Study On The Effect And Mechanism Of Rna Inference Targeting TFF3 Gene On The Proliferation And Invasion Ability Of Thyroid Papillary Carcinoma Cells

Thyroid carcinoma is the common tumor in head and neck, also the most frequently endocrine malignancy. It constitutes 5% of all thyroid neoplasms and account for about 1% of the systemic malignant tumor. It is one of the ten cancer types that are harmful to women’s health.Incidence in women is higher than that of men. The mortality of thyroid carcinoma is one of the most lowerest cancer, with 6.8 case per 100,000. Differentiated thyroid cancers(DTCs), derived from folicular epithelia, divided into two major histological types:papillary thyroid carcinoma(PTC) and follicular thyroid carcinoma according histological types.PTC is the most comman type accounted for about 80% thyroid malignancies.American Thyroid Association(ATA) guidelines recommend fine-needle aspiration biopsy(FNAB) as the most effective diagnosis method for thyroid carcinoma.But many factors may influnce its accuracy and induce 20% of thyroid carcinoma misdiagnosis or missed diagnosis, such as operator-dependent puncture sites and gathering method limit of FNAB detection technology. Therefore, looking for accurate and sensitive diagnostic techniques such as technology based on molecular markers in the diagnosis of individual medical treatment for thyroid cancer is necessary.The standard treatment of thyroid cancer usually includes surgery, thyroid stimulating hormone suppression therapy, radioactive iodine thyroid residue(RAI)treatment,etc. Thyroid carcinoma usually has a good prognosis, with a mortality rate of less than 10%,however, 5% thyroid carcinoma has recurrence and poor prognosis for absent efficency treatment. Effective therapeutic targets and targeted drugs will help to further improve the therapeutic effect of this part of thyroid cancer.Trefoil factor 3 is a small molecules secreted peptides, comprises 42 amino acids containing six cysteine residues, which form disulfide bonds resulting in the characteristic trefoil structure P-domain or trefoil motif.Recent compelling evidence has emergedwhich indicated a pivotal role of TFF3 in oncogenic transformation, growth and metastatic extension of common human solid tumours.TFF3 m RNA and protein were overexpressed in breast carcinoma, gastric carcinoma, prostate cancer; colorectal cancer,endometrial carcinoma and endometrial carcinoma etc. However, Takano found TFF3 down-regulated.in thyroid follicular carcinoma from follicular epithelial cells Then scientists also found TFF3 protein low expression in PTC and anaplastic thyroid carcinoma by immunohistochemical stain. However, the previous work of our teams showed that TFF3 was highly expressed in PTC,and it was related to lymph node metastasis and clinical stage. In this study,we will further explore the role of TFF3 in the occurrence and development of PTC by a series in vitro and in vivo experiments.Part one Expression and clinical significance of TFF3 in humanpapillary thyroid carcinoma specimenObjective:To detect the expression of TFF3 in human papillary thyroid carcinoma specimen, and the relationship with clinical pathological parameters, explore clinical significance of TFF3 in thyroid papillary carcinoma.Methods : Commercial PTC tissue microarray was purchased from Shanghai outdo biotech co.,ltd. Fresh surgical specimens were derived from patients underwent thyroid cancer surgery at the First Affiliated Hospital of Hebei North University in 2014. Patient consent forms were obtained from all patients in accordance with the Declaration of Helsinki. The diagnosis was made by two pathological doctors.Tissue microarray was used to detect TFF3 expression by immunohistochemical stain.Part of surgical specimens were routinely for immunohistochemiscal and in situ hybridization stain(ISH).RT-PCR to detect transcription level of endogenous TFF3 m RNA.Results:1 Expression of TFF3 in microarrayThe typical characteristic of PTC were clearly visible in 31 case carcinoma tissue,such as Papillary structures,ground glass nuclei and irregular karyotype, nuclear grooves.TFF3 positive signal was high expression in the cytoplasm of columnar cells. TFF3 were negative or weakly positive in cytoplasm of cuboidal epithelial cells at paracarcinoma tissue. The positive rates and AOD value of TFF3 in PTC tissues were significantly higher than those in paracarcinoma tissues. The TFF3 were all positive in PTC with lymph node metastasis(15/15), the positive rate was 68.75% in cases without lymph node metastasis(11/16). The expression level(AOD value) of TFF3 in lymph node metastasis cases was significantly higher than that without lymph node metastasis(P<0.05). The strong positive rate of TFF3 in clinical stage III ~IVpatients(17/20) was significantly higher than that in patients with stage I ~II(1/11)(P <0.05).2 Result of TFF3 m RNA by ISHThe positive signals of TFF3 m RNA were blue particles in the cytoplasm.The positive signal of cancer tissue was significantly higher than that of paracacinoma tissues(P < 0.01).3 Results of TFF3 m RNA by RT-PCRThe amplification products of TFF3 m RNA and β-actin were clearly bands between 100 ~ 200 bp and 200 ~ 500 bp, respectively. The ratio of TFF3/β-actin in papillary thyroid carcinoma was significantly higher than that of paracacinoma tissues(P<0.01).Summary:1 TFF3 protein and m RNA were high expression in epithelial cells of papillary thyroid carcinoma.2 Overexpress of TFF3 protein was associated with lymph node metastasis and clinical stage in PTC.Part 2 Construction and screening effective sequence of sh RNAtargeting human TFF3Objective: We constructed sh RNA expression vector and screened the best si RNA sequence targeting human TFF3 by the transient-transfection ofthe lentiviral mediated sh RNA into thyroid carcinoma K1 cells which secrete TFF3 themselves.Methods:1 We designed four different RNA interfere(RNAi) sequences targeting for 132, 170, 258, 537 site of human TFF3 m RNA and one negative control using online design software of Invitrogen company.The sh RNA expression vector were designed according to the principle of Sense+Loop+Antisense+termination signal and synthesized by Gene Copoeia, Guangzhou. After annealing in vitro,insert p LVX-sh RNA-puro vector to construct recombinant plasmid,then enzyme digestion and sequencing analysis.2 The sh RNAs plasmid was transiently transfected into papillary thyroid carcinoma K1 cells. The transfection efficiency was observed by fluorescence microscope at 48 hours after transfection.3 TFF3 m RNA and protein levels in K1 cells were tested by real-time PCR and western blot at 48 hours post transient-transfected.Results:1 Identification of human sh RNA-TFF3 expression vector by Enzyme digestion.p LVX-Sh RNA2-puro-TFF3 plasmid digest with Xho I enzyme digestion.The identification of positive plasmid sequenced by invitrogen company.Results show that the insertion sequences of sh RNA3,4 completed coincidence with the original design sequence.however, genetic mutations in two sequences of sh RNA1~2,so the follow-up study terminated.2 The K1 cells showed obvious green fluorescence under the inverted fluorescence microscopy at 48 hours after transfection,indicated the recombinant plasmid had been successfully transferred into K1 cells. The percent of green fluorescenct cells was(70.45+2.0)%,(79.83+ 2.3)%,(71.7+ 3.0)% in group of sh RNA3,sh RNA4 and sh RNAC,respectively. And the fluorescence intensity was similar in all groups. There was no fluorescence on nontransfected group.3 TFF3 m RNA expression analysisTFF3m RNAexpression significantly decreased in groups of sh RNA3 and sh RNA4 compared with sh RNAC at 48 h after transfection(P<0.01).The silencing efficiency of TFF3 m RNA were 60.67% and 57.33%tested by real-time PCR.4 TFF3 protein expression analysisTFF3 protein content in sh RNA3,sh RNA4 were obviously lower than that of normal groups and sh RNAC(P<0.05). Analysing with TFF3/ β-actin by Image J software.Summary:1 In this part, we successfully designed and synthesized 2 specific targeting TFF3 sh RNA vectors.2 The TFF3 gene transcription and translation level were significantly inhibited after K1 transiently transfected 48 h with recombinant vectors detected by Real-time PCR and Western blot.The results validated the target to TFF3 gene recombinant sh RNA lentiviral vector was successful and effective.Part 3 The Effect of biological activity in human papillary carcinomaTPC-1 cell by lentiviral-mediated sh RNA targeted downregulation TFF3Objective: Construct stable transfected TPC-1 cells targeted down regulation TFF3 to explore silencing TFF3 gene effect on the biological characteristics of TPC-1 cells and the possible mechanism.Methods:1 Lentiviral packaging 293 T cells and viral titer determination.2 In this study, we used the method of lentiviral infection to construct stable transfected TPC-1 cells, and detected the silenced efficiency of TFF3 in stable transfected TPC-1 cells by Quantitative Real-time PCR and western blot.3 Detected silencing of TFF3 effect on biological characteristics of TPC-1 cells.Cell proliferation ability were assessed by clone formation experiment and CCK-8 assay. Scratch assay and transwell cell migration assaywere used to assess cell migration ability.Invasion ability and adhesion ability were also detected.Apoptosis related indexs Bax and bcl-2 expression were detected by Quantitative Real-time PCR and Western blot.Results:1 lentivirus packaging and stable cell line screenedGreen fluorescence were inspired at 493 nm under the fluorescent microscope after sh RNAC and sh RNA-TFF3 packaging 293 T cells. The titer of the r LV-TFF3 was 5.0x107 TU/ml and that of r LV-sh RNAC was1.0x109TU/ml.After several days of screening, sh RNA-TFF3-TPC-1 and sh RNAC-TPC-1 stable cell strain were emitting bright fluorescence and untransfected TPC-1 could not be seen green fluorescence.2 Identification of stable cell linesThe transcription level of TFF3 m RNA were significantly lower in group sh RNA-TFF3 than in the control group(P<0.01). The expression of TFF3 protein in sh RNA-TFF3 was significantly lower than that of control group detected by western blot. TFF3 immunoreactive positive signal was located in the cytoplasm, sh RNA-TFF3 was significantly decreased compared with group TPC-1 and sh RNAC(P<0.01).3 TPC-1 cell proliferation ability decreased silenced TFF3 geneClone formation results show that the average number of clones of TPC-1and sh RNAC cells were(33.7+4.1 and 28.7+3.6) obviously more than that of sh RNA-TFF3 cells(20.7+2.9). Number of clones in sh RNA-TFF3 reduced27.85% and 38.58% compared with that of the TPC-1 and sh RNAC(P< 0.05).si TFF3 significantly inhibited the proliferation of TPC-1 cells by CCK-8assay. Compared with the TPC-1 and sh RNAC cells, the cell proliferation was significantly inhibited in sh RNA-TFF3(P<0.01). Three groups of cells were in logarithmic phase of growth at day2~day4, but OD value of sh RNA-TFF3 cells was significantly lower than that of TPC-1 and sh RNAC at the same time(P <0.01).4 Cell migration ability assayThere were no statistical significant compared the scratch width among TPC-1,sh RNAC and sh RNA-TFF3 at 12 h. The scratch width of TPC-1,TPC-1,sh RNAC and sh RNA-TFF3 were obviously different at 24h(26 ? m,17?m and 75?m, respectively). The cell migration ability of sh RNA-TFF3 groups was significantly lower than that of the other two groups(P<0.01), but no difference between the sh RNAC and TPC-1 group(P> 0.05).Transwell migration assay showed that sh RNA-TFF3 group numbers of transmembrane cells from upper to lower chamber was significantly less than that of sh RNA group and TPC-1 group. Absorbance value of transmembrane cells eluted by acetic 10% acid were differencet among three groups(0.8088+0.0012,1.1590+0.0091 and 1.1950±0.0280; sh RNA-TFF3 group,sh RNAC and TPC-1 in turn). Absorbance value of sh RNA-TFF3 was significantly lower than that of the other groups(P<0.01).5 Ablity of cell invasion assessedThe Transwell invasion experiments results show that cells invasion from upper to lower in sh RNA-TFF3 group were decreased significantly than TPC-1 or sh RNAC(P<0.01). There were no difference between TPC-1 and sh RNAC(P > 0.05).6 Cell adhesion abilityCells of three groups cultured for 2 hours after the abandoned nonadhesion cells and the adherent were used to detect the OD value by CCK-8 assay. The adhesion ability were different in three groups the adhesion rate of sh RNA-TFF3 was 5.16%, which was significantly lower than that of sh RNAC and TPC-1 groups(P<0.05).7 TFF3 gene silenced promotes TPC-1 apoptosis(1) Bcl-2, Bax gene transcriptionThree groups of cells were detected in the expression of bcl-2 m RNA and Bax m RNA. In sh RNA-TFF3 group,Bax was significantly higher than that of sh RNAC, and Bcl-2 was lower than that of the sh RNAC(P < 0.01).(2) The changes of BCL-2 and BAX proteinIn group sh RNA-TFF3 BAX was obvious enhanced and bcl-2 reduced compared with group sh RNAC and TPC-1(P<0.05).(3) Result of TUNEL apoptosisCell apoptosis with nuclear chromatin concentration and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed.TUNEL signals significantly increased in sh RNA-TFF3 group compared with sh RNAC and TPC-1group.Summary:1 Stable knowed down TFF3 expression cell line sh RNA-TFF3-TPC-1was successfully constructed by lentiviral infection.2 The ability proliferation, migration and invasion, anti apoptosis of TPC-1 cells were decreased after TFF3 gene silenced.3 TPC-1cells after silenced TFF3 gene downregulate Bax,but upregulate Bcl-2 expression Part 4 The tumor formation of sh RNA-TFF3- TPC-1 cells in nude miceObjective: TPC-1 cell lines were injected to subcutaneously of nude mice to make a xenograft model for human papillary thyroid carcinoma.Growth of xenograft were observed to assess the effects of TFF3 gene silence.In order to verificate changes of biological characteristics in vivo by sh RNA-TFF3 in TPC-1 cells.Methods:1 Experimental groupsThirty-six athymic nude mice half male and half female were randomly divided into TPC-1, sh RNAC and sh RNA-TFF3 three groups. In their right flank,corresponding cellswere injeceted respectively.2 Cell cultureAll cells were cultured at 37℃ and 5% CO2 in RPMI 1640 supplemented with 10% FBS.Then these cells were collected and suspended at 5× 105cells/ml sterile PBS for implanting.3 Tumor cell suspension implanted and observation of tumor0.2ml cell suspension(5×105) were injected slowly in mice right flankafter skin sterilized with 75% alcohol. The state of mice and xenograft occurrence and growth were observed daily. Time of xenograft formed were recorded, weighing mice and measurement size of tumor using vernier interval of 2 days. Xenograft growth curve were drawed at 28 days.4 The nude mice were removed from the sterile room, after the last tumor size measured and weighing. Then 3 animals of each groups were made small animals in vivo imaging,then euthanasia,take out the tumor samples and take photos immediately, Xenografts were weighed,2/3 of xenografts stored at-80℃freezer,the rest were procuced wax block.5 HE staining and immunohistochemical stainingXenograft were fixed in Bouin’s solution for 12 h, dehydrated by gradient alcohol, transparented by xylene, embedded in paraffin. Sliced section at 5?m thickeness, HE and immunohistochemical stained.6 Total RNA of Xenograft extracted and q PCR for detecting TFF3 m RNA level in three groups.Results:1 Growth status of nude miceXenografts were emerged at the seventh day after PTC-1 and sh RNAC cells implanted into nude mice.The Xenografts were emerged at the tenth dayin the sh RNA-TFF3 group. All three groups of nude mice were in good condition and weight steady growth from 1 to 3 weeks after implanted.The weight of sh RNA-TFF3 group grew slowly or stopped after the 3th weekend.2 Effect of sh RNA-TFF3 on the growth of XenograftXenografts emerged at seventh days on TPC-1 and sh RNAC group and grew fast, However, Xenografts emerged at tenth days at tenth days in sh RNA-TFF3 groups,not only Xenograft formation was delayed, but the growth rate was obviously lagged behind the other two groups. Xenograft emmited green fluorescence by In vivo imaging of small animals. The volume of Xenograft were smaller and fluorescence were weaker than that of sh RNAC group. And male’s Xenograft volume were bigger than female’s.3 Effect on weight of Xenograft by sh RNA-TFF3The tumor weight of sh RNA-TFF3 group was significantly lower than that in TPC-1 and sh RNAC group(P<0.05).4 HE and immunohistochemical staining for XenograftThe xenograft had characteristics of the cancer cells,such as pathological multiple nuclei and nuclear division phase. The yellow or brown color of TFF3 immunoreactive positive signal were located in cytoplasm. TFF3 positive signal in sh RNAC and TPC-1 group were stronger than that of sh RNA-TFF3 group. Compared with the TPC-1and sh RNAC group, the AOD value of TFF3 in sh RNA-TFF3 group significantly lower than that of sh RNAC and TPC-1 group(P<0.01).5 The level of TFF3 m RNA in XenograftThe level of TFF3 m RNA was lower in sh RNA-TFF3 than that of TPC-1and sh RNAC group by q PCR(P<0.01). Tranfected TPC-1 cells by sh RNA-TFF3 in vivo were stable which was similar with that of in vitro cultured condition.Summary:1 We successfully established a Xenograft nude mice model of human papillary thyroid carcinoma of TPC-1cell line.2 In vivo, knockdown of TFF3 gene expression cell can inhibited Xenograft growth.Conclusions:1 High expression of TFF3 in papillary thyroid carcinoma is associated with the lymph node metastasis and clinical stage.2 Knockdown expression of endogenous TFF3 alter the biological characteristics of TPC-1 cells such as inhibited growth, decreased invasive and clone formation ability in vitro etc.All that may be associated with proapoptosis through the of Bcl-2 downregulation and upregulation of Bax.3 TFF3 may be used as a potential therapeutic target in human PTC.