Manipulations In HIWI And ERCC1 Level Exerts Influence On The Proliferation Of Human Non-Small Cell Lung Cancer Cells And It's Clinical Relationship

Lung cancer is the leading cause of cancer-related mortality worldwide, though the molecular imaging such as FDG-positron emission tomography (FDG-PET) has markedly improved the diagnosis and the treatment of lung cancers. HIWI belongs to the human Piwi family, which is well known for their roles in RNA silencing. HIWI has been shown to play essential roles in stem cell self-renewal. Recently, results suggested that HIWI was overexpressed in lung cancers.Recently, the research results showed that HIWI expressed in cancer cells. Current clinical treatment of non-small cell lung cancer was still the main scheme and the principle of surgical was to remove of cancer tissue, chemotherapy after surgery has become an important part of comprehensive treatment for non-small cell lung cancer,but resistance and resistance to chemotherapy became the main problems, was also the important factor affecting the effects of non-small cell lung cancer treatment. Large amounts of DNA repair factor exists in the body's cells, at the same time because of the different types of DNA damage, and in the body's repair played a special role, NER nucleotide excision repair system was an important way to repair genes, including 1 excision repair cross gene ERCC1 namely in repair system played a connection damage identification and the effect of cutting, also found that the platinum chemotherapy drug resistant and sensitive properties could also be the excision repair cross complementary gene expression to display, Relevant excision repair cross complementary relationship between gene 1 and non-small cell lung cancer were reported, but it was not be unified.To explore the HIWI and excision repair cross-complementing 1 (ERCC1) in non-small cell lung cancer tissues, gene expression levels of HIWI and ERCC1 prognostic evaluation, summary basis and value it provides for individualized clinical treatment.Part one:Manipulations in HIWI level exerts influence on the proliferation of human non-small cell lung cancer cellsObjective:To assess HIWI over expression in non-small cell lung cancer specimens and HIWI's influence on the growth of A549 cells.Reveal the HIWI in non-small cell lung cancer cells play an important role in the growth and to provide a basis for the study of new anticancer therapy potential targets.Methods:1.Material: The 57 intratumor specimens and peritumor specimens (as control; at a distance?10 mm from the tumor edge) were resected from these patients before being exposed to radiotherapy and chemotherapy and were immediately frozen in liquid nitrogen and stored at -80 ?. NSCLC A549 cells were cultured.2.RNA extraction and quantitative real-time polymerase chain reaction:Total RNA from the intra-tumor, peritumor specimens, or A549 cells was prepared Real-time quantitative PCR assay (RT-qPCR) was performed to quantify the HIWI mRNA with a SYBR PrimeScript RT-qPCR Kit.3.1mmunoblot analysis for HIWI expression NSCLC cancer specimens were homogenized and subject to the protein isolation with a cell lysis reagent according to the manual. All protein samples were separated by 10% SDS-PAGE gel, and were transferred to a polyvinylidene fluoride membrane and were blocked in tris-buffered saline containing milk (5%). The band of HIWI and ?-actin were respectively detected with the rabbit polyclonal antibody to HIWI 4.Manipulation of HIWI expression in A549 cells:To overexpress HIWI in A549 cells, we amplified the wild HIWI coding sequence and enhanced green fluorance protein (EGFP) and cloned them into the eukaryotic expressing pcDNA3.1(+) vector with a linker of 2A self-cleavage sequence, which facilitate them to transcript into one mRNA sequence and translated into two separate proteins. And the HIWI-2A-EGFP-pcDNA3.1 (+) or control CAT-2A-EGFP-pcDNA3.1 (+) vectors was transfected into A549 cells by lipofectamine 2000 and detected knockdown the HIWI expression.5.Cell count assay, CCK-8 assay and Colony formation assay.Results:1.HIWI is overexpressed in NSCLC specimens:? To investigate the HIWI expression in NSCLC tissues, we determined both mRNA and protein levels of HIWI in NSCLC specimens with RT-qPCR and western blot assay. It was demonstrated that there was a significantly high HIWI level in the intratumor specimens than in the peritumor specimens (mean differences was 0.7642, and the 95% confidential interval is 0.5695 to 0.9590, R2=0.5249 and p<0.01, by paired-samples t-test) from 57 non-small cell lung cancer patients.? Then we re-evaluated the HIWI expression in protein level by western blot assay in 15 intratumor specimens and 15 peritumor specimens, the protein level of HIWI in the intratumor specimens was also significantly higher than in the peritumor specimens (p<0.01, paired-samples t-test). Taken together, we confirmed the overexpression of HIWI in NSCLC specimens. 2.Overexpressed HIWI promotes A549 cell proliferation:? we generate an HIWI and EGFP co-expressing A549 cell line, in which HIWI and EGFP were co-transcribed into a single mRNA, but were translated separately into two HIWI and EGFP. A549 cell clone overexpressing HIWI was selected under the G418 pressure, and propagated serially for five passages. ? The growth difference between A549 HIWI (+) cells and A549 control cells was reconfirmed by the colony formation assay, there were more colonies formed by A549 HIWI (+) cells than A549 control cells,24 or 48 hours post an inoculation of same cell number (p< 0.01 respectively). ? Then we repeated the experiment under the presence of Cisplatin, there were less cells reduced in the A549 HIWI (+) group than in the A549 control group (p<0.05 or p<0.01). The growth difference between A549 HIWI (+) cells and A549 control cells was reconfirmed by the colony formation assay. ? The growth difference between A549 HIWI (+) cells and A549 control cells was reconfirmed by the colony formation assay, there were more colonies formed by A549 HIWI (+) cells than A549 control cells,24 or 48 hours post an inoculation of same cell number (p< 0.01 respectively).Thus, we confirmed in vitro the promotion of overexpressed HIWI to the proliferation of NSCLC A549 cells.3.HIWI knockdown by siRNAs blocks the promotion by overexpressed HIWI to the A549 cell proliferation.We then re- evaluated the promotion by overexpressed HIWI to the A549 cell proliferation with the RNAi method.? we knocked down the HIWI expression with hiwi specific siRNAs in the A549 HIWI (+) cells, A549 HIWI (+) cells post the transfection with 10 nM siRNA-HIWI 1 or siRNA-HIWI 2 expressed a significantly reduced HIWI in both mRNA and protein levels, compared to the siRNA-con transfected cells (p< 0.01 respectively, unpaired t test. ? The cell count assay indicated a significantly reduced cell proliferation in the siRNA-HIWI 1-or siRNA-HIWI 2-transfected cells (p< 0.05 respectively for the 24 hour post transfection, and p< 0.01 respectively for the 48 hour post transfection). ?And the colony formation assay also indicated a significantly reduced colonies formed in both siRNA-HIWI 1 and siRNA-HIWI 2 groups, compared to the control siRNA group (either p< 0.01, by unpaired t test). Thus, we reconfirmed the promotion by the overexpressed HIWI to the proliferation of NSCLC A549 cells by the loss-of-function strategy.Conclution:1.In summary, present study confirmed the HIWI overexpression in NSCLC tissues;2. HIWI improved the growth of non-small cell lung cancer cells in vitro by the functional gain and function loss; 3. It implied the oncogenic role of HIWI in lung cancers.Part two: Non-small cell lung cancer excision repair cross-complementing gene expression and prognosis of studyObjctive:To explore the excision repair cross-complementing 1 (ERCC1) in non-small cell lung cancer tissues, gene expression levels of ERCC1 prognostic evaluation, summary basis and value it provides for individualized clinical treatment.Methods:1.Patients:42 cases of complete surgical resection after pathologic diagnosis of non-small cell lung cancer tissue samples were collected, The case data were selected according to the integrity of data and follow-up.According to the international union of anti-cancer(1997 edition) of the revised criteria of non-small cell lung cancer staging, and preoperative imaging and pathological data of postoperative decide clinical stage I-III a period, la-b period were 18 cases, II a-b period were 11 cases, III period were 13 cases.22 cases were treated with cisplatin postoperative adjuvant chemotherapy (52.38%), the spatients1 survival period were 7-76 months, the average survival period was (35.6±1.5) months.2. Detected in excision repair cross complementary 1 gene and HIWI:42 patients of non-small cell lung cancer specimens were detecteed ERCC1 and HIWI expression by S-P method.ERCC1 and HIWI integrating the histologic grade were used for calculation, the integral was 0 points in (-),1-3 points were represented as weak positive, expressed by (+),3-8 were divided into moderate positive (++),9-12 were divided into strong positive, expressed with (+++), on the basis of the experimental section, HE stain and negative for photos werre judged the result, the final review, above (++) is defined as the positive expression.3. The statistical data obtained according to the clinical stage, histopathological type, age structure, sex, prognosis compared to group. 4. The HIWI ERCC1 and correlation were analysis.5. The relationship between HIWI /ERCC1 expression with lung cancer tumors>5 cm, lobe tumors, tumors burr, triple organ involvement and the relationship between the lung door of mediastinal lymph node enlargement.were studied.Result:1.42 patients, the positive expression rate of HIWI was 55.6%, the positive expression rate (45.24%), and found that the expression levels of ERCC1 and patient age, gender, smoking, clinical stage and pathological conditions no significant correlation (P>0.05), survival comparison, ERCC1 expression is better than negative positive, statistically significant differences (P= 0.003).2. This group of 42 patients with non-small cell lung cancer, survival period for 7-76 months, the average survival period for (35.6+1.5) months. ? positive ERCC1 expression were 19 cases, average survival period was (25.4+1.2) months;ERCC1 negative expression were23 cases, the average survival period was (45.1+2.3) months, through the statistical treatment, according to positive ERCC1 expression and negative expression of man's survival had obvious difference, the negative expression was better than that of positive expression (P=0.003).(2) the HIWI positive expression were 23 cases, the average survival period was 26.2 months;ERCC1 negative expression were 23 cases, the average survival period was 45.1 months, through the statistical treatment, according to positive ERCC1 expression and negative expression of man's survival has obvious difference, the negative expression was better than that of positive expression (P= 0.003).3. The HIWI positively was correlated with the expression of ERCC1, correlation coefficient was 0.841.4. Non small cell lung cancer tumors were greater than 5 cm, lobe tumors, tumors burr, involvement and pulmonary door of mediastinal lymph node enlargement, except lung door of mediastinal lymph node enlargement, ERCC1 and HIW1 positive expression were more the negative expression, there were statistical significance difference.Conclution:1.Excision repair cross-complementing gene 1 (ERCC1) and HIW1 have positive expression rate in non-small cell lung cancer (45.24%)(55.6%), the expression level and age, sex, clinical stage, histological type and smoking had no significant association; 2.1n non-small cell lung cancer, the average survival time than those with negative expression positive expression can be used as prognostic indicators, in the negative expression, independent of gender, age, smoking or prognosis of patients with pathological type, in the positive expression men with clinical stage earlier, the patient's survival has certain advantages, in the negative expression, non-small cell lung cancer can get some survival benefit from chemotherapy; 3.Testing can be used as the reference standard is selected ERCC1 of cisplatin chemotherapy.