| [Background]Renal cell carcinoma(RCC)，comprises a histologically diverse group of solid tumors,arising from different parts of the kidney. The vast majority of RCCs are clear cell renalcell carcinomas (CCRCCs). Because renal cell carcinoma are not sensitive to radiotherapyand chemotherapy, so the treatment of renal cell carcinoma mainly depends on the surgicaloperation. Therefore, the treatment of metastatic renal cell carcinoma is difficult. Andbiological treatment of renal cell carcinoma is increasingly emphasized.The Von Hippel Lindau (VHL) gene is named for the VHL disease(Von Hippel Lindau’disease). VHL disease is an autosomal dominant familial Tumor syndrome. The human VHL gene is located in chromosome3p25-26region, a total length of l5kb, whichcontains3exons and2introns. VHL gene is a classical guardian inhibiting renal tumorinitiation. Defects in the VHL gene are the most common cause of familial CCRCC, andmore than80%of patients with sporadic CCRCC have an inactive VHL gene or loss ofVHL gene. The majority of renal cell carcinomas (RCCs) are characterized by loss offunction of the tumor suppressor gene VHL. VHL gene encodes213amino acids, whenfully translated which produce30KD protein (pVHL30). When translated from54codon,it produces19KD protein (pVHL19). Both pVHL30and pVHL19are collectivelyreferred to as pVHL. pVHL contains two domains, which are alpha domain and betadomain. Alpha domain, Cullin-2, Rbx1, NEDD, the regulatory subunits of transcriptionfactors Elongin subunit B and C (Elongin B and Elongin C) make up E3ubiquitin ligasecomplex. Beta domain targets hypoxia inducing factor alpha (HIF-alpha). Aftercombination, the E3ubiquitin ligase complexes lead to hypoxia inducing factor alphadegradation. In the absence of VHL, HIF-α protein becomes stabilized and contributes totumorigenesis. HIF-α family includes three members, which are HIF-1α，HIF-2α andHIF-3α. And HIF-1α is increasingly emphasized in the renal carcinogenesis, invasion,metastasis and drug resistance of role. Many studies have shown that there areover-expression of HIF-1α in renal cell carcinoma. HIF-1α is a nuclear protein, which hasa broad spectrum of target genes, including hypoxic adaptation, inflammationcarcinogenisis, and so on. Under normal oxygen, HIF-1α binds pVHL through itshydroxylated proline residues and is polyubiquitinated by pVHL, which ultimately leadsto the degradation of HIF-1α via the proteasome. During hypoxia, HIF-1α is nothydroxylated and escapes from pVHL-mediated degradation. The labile HIF-1α thenforms functional transcription factor by associating with the constitutively expressedHIF-1β subunit. Together, this complex binds to DNA motifs referred to as hypoxiaresponse elements to regulate the expression of a number of genes involved in cellproliferation, metastasis and angiogenesis.N-myc Downstream Regulated Gene2(NDRG2), is a member of the NDRG family, isa newly identified tumor suppressor. It has been reported that NDRG2expression is downregulated in a variety of carcinomas, including liver cancer, pancreatic cancer,meningioma and prostate cancer. Our previous study implied that NDRG2upregulated theexpressions of p53and pVHL while down-regulates the expressions of VEGF and HIF-1αin breast cancer cell lines. Recently, it is reported that NDRG2is downregulated inCCRCC tissues compared to adjacent non-neoplastic tissues. Moreover, forced expressionof NDRG2inhibits the growth of clear cell RCC (CCRCC) cell lines and induces cellapoptosis. These findings suggest that NDRG2plays an important role in carcinogenesisof CCRCC. Further exploration on the molecular mechanism of NDRG2modulation mayoffer a novel approach for treating human renal cancer.Mesenchymal stem cells derive from the mesoderm, and they are adult stem cells invarious organs. At present, MSCs can be isolated from bone marrow, fat, amniotic fluidand umbilical cord blood. With the development of the research on tumor etiology andmolecular mechanism, tumor gene therapy develops rapidly. Compared with traditionalsurgery, radiotherapy and chemotherapy, tumor gene therapy has its unique advantagesand broad prospects. But there are some questions in target gene importing and stability,so the treatment effect of gene therapy is not satisfactory. In addition, viral vectormediating gene therapy also has the risk of gene integration and gene mutation. Therefore,seeking a good targeting, stable expressing, lasting effect and little side effect on the othertissues are becoming the focus of current research.[Objective]To compare the expression and correlation of NDRG2, VHL and HIF-1α in renal clearcell carcinoma tissues and normal renal tissues. To investigate the molecular mechanismof NDRG2inhibiting proliferation and metastasis of renal cancer cells in order to providethe theoretical basis for the targeted therapy of renal carcinoma. By infection of BMSCswith adenoviral vector carrying NDRG2gene, we observe the influence of geneticmodification on the biological characteristics of BMSCs. That is to lay a foundation forfurther study of BMSCs in renal tumor targeting therapy. [Materials and methods]1The expression and correlation of NDRG2, pVHL, HIF-1α and HIF-2α in renalclear cell carcinoma tissues and normal renal tissues.1.1The immunohistochemistry staining for detecting the expression of NDRG2, pVHLHIF-1α and HIF-2α in renal clear cell carcinoma tissues and normal renal tissues1.2The results determination of immunohistochemistryThe immunohistochemistry staining was performed according to the manufacturer’sinstructions. Both the intensity and the extent of immunological staining were analyzedsemi-quantitatively. Sections with no labeling or with fewer than5%labeled cells werescored as0. Sections were scored as a1if5-25%of cells were labeled, as a2if25-50%ofcells were labeled, and as a3if50-75%of cells were labeled. Finally, labeling of≥75%ofthe cells was scored as a4. The staining intensity was scored similarly, with0used fornegative staining,1for weakly positive,2for moderately positive and3for stronglypositive. The scores for the percentage of positive cells and for the staining intensity weremultiplied to generate an immunoreactive score for each specimen. The product of thequantity and intensity scores were calculated such that a final score of0indicated noexpression,1-4indicated weak expression,5-8indicated moderate expression and9-12indicated strong expression. The photos were collected through light microscopy(Olympus, Japan).2Inhibition of renal cancer cells’ proliferation, metastasis and VEGF secretion byNDRG2regulating HIF-1α in vitro2.1Western-blot was used to analyzed the effects of Ad-NDRG2on the NDRG2expression in CaKi-1cells.2.2Proliferation ability of CaKi-1cells were tested by MTT assay.Cells were seeded (5×103cells per well) in triplicate in96-well plates. After removingthe medium,40MOI adenovirus expressing NDRG2or the negative control gene LacZ(Ad-LacZ) was added in serum-free RPMI1640, incubated for2h, replaced with freshRPMI1640supplemented with10%FBS. Plates were then incubated for12,24,36,48,60and72h. The number of viable cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and readingthe absorbance at490nm.2.3Invasion and migration ability of CaKi-1cells were tested by Transwell assay.Invasion assay with a Matrigel-coated membrane and migration assay with aMatrigel-uncoated membrane were performed using a24-well chamber system (BDBiosciences, Bedford, MA), according to the manufacturer’s instructions. The cells weretrypsinized and seeded in the upper chamber at2.5×105cells/well in serum-free medium.The vector carrying LacZ or NDRG2, at a multiplicity of infection (MOI) of40, wasadded immediately after cell plating. Medium supplemented with10%FBS (used as achemo-attractant) was placed in the bottom well. Incubation was carried out for24h at37℃in humidified air with a5%CO2atmosphere. The cells were allowed to migratethrough a porous, Matrigel-coated or uncoated membrane (BD Biosciences). After theincubation, the chambers were removed, and invading cells on the bottom side of themembrane were fixed with methanol and stained with Gimsa. The number of invadingcells or migrating cells were determined by counting five high-power fields on eachmembrane and calculated as the mean number of cells per field.2.4Plate clone formation test2.5Annexin V-FITC/PI apoptosis detection assay2.6Morphological observing the effects of NDRG2on CaKi-1cells2.7Wound-healing assayCells were seeded (5×105cells per well) in triplicate in6-well plates. After removingthe medium,40MOI adenovirus expressing NDRG2or the negative control gene LacZ(Ad-LacZ) was added in serum-free RPMI1640, incubated for2h, replaced with freshRPMI1640supplemented with10%FBS. Plates were then incubated for12h. Gently andslowly scratch the monolayer with a cell scraper across the center of the well. Tenscratches locations were chose in advance. Plates were then incubated for12h,24h and48h. Under the inverted phase contrast microscope photograph, get average measurementof each time point scratch sides of cell migration distances.2.8Western-blot was used to analyzed the effects of NDRG2on the protein expression of HIF-1α and VEGF in CaKi-1cells.2.9Immunofluorescence assay was used to analyzed the effects of NDRG2on the proteinexpression of HIF-1α and VEGF in CaKi-1cells.CaKi-1cells were seeded on6-well plates. After removing the medium,40MOIAd-NDRG2or Ad-LacZ was added in serum-free RPMI1640, incubated for2h, replacedwith fresh RPMI1640supplemented with10%FBS. Plates were then incubated asdescribed above for24h. After permeabilization (0.5%Triton X-100), fixation (4%formaldehyde),and blocking (10%normal goat serum and0.5%Triton X-100), mouseanti-NDRG2, HIF-1α and VEGF monoclonal antibody was added to cells for overnight at4℃respectively. Cells were then stained with FITC-conjugated or Cy3-conjugated goatanti-mouse polyclonal antibody at37℃for2h in the dark. The fluorescence stainingintensity was then examined by immunofluorescence microscopy.2.10ELISA was used to analyzed the effects of NDRG2on the secretion of VEGF inCaKi-1cells.Cells were seeded (5×105cells per well) in triplicate in24-well plates. After removingthe medium,40MOI Ad-NDRG2or Ad-LacZ was added in serum-free RPMI1640,incubated for2h, replaced with fresh RPMI1640supplemented with10%FBS.Conditioned media of the24h cell cultures were assayed for VEGF by using commercialsandwich ELISA kits (Endogen, Woburn, MA, USA) according to manufacturer’sinstructions. Each sample was tested in duplicate. Data were expressed in VEGF (pg/ml).3NDRG2inhibiting renal cancer cells’ proliferation in vivo by up-regulating pVHLand down-regulating HIF-1α and VEGF3.1Growth inhibition assays in vivo.The Six-week-old BALB/C athymic (nu/nu) mice were provided by the ExperimentalAnimal Center of the Fourth Military Medical University (Xi’an, China). All of theprotocols were approved by the Animal Care and Use Committee at the Fourth MilitaryMedical University and were performed in accordance with the animal care rules set forthby the university. All efforts were made to reduce the number of animals used and tominimize their suffering. CaKi-1cells were harvested and resuspended in sterile PBS. CaKi-1cells (5×106cells) in0.2ml were injected subcutaneously into the left flanks ofthe6-week-old nude mice. When the mean size of tumors reached200mm3, all the micewere divided into three groups randomly (Ad-NDRG2, Ad-LacZ and PBS Control groups,n=10per group). Intratumoral injections of adenovirus (1×109pfu in100ul PBS) weremade every3d for a total of seven times. Tumor volumes were calculated based on calipermeasurements of the length (a) and width (b) of the lesions using the following formula: V=ab2/2. The growth curve was then derived from these data. The conditions of the micewere monitored every day, and mouse survivals were recorded throughout theexperimental period. The mouse was sacrificed by cervical dislocation, when it appearedcachexia, which was characterized as an overall state of ill health, accompanied by a lossof lean body mass and fat mass, weakness, fatigue, sluggishness, sharply decreased foodintake, ascite and anorexia. All sacrifices were performed under anesthesia (130mgketamine/8.8mg xylazine/kg body weight). Then tumor specimens were removed toprepare paraffin sections for histological staining.3.2The immunohistochemistry staining for detecting the expression of NDRG2, pVHL,VEGF and HIF-1α in tumor tissues of tumor-bearing Mice4The research of isolating, culturing, identifying and renal tumor tracking abilitiesof SD rat BMSCs4.1Cells isolating, culturing and identifying of SD rat BMSCs4.2MTT assay was used to test the proliferation abilities of BMSCs4.3Transwell assay was used to test the renal tumor tracking abilities of BMSCs4.4Immunofluorescence assay was used to test the effect of NDRG2gene on BMSCs’surface markers4.5Transwell assay was used to test the effects of NDRG2gene on BMSCs’ renal tumortracking abilities.[Results]1The expression and correlation of NDRG2, pVHL, HIF-1α and HIF-2α in renalclear cell carcinoma tissues and normal renal tissues.The rate of NDRG2positive expression in CCRCC tissue specimens was30.0%, which was significantly lower than that in normal renal tissue60.0%(P <0.01). And the rate ofpVHL positive expression in CCRCC tissue specimens was also significantly lower thanthat in normal renal tissue (26.9%v.s.66.2%, P <0.01). On the contrary, the rate ofHIF-1α positive expression in CCRCC tissue specimens was significantly higher than thatin normal renal tissue (55.4%v.s.33.7%, P <0.01). And the rate of HIF-2α positiveexpression in CCRCC tissue specimens was significantly higher than that in normal renaltissue (57.7%v.s.35.4%, P <0.01). Furthermore, the expression levels of NDRG2werepositively correlated with those of pVHL in CCRCC tissue specimens（r=0.749，P <0.01）, while the expression levels of NDRG2were negatively correlated with those ofHIF-1α in CCRCC tissue specimens（r=-0.331，P <0.01）. And the expression levels ofNDRG2were also negatively correlated with those of HIF-2α in CCRCC tissuespecimens(r=-0.253，P <0.01).2Inhibition of renal cancer cells’ proliferation, metastasis and VEGF secretion byNDRG2regulating HIF-1α in vitro2.1The expression of NDRG2protein were analysed by Western-blotAfter infection for24h, strong expression of the NDRG2protein in CaKi-1cells wasconfirmed by Western-blot analysis using a monoclonal antibody against NDRG2.2.2NDRG2overexpression inhibited proliferation of human renal cancer cells in vitroCell proliferation was significantly inhibited in Ad-NDRG2group CaKi-1cells,respectively, comparing to Ad-LacZ group and Control group.2.3Invasion and migration of CaKi-1cells were measured by Transwell chamber assayThe results showed that the number of CaKi-1cells that invaded the inferior chamber inthe Ad-NDRG2group (22.6±5.70) was significantly decreased as compared with theControl group (37.8±6.42)，(P＜0.01) and the Ad-LacZ group (36.2±5.56)，(P＜0.01).The results showed that the number of CaKi-1cells that migrated the inferior chamberin the Ad-NDRG2group (25.1±5.41) was significantly decreased as compared with theControl group (52.3±6.39)，(P＜0.01) and the Ad-LacZ group (51.2±5.66)，(P＜0.01). 2.4Plate clone formation testThe clone formation rate of cells infected with Ad-NDRG2was (34.3±4.64)%significantly higher than Control group (61.8±6.13)%and Ad-LacZ group (61.7±6.67)%,(P＜0.05).2.5Annexin V-FITC/PI apoptosis detection assayThe apoptosis rate of cells infected with Ad-NDRG2was (18.7±1.83)%significantlyhigher than Control group (7.1±1.26)%and Ad-LacZ group(6.9±0.98)%,(P＜0.05).2.6Morphological observing the effects of NDRG2on CaKi-1cellsUnder light microscope，the Ad-NDRG group CaKi-1cells became round and weresicker than Control group and Ad-LacZ group.2.7Wound-healing assayWound scratch assay showed that migration distances of Ad-NDRG2group CaKi-1cells were less than Control group and Ad-LacZ group, at12,24,48h.2.8Western-blot was used to analyzed the effects of NDRG2on the protein expression ofHIF-1α and VEGF in CaKi-1cells.The levels of NDRG2protein in Ad-NDRG2group CaKi-1cells were significantlyhigher than that in Control group and Ad-LacZ group. However the levels of HIF-1α andVEGF protein in Ad-NDRG2group CaKi-1cells were significantly lower than that inControl group and Ad-LacZ group.2.9Immunofluorescence assay was used to analyzed the effects of NDRG2on the proteinexpression of HIF-1α and VEGF in CaKi-1cells.Immunofluorescence assay results showed that the levels of NDRG2protein inAd-NDRG2group CaKi-1cells were significantly higher than that in Control group andAd-LacZ group. However the levels of HIF-1α and VEGF protein in Ad-NDRG2groupCaKi-1cells were significantly lower than that in Control group and Ad-LacZ group.2.10ELISA was used to analyzed the effects of NDRG2on the secretion of VEGF inCaKi-1cells.Under condition of normoxia and hypoxia, the levels of VEGF in Ad-NDRG2groupCaKi-1cells were significantly lower than that in Control group and Ad-LacZ group. 3NDRG2inhibiting renal cancer cells’ proliferation in vivo by up-regulating pVHLand down-regulating HIF-1α and VEGF3.1Growth inhibition assays in vivoThe tumor sizes of tumor-bearing mice in Ad-NDRG2group were significantly smallerthan that in Control group and Ad-LacZ group(P＜0.01). And no significant differenceswere observed between Control group and Ad-LacZ group.3.2Kaplan-Merier survival analysisIntratumoral injection of Ad-NDRG2significantly prolonged mice survival times(P＜0.01). And no significant differences were observed between Control group and Ad-LacZgroup.3.3ImmunohistochemistryThe results showed that the expression levels of NDRG2and pVHL in tumors ofAd-NDRG2group tumor-bearing mice were significantly higher than that in Controlgroup and Ad-LacZ group. And the expression levels of HIF-1α and VEGF in tumors ofAd-NDRG2group tumor-bearing mice were significantly lower than that in Control groupand Ad-LacZ group. ImagePro Plus soft analysis showed that IOD values of NDRG2andVHL in Ad-NDRG2group were significantly higher than that in Control group andAd-LacZ group. And IOD values of HIF-1α and VEGF in Ad-NDRG2group weresignificantly lower than that in Control group and Ad-LacZ group. And no significantdifferences were observed between Control group and Ad-LacZ group.4Research of in vitro isolation technology, cultivation technology, identificationtechnology and renal tumor tracking abilities of rat bone marrow mesenchymal stemcells.4.1We use density gradient centrifugation method to isolate BMSCs and culture cells with10%fetal bovin serum L-DMEM. Immunofluorescence assay showed negative expressionof CD34, CD45and positive expression of CD44,CD90in obtained BMSCs. After14days of adipogenic induction and25days of osteogenic induction, obtained cellsperformed adipocyte and osteobast morphology and can be satined by Oil-Red-O andAlizarin-Red. 4.2MTT showed that proliferation abilities of P0-BMSCs was less than P3-BMSCs andP6-BMSCs. And there was no difference between P3-BMSCs and P6-BMSCs in theproliferation abilities.4.3Transwell showed the number of BMSCs cells that invaded the inferior chamber in theCaKi-1group was significantly more than the control group and the Ad-LacZ group（42.4，15.6and16.1respectively)4.4Immunofluorescence assay showed that NDRG2gene modification had no effect onBMSCs’ surface markers.4.5Transwell showed that NDRG2gene modification had no effect on BMSCs’ renaltumor tracking abilities.[Conclusions]1. The rate of NDRG2and pVHL positive expression in CCRCC tissue specimens wassignificantly lower than that in normal renal tissue. On the contrary, the rate of HIF-1α andHIF-2α positive expression in CCRCC tissue specimens was significantly higher than thatin normal renal tissue. Furthermore, the expression levels of NDRG2were positivelycorrelated with those of pVHL in CCRCC tissue specimens, while the expression levels ofNDRG2were negatively correlated with those of HIF-1α and HIF-2α in CCRCC tissuespecimens.2. NDRG2could inhibit proliferation, invasion and migration of human renal cancer cellsin vitro.3. Ad-NDRG2could up-regulate the expression of NDRG2and down-regulate theexpression of HIF-1α and VEGF, and NDRG2could inhibit the secretion ability of VEGFin CaKi-1cells.4. NDRG2could inhibit the proliferation ability of CaKi-1cells in vivo, what is more,intratumoral injection of Ad-NDRG2significantly prolonged mice survival times.5. NDRG2could up-regulate the expression of pVHL in tumors of tumor-bearing mice,furthermore NDRG2could down-regulate the expression of HIF-1α and VEGF in tumorsof tumor-bearing mice.6. NDRG2gene modification had no effect on BMSCs’ surface markers and renal tumortracking abilities. And BMSCs could be used to treat renal cancer as gene carriers. |
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