| Background: High postoperative recurrence rate is the main factors affecting theprognosis of patients with liver cancer. How to effectively predict and assess the risk ofliver cancer patients with postoperative recurrence and metastasis and effectively interveneis the problem needed to be settled in the field of liver cancer research. The screeningresearch of current risk factors based on the characteristics of tumor biology has made a lotof progress. But in the field of molecular biology, traditional screening researches based ontumor signal transduction pathway of a single channel or target are inefficient anduncertain. In recent years, taking advantage of the chip technology to high-throughputscreen related mRNA, protein and miRNAs, also mapping the molecular of recurrence andmetastasis has brought the dawn for the field. The previous research by gene chip analysisdemonstrated that in terms of the level of gene SEC62in peripheral blood mononuclearcells (PBMC) and its mRNA and protein in liver cancer, one-year recurrence group washigher than three-year non-recurrence group. And SEC62may be an independent riskfactor affecting the postoperative recurrence of liver cancer. Sec62located on theendoplasmic reticulum is a transmembrane protein that acts on the ribosome andcoordinatingly translocates proteins to the endoplasmic reticulum for folding processing.Also, Sec62can affect the endoplasmic reticulum stress (ERS), and ERS is closely relatedwith oncogenesis. We assume whether Sec62becomes one of the key factors that affect theearly postoperative recurrence of liver cancer? At the same time, our research group hasthe research foundation and accumulated rich experience in the prevention of postoperativemetastasis of liver cancer with Chinese medicine. The preliminary work, perspectivemulticenter clinical study has witnessed that Chinese medicine Cinobufacini injectioncould reduce the postoperative recurrence of liver cancer, and through the experimentalstudy we confirm that bufalin which is the main effective component can promote theapoptosis of tumor cells in vitro, but specific targets remain to be further discussed.Therefore, regulating Sec62whether the bufalin inducing the apoptosis of tumor cells isone of the targets of prevention and treatment of hepatocellular carcinoma recurrence? Thisis the second question we want to argue with.Objective:1.Through the experiment in vitro, observe Sec62effects on biologicalcharacteristics, such as proliferation and apoptosis, of the liver cancer cells in adherent andanoikis state.2. Study whether Sec62could adjust ERS inducing apoptosis of liver cancercells.3. Explore whether bufalin could promote the apoptosis of hepatocelluar carcinoma cells by regulating Sec62.Methods:1. Screen the one with high expression of Sec62in these cell lines whichare MHCC-97H, MHCC-97L, MHCC-LM3, HepG2, Huh7, Hep3B through RT-PCR andWestern Blot.2. Screen the most effective Sec62-shRNA and construct lentivirus with this shRNA.Infect the selected cell line with the lentivirus to build a new cell line with Sec62stablysilenced.3. With CCK-8, we detect the proliferation of cell lines of adherent or anoikismodels and treated with or without thapsigargin inducing ERS to observe effects ofsilenced Sec62on the proliferation.4. Through plate clone formation assay and soft agar formation assay, it can be seenthat the effect of silenced Sec62on the malignance and anchor independent growth ofliver cancer cell lines.5. Utilizing flow cytometry technique, we detect the silenced Sec62on the apoptosisof cell lines in anoikis state with or without thapsigargin inducing ERS.6. By western blot, we detect the ERS related proteins levels which are IRE1alpha,PERK, Bip and CHOP and apoptosis related proteins levels which are P-JNK, JNK, Bim,Bcl-2, Caspase3, Caspase9and Caspase12. Preliminarily discuss whether silencing Sec62induces apoptosis by adjusting the ERS.7. We utilize western blot to detect the effects of bufalin on Sec62protein expression.And after the Sec62highly expressed and the silenced liver cell line treated with bufalin,we utilize flow cytometry technique to detect the effects of bufalin on the apoptosis.Results:1.The selected liver cancer cell lines with highly expressed Sec62are Huh7,HepG2and Hep3B. And Sec62protein level of Huh7is the highest one.2. Successfully get the effective shRNA sequence and construct the lentivirus needed.After infection and screening, we get the stably silenced cell line calledHuh7/LV-SEC62-shRNAi and negative control called Huh7/LV-SEC62-NC.3. In the adherent state, there is no significant difference between inhibition rates ofHuh7/LV-SEC62-shRNAi group and Huh7/LV-SEC62-NC group at the same time pointtreated with or without thapsigargin (P>0.05, P>0.05). We use the plates pretreated withpolyHEMA to establish the anoikis model in vitro. There is no significant difference ofinhibition rate of proliferation between silenced Sec62and control group (P>0.05). Butsilencing Sec62predominantly inhibits proliferation when treated with thapsigargin after 48h and72h. The inhibition rate of Huh7/LV-SEC62-shRNAi group is significantlyhigher than Huh7/LV-SEC62-NC group’s (P<0.05).4. For Huh7/LV-SEC62-shRNAi group, the number of soft agar clone is less thanHuh7/LV-SEC62-NC group and wild type Huh7group (P<0.05). And there is nosignificant difference between Huh7/LV-SEC62-NC group and wild type Huh7group(P>0.05). For Huh7/LV-SEC62-shRNAi group, plate clone number is also less thanHuh7/LV-SEC62-NC group and wild type Huh7group (P<0.05). And there is nosignificant difference between Huh7/LV-SEC62-NC group and wild type Huh7group(P>0.05).5. After establishing the anoikis model, using flow cytometry technique to detect theapoptosis of wild type Huh7group, Huh7/LV-SEC62-shRNAi group andHuh7/LV-SEC62-shRNAi group. The apoptosis rates of them are7.09%,8.21%and16.18%while treated with thapsigargin they are7.42%,10.08%and24.37%.6. Comparing Huh7/LV-SEC62-shRNAi group with Huh7/LV-SEC62-NC groupafter treated with thapsigargin, we find that its protein levels of ERS related proteinincluding IRE1α, PERK, Bip and CHOP are upregulated. And in the anoikis model,silencing Sec62can activate IRE1α and enhance the expression of P-JNK. Upregulatedownstream pro-apoptosis proteins Bim and downregulate anti-apoptosis protein Bcl-2and simultaneously activate Caspase12, Caspase9, Caspase3.7. With western Blot, Bufalin can downregulate the wild type Huh7’s Sec62proteinlevel and is time-dependent. After establishing the anoikis model, using flow cytometrytechnique to detect the apoptosis of wild type Huh7group, wild type Huh7group withBufalin, Huh7/LV-SEC62-shRNAi group with Bufalin. The apoptosis rates of them are12.06%,15.18%and33.28%.Conclusion:1. Silencing Sec62can improve the sensitivity of anoikis of livercancer cell Huh7. The mechanism may be through the route that regulating ERS andactivating IRE1α and phosphosphorylating JNK subsequently to activate Caspase12andin the end inducing apoptosis.2. Bufalin may weaken protective effect of Sec62on thetumor cells and increase the rate of anoikis by reducing Sec62protein level. |
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