KIF15 is An Oncogene of Prostate Cancer and is A Key Prognostic Factor in Patients with Prostate Cancer

KIF15, a member of kinesin superfamily proteins, has been found that play a of vital role in the carcinogenesis of various malignant tumor. But whether KIF15 can facilitate the evolution of prostate cancer (PCa) is still unknown. This study aims to explore its biological function in PCa cells and its relevance to prognosis and clinical features in PCa patients. KIF15 expression at mRNA and protein level in tumor and normal tissues was detected by quantitative real-time PCR (RT-PCR) and immunohistochemistry. Then the correlations between KIF15 expression and PCa patients’ clinical characteristics was analyzed. After inhibiting the expression of KIF15 by shRNA, the role of KIF15 on proliferative capacity of PCa was evaluated by using MTT assay. The function of KIF15 on metastatic potential of PCa was determined by using transwell assay. The prognostic value of KIF15 was determined by using bioinformatics analysis.


Introduction
Prostate cancer is still a common malignant tumor in male in developed countries, and its morbidity and mortality have gradually increased in recent years [1][2][3]. In addition, the increasing economic burden and care costs for treating prostate cancer each year have become global issues [4]. Due to the increasing popularity of PSA screening in China, the morbidity of PCa in Chinese male is also increasing, which form a problem that cannot be ignored [5]. Although prostate cancer patients can bene t from radical prostatectomy and androgen deprivation treatment, and the overall survival (OS) of PCa patients has continued to increase in recent years, PCa patients inevitably have recurrence and metastasis 3-5 years after receiving the initial treatment. Eventually it progressed to castration-resistant prostate cancer [6]. So far, the molecular mechanism of the occurrence and development of PCa hasn't been fully understood [7,8]. At the same time, prostate cancer is a heterogeneous disease [9]. With the development of precision medicine, it is urgent to nd an oncogenic factor that promotes the pathological development of prostate cancer to provide better treatment strategies for urologists [10].
Kinesin superfamily proteins was known as "hub" protein in microtubule depolymerizing, chromosome segregation and organelle transport [11]. So these proteins play an essential role in cellular morphology, mitosis and meiosis [12,13]. In recent years, more and more studies have pointed out that these proteins are closely related to the tumorigenesis and occurrence of cancer [14,15]. Xiong et al. demonstrated that the upregulation of KIF20A promoted cell proliferation and drug resistance via JAK/STAT3 pathway in colorectal cancer [16]. KIF15, which belongs to the Kinesin-12 family, is thought to be related to the proliferation, invasion, metastasis and poor prognosis of various tumor cells [17]. For instance, Wang et al found that suppressing KIF15 expression inhibit proliferation ability of pancreatic cancer effectively by activated MEK-ERK pathway and KIF15 promotes the transformation of tumor cells from G1 phase to S phase by regulating cell cycle related proteins [18]. Except that, Millic et al highlighted that KIF15 inhibitor combined with Eg5 inhibitor is an effective strategy to solve the problem of chemotherapy resistance [19].
But there is few researches with regard to the characterization of KIF15 in the tumorigenesis of prostate cancer.
Therefore, in the present research, we compared the KIF15 expression in PCa tissues and prostatic tissues using immunohistochemical methods. Furthermore, the correlation between the KIF15 expression and the PCa patients prognosis was analyzed by means of bioinformatics. Finally, we used MTT, Transwell, and western blot assay to invest the function of KIF15 in the proliferation and invasion abilities of PCa in vitro. This study aims to prove that KIF15 can be a original therapeutic target for PCa patients and a predictor for prognostic analysis of PCa patients.

Patients information and tissues specimen
We collected clinical information and tissue samples of patients undergoing radical prostatectomy in our institution from 2016 to 2019. Clinical information includes age, PSA level at initial diagnosis, clinical stage, Gleason score, and whether the seminal vesicles and lymph nodes are invaded. None of these patients received androgen deprivation therapy, chemotherapy or radiation before surgery. All patients signed informed consent. This study was approved by the Ethics Committee of Tangshan Gongren Hospital. The clinical trial registry number is GRYY-LL-2019-40.
According to the instructions, a mixture of 100 nM shRNA and lipofectamine 3000 Transfection Reagent (Invitrogen,L3000015,USA) was added to the serum-free RPMI 1640 medium. Detection of KIF15 knockdown e ciency by Western blot after 48 hours Quantitative real-time PCR Total RNA was extracted from tumor tissues and normal tissues by using Trizol reagent (Invitrogen,15596018,USA).According to the manufacturer's protocol, Reverse Transcription system, which included DEPC water, primer-mix, dNTP-mix, DTT and 5×PrimeScript buffer, was used to reversetranscribed total RNA to generate cDNA. SYBR Premix Ex Taq was performed to quanti ed the expression of KIF15 at mRNA level and the expression of GAPDH was used as internal reference. The primers of KIF15 and GAPDH used for qPCR were as follows: Western blotting RIPA buffer (Invitrogen,89900,USA) was used to lyse the cell lines to obtain protein. The concentration of extracted protein was measured by using BCA kit (Solarbio,PC0020,Beijing). 25ug protein samples was separated by SDS-PAGE and then transferred to the PVDF membrane. Subsequently, the membrane was incubated with the KIF15 antibody (Abcam,ab272615,UK), GAPDH antibody (Abcam, ab181602,UK), Ki67 antibody (Abcam, ab16667,UK), or MMP9 antibody (Abcam, ab219372,UK) at 4ºC overnight. At last, the membrane was incubated with secondary antibodies for 1 hour and the bands was displayed by using ECL kit (Solarbio,PE0020,Beijing). ImageJ was performed for the semi-quantitative analysis of bands.

MTT assays
The cells in the logarithmic growth phase, which transfected with shCON or shKIF15, were collected and seeded on 96-well plates with 800 cells/well. After 6 days, MTT reagent (Solarbio,M1020,Beijing) was added into every well and cultured with cells for 2 hours. Then remove the MTT reagent, incubate with the cells for half an hour after adding DMSO (Solarbio,D8371,Beijing), and detect the absorbance by using a microplate reader at 570 nm.

Transwell assay
The matrigel (Corning,354234,USA) and the pre-chilled serum-free 1640 medium were mixed on ice at a ratio of 1 to 8. 60 ul diluted matrigel was added to the upper chamber (Corning,3496,USA) of transwell and 1640 medium containing 20% serum was added below the chamber. Add 9,000 cells to each chamber in a 24-well plate and incubate in the incubator for 30 hours. Discard the supernatant and stain the cells with crystal violet. Remove the remaining cells in the upper chamber at the same time. Take a photo under the microscope and Perform a cell count.

Immunohistochemistry
The tissue-embedded para n block was cut into sections with a thickness of 4 um. The sections were subjected to dewaxing, hydration, and antigen retrieval in order. A peroxidase blocker was added to the sections to block the effect of peroxidase. After washing with PBS, the sections were incubated with the primary antibody (KIF15, Abcam,ab272615,1:50,UK) at 4ºC overnight. Sections were then incubated with secondary antibodies for 1 hour at 37 ºC. DAB kit (Solarbio,DA1010,Beijing)was used for coloration and then hematoxylin staining was added. Finally, the slices were dehydrated and transparently treated, and neutral gum was used for sealing. Observe and take pictures under the microscope.
Bioinformatics analysis GEPIA(http://gepia.cancer-pku.cn/) was used to analyze the PCa patients' data from TCGA(The Cancer Genome Atlas) dataset. Using the median value of KIF15 mRNA expression level in PCa patients as the cutoff value, the patients were divided into a KIF15 high expression group and a KIF15 low expression group. Kaplan-Meier method was performed to analyze the overall survival and disease-free survival in the two groups of patients. P <0.05 was considered statistically signi cant.

Statistical analysis
Statistical analysis was performed by using SPSS 22.0 software. The correlation between KIF15 expression and the clinical feature of PCa patient was analyzed by using Chi-square test. Paired t test and rank sum test were used to analyze the differences between two groups. P < 0.05 is considered statistically signi cant. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and n.s. indicates no signi cant difference.

KIF15 was highly expressed in PCa
In order to evaluate the KIF15 expression in PCa, at rst, we collected tissues from 7 PCa patients undergoing radical prostatectomy, and tested the expression level of KIF15 in PCa tissues and prostatic tissues by using qPCR assay. As shown in Fig. 1A, KIF15 expression at mRNA level in PCa tissues is higher when compared with prostatic tissues, and it has statistical signi cance. Then we detected the expression at protein level of KIF15 in 3 PCa cell lines (C4-2, Lncap, and PC3) and 1 prostatic hyperplasia cell line (BPH-1) by using western blot, and found the expression of KIF15 at protein level in PCa cell lines was higher when compared with prostate hyperplasia cell line (Fig. 1B). Except that, the IHC staining was conducted to explore the KIF15 expression in 84 para nized prostate cancer tissues and 15 normal prostate tissues. As shown in Fig. 1C, KIF15 is mainly located in the cell cytoplasm. Compared with normal tissues, KIF15 expression levels in prostate cancer tissues are signi cantly increased (P < 0.001).
In summary, KIF15 is abnormally highly expressed in PCa cells at transcription and translation levels.

Suppress KIF15 expression inhibits the proliferation and invasion of PCa cells
In order to further explore the biological effect of KIF15 in the carcinogenesis of PCa, we successfully established 2 PCa cell lines (C4-2 and Lncap) with stable knock-down of KIF15 by using shRNA. As shown in Fig. 2, veri cation by qPCR and WB assay found that the KIF15 expression of two PCa cell lines were successfully knocked down in both mRNA level (P < 0.01) and protein level (P < 0.01), and the knockdown e ciency is greater than 70%. Previous studies have demonstrated that KIF15 was involved in the proliferation, cell apoptosis and served as novel therapeutic targets in various cancers such as osteosarcoma, melanoma and lung adenocarcinoma [17,[20][21][22]. Hence, we used two successfully constructed stable knockdown KIF15 cell lines to verify whether KIF15 affect the proliferation and invasion ability of prostate cancer. As shown in Fig. 3A, the MTT assay demonstrated that the proliferative capacity of PCa cells decreased signi cantly in two shKIF15 cell lines (P < 0.001). At the same time, we found that the proliferation-related marker Ki67 expression was signi cantly reduced in two shKIF15 cell lines by using WB assay when compared with shCON group (P < 0.001, Fig. 3D and 3E). The results of transwell invasion assay informed that the number of PCa cells migrating to the lower transwell chamber through matrigel decreased signi cantly after inhibiting KIF15 expression (P < 0.001, Fig. 3B and Fig. 3C).Meanwhile, the invasion-related marker MMP-9 expression was also signi cantly reduced after knocking down KIF15 by using western blot assay (P < 0.001, Fig. 3F and 3G). The results of in vitro experiments showed that knocking out KIF15 can effectively inhibit the proliferation and invasion ability of PCa cells.
High expression of KIF15 at protein level was related with progression of Prostate cancer According to results of immunohistochemistry assay, the 84 PCa patients from our institution was divided into high-and low-expression KIF15 groups. Chi-square test was conducted to explore the correlation between the clinicopathological characters of PCa patients and the KIF15 expression (Table 1). Interestingly, the high KIF15 expression was related clinical T stage (P = 0.004), seminal vesicle invasion (P = 0.02) and lymph node metastasis of PCa (P = 0.03). However, the correlation between the KIF15 expresion and the age (P = 0.54), perioperative PSA level (P = 0.87) and Gleason score (P = 0.08) of PCa patients was not statistically signi cant.

Overexpression of KIF15 was related with adverse prognosis of PCa patients
Kaplan-Meier method was conducted to analyze the value of KIF15 in predicting the PCa patients prognosis. The overall survival (OS) and disease-free survival (DFS) was analyzed in 498 PCa patients from the cancer genome atlas (TCGA) database. The median value of KIF15 expression was used as the cutoff value to divide PCa patients into high KIF15 expression group and low KIF15 expression group. As shown in Fig. 4B, the DFS of high KIF15 expression was poorer than the low KIF15 expression group signi cantly (P = 0.0076). However, the OS didn't have signi cant difference between the two groups (P = 0.17, Fig. 4A). The results demonstrated that KIF15 expression may be a novel prognostic factor for the DFS in PCa patients which was closely related with the clinical outcomes of PCa patients.

Discussion
PCa is the most familiar malignant tumor with high morbidity and mortality among men in developed countries [23,24]. With the popularization of PSA screening in China, the number of patients with prostate cancer has increased year by year, causing a huge economic and health burden to the society [25]. Due to prostate cancer is a heterogeneous multifocal solid tumor, it is di cult to accurately predict the prognosis of each PCa patient. At the same time, the tumorigenesis and occurrence of PCa has not been fully understood. Early diagnose and accurate classi cation of prostate cancer is of vital for urologists to develop therapeutic schedule. Therefore, it is urgent need to identify prognostic factors and potential therapeutic targets for PCa patients.
Previous studies has showed that the KIF15 expression was up-regulated in various types of malignant tumors such as osteosarcoma [17], melanoma [21], lung adenocarcinoma [22], pancreatic cancer[18] and so on. Song et al identi ed thirteen kinesin superfamily associated genes included KIF15 were enriched in the stage of DNA replication and cell cycle in breast cancer by using bioinformatic analysis. The overexpression of KIF15 was also associated with P53 signal pathway and mismatch repair[26]. Yu's study also con rmed that KIF15 was related with tumorigenicity of melanoma. They demonstrated that KIF15 could promote tumor growth and inhibit apoptosis of tumor cells. And the bioinformatic data found three potential targets of KIF15 included BIRC59, CDK4, and WNT5A [21] .
To our knowledge, this is rst time to explore the biological effect of KIF15 in the carcinogenesis of PCa. In present research, we found that KIF15 was overexpressed in PCa when compared with normal prostatic tissues. Interestingly, the the expression of KIF15 at protein level was related with the progression of PCa such as higher clinical T stage, seminal vesicle invasion and lymph node metastasis. And the bioinformatic analysis con mred that the KIF15 high expression group in patients with PCa have an shorter DFS. Except that, the results of in vitro experiments con rmed that KIF15 can promote the proliferation and invasion ability of prostate cancer cells. This result further con rmed that KIF15 played a vital role in promoting the carcinogenesis of PCa at the molecular level. In a conclusion, this preliminary mechanism study con rmed that KIF15 can served as a novel therapeutic target for patients in PCa. Recently study showed that they developed four drug candidates targeting KIF15 by using dihydropyrrole and dihydropyrazole derivant [20]. Therefore, we believe that KIF15 inhibitors will have a promising prospect in the precise treatment of PCa patients in the near future.

Conclusion
In this study, we found that KIF15 was overexpressed in PCa. Except that, high KIF15 expression was correlated with clinical stage, seminal vesicle invasion, lymph node metastasis, and adverse DFS in PCa patients. Finally, we con rmed that KIF15 promoted proliferation and invasion of PCa cells. This study also has some limitations. The underlying mechanism of how KIF15 affects the proliferation and invasion of PCa cells should be elucidated in detail. Due to the short follow-up time and small samples of patients in our institution, no survival analysis was performed on these PCa patients. Large-scale randomized clinical controlled studies was needed to further con rm the relationship between KIF15 expression and PCa patient prognosis. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate: Written informed consents were obtained from all participants, and this study was permitted by the Medical Ethics Committee of Tangshan Gongren Hospital.   Knockdown of KIF15 inhibits the proliferation and invasion abilities of PCa cells. A) MTT assay was used to estimate the growth in both C4-2 and Lncap cell lines after knocking down KIF15. The absorbance value was measured at 490 nm (***P<0.001). B) Transwell assay was used to estimate the invasion ability of C4-2 and Lncap cell lines after knocking down KIF15. C) Histogram was used to analyze the number of invasion cells (***P<0.001). D) The expression levels of Ki67 was detected by western blot after knocking down KIF15 in both C4-2 and Lncap cell lines. E) The histogram is a semi-quantitative analysis of the Ki67 expression at protein level(***P<0.001). F) The expression levels of MMP9 was detected by western blot after knocking down KIF15 in both C4-2 and Lncap cell lines. E) The histogram is a semi-quantitative analysis of the MMP9 expression at protein level(***P<0.001).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table1.xlsx