transfection reagent instructions).Take photos of Fluorescence to detect transfection of NC-siRNA-FAM; RT-q PCR was used to detect the expression of UC001 kfo and a-SMA; immunohisto chemistry was used to detected the change of cytoskeleton; Transwell chamber experiments was used to detecte cell invasion, cell scratch assay was used to detect cell migration; MTT and cell clonogenic assay were used to detect cell proliferation; flow cytometry was used to detect cell apoptosis.Statistical methods Using SPSS18.0 statistical software to analyze, measurement data were expressed as mean±standard deviation( x ±s), two groups of quantitative data were compared with t-test. α = 0.05 as inspection standard.Result1 RT-q PCR was used to detect the expression of UC001 kfo and a-SMA(1) UC001kfo: after 48 h UC001kfo-pCDNA group, pCDNA group, HepG2 group, UC001kfo-si RNA group, NC-si RNA group were 1924.14±238.69, 1.87±0.43, 1.00±0.29,0.01±0.00,0.85±0.25 respectively.(2)α-SMA: after 48 h UC001kfo-p CDNA group, p CDNA group, Hep G2 group, UC001kfo-si RNA group, NC-si RNA group were 6.81±1.39, 0.82±0.14, 1.00±0.54, 0.59±0.04, 1.47±0.15 respectively. The results of t-test: up-regulated test(1) UC001kfo: after 48 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05).(2)α-SMA: there was statistical difference between UC001 kfop CDNA group and control groups(negative control group, blank group)(Both P<0.05). after 48 h. Down-regulated experiments:(1) UC001kfo: there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05).(2)α-SMA: there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05).2 IA values48h UC001kfo-p CDNA group, p CDNA group, Hep G2 group, UC001kfo-si RNA group,NC-si RNA group were 972.53±59.10, 623.11±57.66,697.98±51.29, 399.03± 64.47,633.94±44.44 respectively. T-test results: up-regulated test: there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01); Down-regulated test: there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P <0.01).3 Transwell chamber assay was used to detect cell invasionAfter 48 h UC001kfo-p CDNA group, p CDNA group, Hep G2 group, UC001 kfosi RNA group NC-si RNA group were 213±16.17, 96±4.58, 128±3.21, 28±9.64, 80±4.04 respectively; after 96 h UC001kfo-p CDNA group, p CDNA group, Hep G2 group,UC001kfo-si RNA group,NC-si RNA group was 201±19.09,97±5.69, 116±11.53, 40±3.79, 72±10.21 respectively. T-test results: up-regulated test: after 48 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01); after 96 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01). Down-regulated test: after 48 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01); after 96 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01).4 Cell migration assay was used to detect cell migration48h after transfection:24h from scratch:UC001kfo-pCDNA group,pCDNA group, Hep G2 group, UC001kfo-si RNA group, NC-si RNA group were 32.56±5.56, 31.05±7.12, 35.77±7.28, 25.72±5.13, 32.59±3.74 respectively; 48 h from scratch: UC001kfo-p CDNA group, p CDNA group, Hep G2 group, UC001kfo-si RNA group, NC-si RNA group was 63.57±5.58, 63.41±6.04, 56.77±7.54, 40.76±4.74, 54.76±0.57 respectively;From scratch 72h: UC001kfo-p CDNA group, p CDNA group, Hep G2 group, UC001kfo-si RNA group, NC-si RNA group was 98.34±2.88, 87.90±0.83, 92.43±2.19, 60.98±1.41, 80.34±1.05 respectively. T-test results: up-regulated test: 48 h after transfection: 24 h from scratch there was no statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P>0.05); from scratch 48 h there was no statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P>0.05); 72 h from scratch: there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Botn P<0.05). Down-regulated test: 24 h from scratch: there was no statisticaldifference between UC001kfo-pCDNA group and control groups(negative control group, blank group)(Both P>0.05); from scratch 48h:there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Boyh P<0.05).; from scratch 72h:there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05).5 MTT and colony formation assay were used to detect cell proliferation1.MTT show that 24 h after transfection, p CDNA3.1-UC001 kfo group, Hep G2 group, pc DNA3.1 group, UC001kfo-si RNA group, NC-si RNA group were 0.72±0.02, 0.67±0.04,0.71±0.02, 0.56±0.04,0.7±0.02 respectively;after 48 h p CDNA3.1- UC001 kfo group, Hep G2 group, pc DNA3.1 group, UC001kfo-si RNA group, NC-si RNA group were 1.22± 0.07, 1.03±0.03, 1.09±0.04, 0.8±0.03, 0.97±0.07 respectively; after 96 h p CDNA3.1-UC001 kfo group, Hep G2 group, pc DNA3.1 group, UC001 kfosi RNA group, NC-si RNA group were 1.63±0.07, 1.39±0.07, 1.47 ±0.03, 1.05±0.04, 1.38±0.02. T-test results: up-regulated test: 24 h :there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05); after 48h: there was statistical difference between UC001 kfop CDNA group and p CDNA group(P <0.05), there was significant statistical difference between UC001kfo-p CDNA group and Hep G2 group(P<0.01); after 96 h there was statistical difference between UC001kfo-p CDNA group and p CDNA group(P<0.05); there was significant statistical difference between UC001kfo-p CDNA group and Hep G2 group(P<0.01); Down-regulated test: 24 h there was no statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P>0.05); 48 h there was statistical difference between UC001kfo-p CDNA group and p CDNA group(P <0.05), there was significant statistical difference between UC001kfo-p CDNA group and Hep G2 group(P<0.01); 96 h there was significant statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01).2.Colony formation assay showed that: clone colony formation rate at 48h(%) pc DNA-UC001 kfo group, Hep G2 group, pc DNAgroup, UC001kfo-si RNA group,NC-si RNA group were 34.94±4.24, 25.73±2.32, 26.80±1.91, 13.20±0.92, 24.40±2.31 respectively; 96 h p CDNA3.1-UC001 kfo group, Hep G2 group, pc DNA3.1 group, UC001kfo-si RNA group, NC-si RNA group was 40.93± 4.41, 30.40 ±5.14, 25.80±3.67, 13.60±1.83, 22.73 ±1.62 respectively. T-test results: up-regulated test: afer 48 h there was statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.05). After 96 h there was significant statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01); Down-regulated test: after 48 h there was significant statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01); after 96 h there was significant statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P<0.01)).6 Flow cytometry was used to detect apoptosisFlow cytometry showed that p CDNA-UC001 kfo group, p CDNA group, UC001kfo-si RNA group, NC-si RNA group were 3.5± 2.36, 6.3 ±1.15, 3.7± 2.76, 3.83±2.46,3.53±2.28 respectively,96 h after transfection:Hep G2 group,p CDNA-UC001 kfo group, p CDNA group, UC001kfo-si RNA group, NC-si RNA group were 3.12±0.58,4.97±1.16, 4.03±1.04, 2.3±0.56, 3.6±1.78 respectively. T-test results: up-regulated test: 48 h, 96 h there was no statistical difference between UC001 kfop CDNA group and control groups(negative control group, blank group)(Both P>0.05); Down-regulated test: 48 h, 96 h there was no statistical difference between UC001kfo-p CDNA group and control groups(negative control group, blank group)(Both P>0.05)..Conclusion1.UC001 kfo can increase formation of cancer cells’ pseudopod and cytoskeletal proteins to promote invasion and metastasis, and the occurrence of epithelial-mesenchymal transition.2.UC001 kfo can promote the proliferation of liver cancer cells.3.UC001 kfo was not involved in the regulation of apoptosis of liver cancer cells. |
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