The Influences Of Tmub1 RNAi On The IL-6 Induced STAT3 Expression And Activation

Background and objective: As the most important cytokine of stimulating hepatocyte proliferation, IL-6 can trigger or regulate the process of liver regeneration through JAK/STAT3 (Signal transducer and activator of transcription-3) signal pathway. In the regeneration process of injury or lobectomy of liver, IL-6 expression increases and activates STAT3, namely the phosphorylated STAT3 (pSTAT3) can enter the nucleus and induce immediate early gene expression rapidly. Thus, gene expression related to inflammation, cell proliferation and acute phase reactions can be regulated and hepatocyte proliferation and liver regeneration can be promoted. Tmub1 (transmemebrane and ubiquitin-like domain containing 1) gene was first reported in 2005 by Della Fazia etc. in the wake of analyzing hepatocyte's cDNA library after hepatectomy and compared with hepatocyte in the control group. At that time, it was named hepatocyte odd protein shuttling (Hops). The results showed that Tmub1 mRNA in hepatocytes increased significantly after PH (partial hepatectomy); at the same time, over-expression of Tmub1 can inhibit the proliferation of H-35 rat hepatoma carcinoma cell (HCC). Moreover, cell proliferation of the NIH-3T3 cells, resulted from Tmub1 RNA interference (RNAi) through the application of appropriate retroviral vector, also can be inhibited by Tmub1 over-expression. The study also found that the combination of HOPS/Tmub1 with eEF-1A (Elongation Factor 1-A) interfere the protein synthesis. Therefore, we speculate that Tmub1 plays a very complex and important role in hepatocyte proliferation and liver regeneration process. However, if Tmub1 could transmit the signal and control the key point of liver regeneration or proliferation have not been reported yet. Demonstration of the transcriptional regulation mechanism of Tmub1 in hepatocyte proliferation will be an important discovery for the hepatocyte proliferation and liver regeneration mechanism research.In this experimental study, we have constructed the lentivirus vector of Tmub1 gene RNA interference. With the RNA interference technology, we silence the expression of Tmub1, which means that Tmub1 gene in rat hepatocyte BRL-3A can't function. Thus, we can explore the function of Tmub1 in IL-6-induced STAT3 expression and activation, and get a more profound insight into the correlation between Tmub1 and the signal transduction pathway of IL-6/JAK/STAT3, which will provide a new theoretical basis for the treatment and research of hepatic failure in the future.Method:1. With the help of RNA interference (RNAi) technology, we construct the lentivirus vector of Tmub1. 4 pairs of oligonucleotide sequences of the Tmub1 gene, coding hairpin RNA (shRNA) are designed and synthesized. They are cloned into pll3.7 plasmid after annealed, then digested by restriction enzyme and sequenced. 293T cells is transfected by the four plasmid lentivirus vector system composed of pRsv-REV, pMDlg-pRRE, pMD2G and interference plasmid (Pll3.7). Then we package the four Tmub1 interference plasmid vector and detect the efficiency of lentivirus infection in the rat hepatocyte BRL-3A with an inverted fluorescence microscope, as well as the RNA interference effect of Tmub1 expression in BRL-3A cells with Western-Blot.2. We package the plasmid with the strongest interference effect and mass-produce Tmub1 RNAi lentivirus vector LV256; Subsequently, we measure the virus titer with the help of Hela cells and flow cytometry; Finally, BRL-3A/256, stably infected cells line of Tmub1 RNAi lentivirus, will be selected with G418 antibiotic. And with the help of RT-PCR and Western Blot, we will detect the Tmub1 expression in the BRL-3A/256 cells line, in preparation for the follow-up experiment.3. In order to study the relationship between Tmub1 and IL-6 in hepatocytes, we will set 5 groups in all of the following researches: Control group, Negative group, IL-6 group, LV256 group and IL-6+LV256 group.In the Negative group, BRL-3A cells are only infected with empty vector plasmid Pll3.7; IL-6 group, BRL-3A cells will be induced by 5ng/ml recombinant rat IL-6 according to the reported documental method; LV256 group is the BRL-3A/256 stable infected cells line (Tmub1 expression is effectively suppressed); BRL-3A / 256 cells will be applicated with 5ng/ml of the rat recombinant IL-6 in the IL-6+LV256 group. RT-PCR and Western Blot techniques will be adopted to detect Tmub1 transcription and translation level of all groups.4. In order to explore the function of Tmub1 RNA interference on IL-6-induced STAT3 expression and phosphorylation further, we also adopt the method of experimental group setting. With the help of RT-PCR and Western Blot Detection, STAT3 and phosphorylated STAT3 (pSTAT3) expression changes of each group are verified for the purpose of exploring the relationship between Tmub1 and IL-6/JAK/STAT3 signaling pathways.Results:1. The four designed and synthesized shDNA templates went through annealing, lineage and connection response; and plasmid were extracted with the alkaline lysis method. What's more, we were engaged in the double enzyme digestion and sequencing for the four recombinant vectors (C0019, C0020, C0021, and C0022) respectively with Xba I and Nhe. It is confirmed that the Tmub1 shRNA oligodeoxynucleotide sequences were inserted correctly, and Tmub1 shRNA lentivirus vectors were constructed successfully.2. Packaging the lentivirus vector with the packaging system consisting of pRSV-Rev, pMDLg-pRRE, and pMD2G and interference plasmid and then infect the Tmub1 BRL-3A cells with packaged lentivirus vector. After a 72-hour's culturing, we collected cells and extracted cell protein for Western Blot test. The results showed that C0020 Sh2-Hops-256 had the best interference effects. We named the lentivirus vector containing the plasmid Tmub1 RNAi as LV256 and made a lot of packagings. With serial dilution of the diluted virus solution and the infection of Hela cells after 96 hours of FACS, we detected the cell's GFP-positive rate and the titer of the virus was 2.3×108TU/ml. With different MOI values (20,30,50,70), we tried our best to make our targeted cells to achieve the best MOI value in transfection process. After using the lentivirus infected BRL-3A with the MOI equals to 70, the transfection efficiency can be up to 90% with an inverted fluorescent microscope and the cells grew well.3. In the experiment, we used the G418 to select the stable transfected cells BRL-3A/256. In this cell line (LV256 group), Tmub1 mRNA and protein expression were significantly lower (* p <0.05) than those in Control and Negative group. So Tmub1 RNAi lentivirus infected cells BRL-3A/256 was constructed stably and successfully, which can be used for future research.4. RT-PCR and Western Blot were used to detect the expression of IL-6 on Tmub1 mRNA and protein. With the help of 5ng/ml reorganized IL-6, we can induce cell Tmub1 BRL-3A expression increase significantly (# p <0.05); in BRL-3A/256 cell line, Tmub1 expression level was significantly lower (* p <0.05), and IL-6 could stimulate and partially reverse the interference effects of RNA of the Tmub1, resulting in Tmub1of BRL-3A/256 cells expression increases (# * p <0.05).5. In the experimental study of"the influences of Tmub1 RNA interference on the expression of IL-6 induced STAT3", we found that IL-6 could significantly increase the expression level of STAT3 in BRL-3A and BRL-3A/256 cells (# p <0.05, # * p <0.05); in LV256 group, compared with the control group (* p <0.05), STAT3 expression in BRL-3A/256 cells also significantly increased. These results suggest that IL-6 and Tmub1 RNAi can make BRL-3A cells STAT3 expression increased, and the interaction between the cells can further enhance the expression of STAT3. Meanwhile, Tmub1 may, to some extent, prohibit STAT3's expression.6. With the same method, we have further examined the pSTAT3 expression level in cells. Experimental results show that in Control group, Negative group and LV256 group (no IL-6 stimulation); pSTAT3 expression in cells was very low. But, both for BRL-3A cells which stimulate IL-6 or BRL -3A/256 cells interfered by Tmub1 RNA, the pSTAT3 expression was significantly increased (# p <0.05, # * p <0.05). More noteworthy is that, with the joint effect of IL-6 and Tmub1 RNAi, the expression of pSTAT3 can further increase (# * p <0.05), but when only IL-6 was stimulated, pSTAT3 expression level was lower than that of the two roles'expression (# p <0.05). This shows that there may be some correlation between IL-6 and Tmubl, and the later may inhibit IL-6-induced STAT3 activation in the cells.Conclusion:1. Through successfully constructing the eukaryotic expression of the lentivirus vector of Tmub1 and infecting with rat BRL-3A of hepatocyte, or building Tmub1 RNAi lentivirus infection stable cell line, we can interfere with the expression of Tmub1 mRNA and protein in hepatocytes effectively.2. IL-6 can induce Tmub1expressiong in hepatic BRL-3A cells increase; it can also partially reverse the expression of inhibitory effect of Tmub1 after the Tmub1 RNA being interfered.3. IL-6 can induce STAT3 expression in BRL-3A hepatocyte of rat increase and STAT3's expression of Tmub1 RNAi lentivirus infected cells in BRL-3A/256 was also significantly increased. While with the joint function of the two, STAT3 expression was further enhanced, which means that Tmub1 can inhibit STAT3 expression.4. In the experimental group without IL-6 stimulation, there wasn't any phosphorylated STAT3 (pSTAT3) expression by RT-PCR and Western Blot, or expression was very low (LV256 group). IL-6 can significantly enhance the BRL-3A and BRL-3A/256 cells'pSTAT3 expression. When IL-6 and Tmub1 RNAi jointly functioned (IL-6 + LV256), the pSTAT3 expression was stronger than that with IL-6 stimulation. It showed that Tmub1 may be involved in IL-6-induced STAT3 activation process. What's more, it prohibited the phosphorylation of STAT3 to a certain extent.5. It is safely to conclude from above that Tmub1, to some extent, inhibited IL-6 induced STAT3 expression and activation, and it plays an important regulatory role in IL-6 induced proliferation of hepatocytes to prevent excessive cell proliferation.