| Background:Hepatocellular carcinoma (HCC) is one of the malignant tumors of highmalignancy and poor prognosis. Annually, approximately0.5to1million people diedfrom liver cancer. Resection is universally recognized as the best approach to cureliver cancer, but the total5-year survival rate after the surgery is low with only34.6%in terms of large hepatocellular carcinoma and62.9%in terms of small hepatocellularcarcinoma. μ-opioid receptor (MOR) is the main member of the opioid receptorsuper-family that is extensively distributed in the body. It is found in studies thatMOR activation promotes the proliferation of many kinds of cells and facilitates thegrowth of some malignant tumors such as lung cancer, breast cancer, neuroblastomaand colon cancer. This means MOR plays an important role in the proliferation anddifferentiation of various kinds of cells. MOR is found to have apparently higherexpression in HCC cells than in the normal liver cells. However, the role that MORplays in HCC progress has not been reported. In addition, little is known about themechanism by which MOR promotes tumor growth.Objective:It was found in our previous studies that MOR was expressed in the tissue andcells of human liver cancer The purpose of this study was to investigate the impact ofdownregulating MOR on human liver cancer progress in vivo and in vitro and toexplore its possible molecular mechanism.Methods:1. RT-PCR and western blotting were employed to analyze the expression ofMOR mRNA and protein in human hepatoma carcinoma cells HepG2, BEL7402and normal liver cell line LO2. Sixty cases of HCC tissue excised in the surgery of FirstHospital of Jilin University from2009to2010were selected for the experiment whichhad all been pathologically confirmed to be HCC and the patients had not receivedany treatment prior to the surgery. At the same time,20cases of normal liver tissuesfrom liver trauma and hepatolithusnext were selected for comparative study. Thetissues were sliced for embedding paraffin, immunohistochemistry was used toanalysis MOR expression in human hepatoma carcinoma tissues and normal livertissues.2. After being cultured for the time HepG2cells initiated logarithmic growth,MOR siRNA and negative control were used to transfect siRNA interfering efficiencywas measured at mRNA and protein level by RT-PCR and Western blot, respectively;In order to verify the impact of downregulating MOR on human hepatoma carcinomacell growth, Naloxone of various concentrations and MOR siRNA of variousconcentrations were administered to treat human hepatoma carcinoma HepG2cellsfor24,48and72h before MTT analysis on cell viability; In order to explore whetherdownregulating MOR results in apoptosis of human hepatoma carcinoma cells, weused Hoechst33342stain and flow cytometry to observe apoptosis of humanhepatoma carcinoma cells, also, we used flow cytometry to analyze the cell cycle tostudy whether downregulating MOR has any impact on the progress of the cycle ofhuman hepatoma carcinoma cells; In order to verify the molecular mechanism fordownregulating MOR to inhibit the growth of human hepatoma carcinoma cells, weused RT-PCR and Western blot, respectively, to test the MKK7mRNA and MKK7proteins and their phosphorylation levels. In order to demonstrate whether MKK7plays a key role in the inhibition of the growth of human hepatoma carcinoma cellsvia downregulation of MOR, we used RNA interference to silence MKK7expressionand observed the growth of human hepatoma carcinoma cell.3. We used the nude mouse model to establish the in vitro tumors. The animalswere vaccinated with the cells of control group and MOR siRNA group, respectively.The growth of the nude mice was observed every day, and after four weeks the mice were sacrificed. The subcutaneous transplanted tumor tissue was cut, under asepticcondition, for index analysis.Results:1. RT-PCR and western blot were employed by us to analyze the expression ofMOR mRNA and protein in human hepatoma carcinoma cells. It was found that therewas high expression of MOR mRNA in HepG2cells and BEL7402cells. Theexpression of MOR protein in HepG2cells and BEL7402cells was similar to that ofmRNA. Moreover, it was found by the immunohistochemical analysis that MORexpression in human hepatoma carcinoma tissues was obviously higher than that innormal human liver tissues.2. We used RNA interference technology to silence MOR expression, we foundthat45nM of MOR siRNA transfection for24h could significantly reduce theexpression level of MOR mRNA, and the expression level of MOR protein declinedaccordingly. This efficiency can endure at least for72h. This indicates that MORsiRNA transfection could effectively silence the MOR gene and affect the expressionof MOR expression in turn; Naloxone of various concentrations and MOR siRNA ofvarious concentrations were administered to treat human hepatoma carcinoma HepG2cells，as indicated in the results, with the increase of the concentrations of Naloxoneand MOR siRNA, A490value of HepG2cells reduced. Such a result means thatdownregulating MOR can inhibit the growth of human hepatoma carcinoma cells; weused Hoechst33342stain and flow cytometry to observe apoptosis of humanhepatoma carcinoma cells. The results indicated that after the administration ofNaloxone and MOR siRNA to treat human hepatoma carcinoma HepG2cells, thecell’s chromatin concentrated with the brightness obviously higher than the normalcontrol group. The apoptosis rate was18.36%and22.99%, respectively, which areboth higher than the6.6%of the control group. This indicates that downregulation ofMOR can promote apoptosis of human hepatoma carcinoma cell to inhibit the growthof human hepatoma carcinoma cells in turn; In order to verify the molecularmechanism for downregulating MOR to inhibit the growth of human hepatomacarcinoma cells. We found that after MOR was downregulated, the total protein expression level of MKK7mRNA and MKK7was not obviously changed, while theMKK7phosphorylation level was significantly increased and was higher than that ofthe control group. Moreover, we found JNK and its phosphorylation level alsoincreased drastically. These results show that downregulating MOR can increase theexpression level of phosphorylation MKK7, which results in JNK and itsphosphorylation level. This may be its molecular mechanism to inhibit the growth ofhepatoma carcinoma cells. In order to demonstrate whether MKK7plays a key role inthe inhibition of the growth of human hepatoma carcinoma cells via downregulationof MOR, we used RNA interference to silence MKK7expression and observed thegrowth of human hepatoma carcinoma cell. The results showed that after MKK7wassilenced, A490nm value of the hepatoma carcinoma cells was apparently on theincrease as compared to that in the group with downregulating MOR. At the sametime, JNK and its phosphorylation level dropped significantly.3. We used the nude mouse model to establish the in vitro tumors. The five nudemice of the normal control group all had tumors with the tumor formation rate of100%. Four nude mice in the MOR siRNA group had tumors with the tumorformation rate of80%. In addition, the tumors of the nude mice in the control groupgrew faster than those in the MOR siRNA group.Conclusion:1. MOR was highly expressed in the tissue and cells of human liver cancer.2. Opioid receptor antagonist naloxone and MOR siRNA transfection couldeffectively downregulate the expression of MOR. Downregulation of MOR couldpromote apoptosis of human hepatoma carcinoma cell, block the cell cycle in G0/G1stage and activate JNK signal transduction pathway via regulating MKK7phosphorylation level.3. The hepatoma carcinoma cell after downregulation of MOR could decline thetumor formation rate, inhibit the proliferation of hepatoma carcinoma cells and causetumor growth retardation. The molecular mechanism need to be explored. |
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